Therapeutic compositions and methods for producing and using the same

Abstract

Disclosed herein are capped RNA molecules comprising one or more modified nucleotides at position +3 or higher with reference to a 5' terminus of the RNA molecule, and methods of making the same. The capped RNA molecules may be made by ligating a 5'-capped modified RNA oligonucleotide to the 3' terminus of an RNA molecule. Also provided are compositions comprising one or more of the capped RNA molecules provided herein, and methods of using said compositions for therapeutic applications, such as in the treatment or prevention of a disease in a subject, such as SARS-CoV-2.

Description

THERAPEUTIC COMPOSITIONS AND METHODS FOR PRODUCING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 640,605, filed April 30, 2024, the entire contents of which are incorporated herein by reference in their entireties.BACKGROUND

[0002] The following discussion is merely provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.

[0003] Messenger RNA (mRNA) technology is an emerging alternative to conventional small molecule, DNA, and protein therapeutics and conventional vaccine approaches because it is potent, programmable, and capable of rapid production of mRNA with desired sequences. mRNA therapy is a rapidly developing field and has been used for the expression of therapeutic proteins, ranging from vascular regeneration factors (e.g., vascular endothelial growth factor A (VEGF-A), erythropoietin (EPO), GATA Binding Protein 4 (GATA4), Myocyte Enhancer Factor 2C (MEF2C), T-Box Transcription Factor 5 (TBX5), Myocardin (MYOCD)), to vaccines for COVID-19, influenza, and Zika virus. Despite recent clinical successes, mRNA therapy still faces challenges of instability, toxicity, short-term efficacy, and potential immunological responses. Increasing the stability and translation efficiency of mRNAs to enhance their efficiency in vivo remains an important problem that must be solved to increase the feasibility of mRNA therapeutics for clinical applications.SUMMARY

[0004] In one aspect the present disclosure provides a composition comprising: (a) an RNA molecule comprising (i) one or more modified nucleotides at position +3 or higher with reference to a 5’ terminus of the RNA molecule, (ii) at least one 5’ cap, (iii) and an open reading frame (ORF), and (b) a delivery agent. In some embodiments, the RNA molecule comprises two or more 5’ caps. In some embodiments, the two or more 5’ caps are conjugated to a 5’ UTR of the RNA molecule. In some embodiments, the two or more 5’ caps are conjugated to the RNA molecule via click chemistry.

[0005] In some embodiments, the one or more modified nucleotides comprises a modified sugar. In some embodiments, the modified sugar is selected from the group consisting of 2'-deoxy fluoro (2FA), Z-adenosine (ZA), 2 '-deoxy adenosine (dA), locked nucleic acid (LNA), 2'- methoxy (2OMe), 2 '-methoxy ethoxy (2M0E), 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 '-di deoxyribose, 3 '-azido-2', 3'- dideoxyribose, 3 '-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'- aminoribose, 5 '-thioribose, 5-nitro-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C- methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose. In some embodiments, the composition comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified sugars. In some embodiments, the composition comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified sugars.

[0006] In some embodiments, the one or more modified nucleotides comprises a modified phosphate. In some embodiments, the modified phosphate is selected from the group consisting of 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, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. In some embodiments, the composition comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified phosphates. In some embodiments, the composition comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100,at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified phosphates.

[0007] In some embodiments, the one or more modified nucleotides comprises a modified nucleobase. In some embodiments, the modified nucleobase is selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3- Indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5- bromocytosine, 5 -carboxy cytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5 -hydroxy cytosine, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5 -hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5- methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5- propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6- chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7- propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8- chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin- 16-aminoallylcytosine, biotin-16- aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3 -aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, Nl- methoxymethylpseudouracil, N1 -methyladenine, N1 -methylpseudouracil, Nl- 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- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, 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-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, the composition comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified nucleobases. In some embodiments, the composition comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified nucleobases. In some embodiments, the one or more modified nucleotides comprise one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof.

[0008] In some embodiments, the 5’ cap is selected from the group consisting of 7- methyguanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7G-3’m- ppp-G), N7,2’-O-dimethyl-guanosine-5’ -triphosphate-5 ’-guanosine (m7Gm-ppp-G), 7- benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G- LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4-chlorophenoxyethyl)-guanosine, 7- ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7- (carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2-PhEt)G], 7-(l- phenylethyl) guanosine [7-(l-PhEt)G], m7GpppBH3G (DI and D2 stereoisomers), m7GppBH3G (DI and D2 stereoisomers), m7GpBH G (DI and D2 stereoisomers), m7GppBH3pm7G, m27’2' ^GpppBffiG (DI and D2 stereoisomers), m272'^GppBHspG (DI and D2 diastereomers), m27-2' °GppspG (DI and D2 diastereomers), N-Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate,phosphorodithioate, capl, cap2, cap3, cap4, ARCA, modified ARCA, inosine, N1- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0009] In some embodiments, the composition further comprises at least one poly-A tail. In some embodiments, the at least one poly-A tail comprises between 25 and 500 nucleotides. In some embodiments, the at least one poly-A tail comprises between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 and 500 nucleotides. In some embodiments, the at least one poly-A tail comprises 10 or more adenosine nucleotides. In some embodiments, 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70- 100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100% of nucleotides of the at least one poly-A tail are adenosine nucleotides.

[0010] In some embodiments, the 5’ cap is added to the RNA molecule through a chemical capping method. In some embodiments, the chemical capping method is an anhydrous reaction between a 5 ’-phosphorylated RNA molecule and a capping nucleotide conjugated to imidazole in the presence of 1 -methylimidazole.

[0011] In some embodiments, the RNA molecule further comprises a 5’ untranslated region (5’ UTR). In some embodiments, the 5’ UTR comprises a promoter. In some embodiments, the RNA molecule further comprises a 3’ untranslated region (3’ UTR). In some embodiments, the 3’ UTR comprises at least one exonuclease-resistant modification. In some embodiments, the exonuclease-resistant modification is selected from the group consisting of phosphorothioate (PS) linkage, 2’-O-methyl (2OMe), 2’ Fluoro, inverted deoxythymidine (dT), inverted dideoxythymidine (ddT), 3’ phosphorylation, C3 spacer, 2'-O-methoxy-ethyl (2'-M0E), G- quadruplex, and 2'-3'-dideoxy nucleotide (ddN).

[0012] In some embodiments, the RNA molecule comprises two or more 5’ caps. In some embodiments, the RNA molecule comprises two or more poly-A tails.

[0013] In some embodiments, the RNA molecule further comprises an open reading frame (ORF). In some embodiments, the ORF encodes a protein. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antigen. In some embodiments, the antigen is a SARS-CoV-2 spike protein or fragment thereof. In some embodiments, the ORF comprises a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In someembodiments, the ORF encodes an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the RNA molecule further comprises a sequence encoding a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA. In some embodiments, the RNA molecule is a circular RNA molecule. In some embodiments, the RNA molecule comprises a stem oligo modification having the sequence of SEQ ID NO: 1. In some embodiments, the RNA molecule comprises a branch oligo modification having the sequence of SEQ ID NO: 2. In some embodiments, the RNA molecule comprises a 5’UTR having the sequence of SEQ ID NO: 3. In some embodiments, the RNA molecule comprises a 3’UTR having the sequence of SEQ ID NO: 5. In some embodiments, the RNA molecule comprises a polyA tail modification having the sequence of SEQ ID NO: 6. In some embodiments, the RNA molecule comprises two 5’ caps, wherein each of the two 5’ caps is LNAm7G. In some embodiments, the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle. In some embodiments, the nanoparticle or microparticle is a lipid nanoparticle or a lipid microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle. In some embodiments, the nanoparticle is a lipid nanoparticle.

[0014] In some embodiments of a composition described herein, (a) the RNA molecule comprises two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF comprising a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6; and (b) the delivery agent comprises a lipid nanoparticle. In some embodiments of a composition described herein, (a) the RNA molecule comprises two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF encoding an antigen comprising an amino acid sequencing having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; a 3’UTR having the sequence of SEQ ID NO: 5; and a poly A tail modification having the sequence of SEQ ID NO: 6; and (b) the delivery agent comprises a lipid nanoparticle. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

[0015] In one aspect, the present disclosure provides an RNA molecule comprising two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF comprising a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6.

[0016] In one aspect, the present disclosure provides an RNA molecule comprising two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF encoding an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6.

[0017] In one aspect, the present disclosure provides a vector comprising the RNA molecule of any of the foregoing embodiments. In one aspect, In one aspect, the present disclosure provides a cell comprising the RNA molecule the vector according to any of the foregoing embodiments. In some embodiments, the cell is a mammalian cell.

[0018] In one aspect, the present disclosure provides a method of preventing or treating a disease in a subject, comprising administering to a subject an effective amount of a composition, RNA molecule, or vector of disclosed herein (e.g., a composition, RNA molecule, or vector of any of the foregoing aspects or embodiments). In another aspect, the present disclosure provides a method of reducing the risk of a disease in a subject, comprising administering to the subject an effective amount of a composition, RNA molecule, or vector of disclosed herein (e.g., a composition, RNA molecule, or vector of any of the foregoing aspects or embodiments). Insome embodiments, the subject is a human subject. In some embodiments, the disease is a bacterial or viral infection, such as a SARS-CoV-2 infection.

[0019] In one aspect, the present disclosure provides a kit comprising the composition according to any of the foregoing embodiments, a device for administering the composition to a subject, and / or instructions for administering the composition to the subject.

[0020] The foregoing general description and following detailed description are examples and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.

[0021] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are provided as being part of the inventive subject matter disclosed herein and may be employed in any combination to achieve the benefits described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGS. 1A-1L show that chemically modified dual-capped mRNA enhances SARS-CoV- 2 mRNA vaccine efficacy in mice. FIG. 1A shows a schematic depicting an mRNA vaccine designed to encode the Receptor Binding Domain (RBD) of Spike Glycoprotein (Swiss-Prot ID: P0DTC2, region: 319-541) of SARS-CoV-2 (Severe Acute Respiratory Syndrome-Related Coronavirus 2) with a trimerization domain. The control mRNA contained mono-m7G-rG cap, and the optimized dual-capped mRNA contained two LNAm7G caps, LN A-2'-(9-m ethyl modified 5' UTR, and phosphorothioate_2'-(9-methoxyethyl_dideoxy cytidine (PS-2MOE_ddC) modified polyA tail. mRNA was encapsulated in lipid nanoparticles (LNP) and delivered through intramuscular injection following a prime and boost injection scheme separated by 14 days, with 800 ng polyC / control / optimized mRNA. FIG. IB is a graph depicting concentrations of RBD-specific antibodies in peripheral blood 7, 14, or 21 days after the first injection were quantified by ELISA, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. 1C is a schematic depicting an experimental workflow for experiments described herein. 24 hrs after booster injection, the mice were sacrificed and inguinal lymph nodes were harvested. The splenocytes / lymphocytes were profiled in intact tissues where nascent mRNAs were detected by STARmap for cell typing and translating RBDmRNA was detected by RIBOmap. FIG. ID is a plot showing differential gene expression indicating higher activation of antigen presenting B cells / dendritic cells / macrophages and RBD mRNA translation by the optimized dual-capped mRNA. FIG. IE shows a representative spatial map of tissue region, major cell types, cells with RBD mRNA translation, and antigen presenting cells in mouse lymph node 24 hrs post boost injection. FIG. IF shows a schematic illustrating that 7 days after boost injection (day 21 after first injection), mouse spleens were harvested and dissociated. Splenocytes were stimulated with Spike peptide pool (S-peptides), or DMSO (negative control), and immuno-typed by Fluorescence-activated Cell Sorting (FACS). Cytokine secretion triggered by S-peptide stimulation was quantified by ELISA. FIG. 1G shows a graph depicting FACS analysis results showing the percentages of CD4+T effector memory (Tem) cells producing IFN-y after stimulation with Spike peptide pools, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. IH shows a graph depicting FACS analysis results showing the percentages of CD4+T effector memory (Tem) cells producing IL2 after stimulation with Spike peptide pools, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. II shows a graph depicting FACS analysis results showing the percentages of CD4+T effector memory (Tem) cells producing TNF-a after stimulation with Spike peptide pools, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. 1 J shows a graph depicting FACS analysis results showing the percentages of CD8+Tem cells producing IFN-y after stimulation, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. IK shows a graph depicting FACS analysis results showing the percentages of CD8+Tem cells producing IL2 after stimulation, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. IL shows a graph depicting FACS analysis results showing the percentages of CD8+Tem cells producing TNF-a after stimulation, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test.

[0023] FIGS. 2A-2H show a spatial transcriptomics analysis of mouse lymph nodes after booster injection. FIG. 2A shows the preprocessed lymph node samples clustered by regions with SPIN to identify T / B / margin / capsule regions, and subsequently clustered by A'-means. FIG. 2B shows a dot plot quantification of cell type specific gene expression for major cell types present in the lymph nodes. FIG. 2C shows a graph depicting a quantification of the percentageof B cells as antigen presenting cells (APCs) in a specified lymph node region (i.e. APC counts in region / total counts of specific cell types), n = 4, biological replicates. Mean ± s.e.m. P values were calculated by ordinary one-way ANOVA. FIG. 2D shows a graph depicting a quantification of the percentage of cDCls as APCs in a specified lymph node region, n = 4, biological replicates. Mean ± s.e.m. P values were calculated by ordinary one-way ANOVA. FIG. 2E shows a graph depicting a quantification of the percentage of cDC2s as APCs in a specified lymph node region, n = 4, biological replicates. Mean ± s.e.m. P values were calculated by ordinary one-way ANOVA. FIG. 2F shows a graph depicting a quantification of the percentage of other dendritic cells (DCs) as APCs in a specified lymph node region, n = 4, biological replicates. Mean ± s.e.m. P values were calculated by ordinary one-way ANOVA. FIG. 2G shows a graph depicting a quantification of the percentage of activated macrophages (act. mph.; i.e., macrophages with elevated CD68 expression) as APCs in a specified lymph node region, n = 4, biological replicates. Mean ± s.e.m. P values were calculated by ordinary one-way ANOVA. FIG. 2H shows a graph depicting a quantification of the percentage of mph. as APCs in a specified lymph node region, n = 4, biological replicates. Mean ± s.e.m. P values were calculated by ordinary one-way ANOVA.

[0024] FIGS. 3A-3H show a quantification of SARS-CoV-2-RBD-specific T cells in mice after vaccination. FIG. 3A shows a gating strategy for single and viable T cells in splenocytes. CD4+or CD8+T-effector memory (Tern) cells (CD44 CD62L ) were further analyzed to detect the expression of cytokines stimulated by corresponding RBD peptide pools FIG. 3B shows representative flow plots for specific CD4+T cell response. FIG. 3C shows representative flow plots for specific CD8+T cell response. FIG. 3D is a graph depicting measurement of the level of IL-2 in the supernatants of peptide pool-stimulated splenocytes with ELISA, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. 3E is a graph depicting measurement of the level of IL-4 in the supernatants of peptide pool-stimulated splenocytes with ELISA, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. 3F is a graph depicting measurement of the level of IL- 13 in the supernatants of peptide pool-stimulated splenocytes with ELISA, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test. FIG. 3G is a graph depicting measurement of the level of TNF-a in the supernatants of peptide pool-stimulated splenocytes with ELISA, n = 4, biological replicates. Mean ± sem. P values were calculated by unpairedtwo-sided / -test. FIG. 3H is a graph depicting measurement of the level of IFN-y in the supernatants of peptide pool-stimulated splenocytes with ELISA, n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided / -test.DETAILED DESCRIPTION

[0025] Provided herein are compositions comprising modified mRNAs comprising modified 5’ cap regions comprising one or more modified nucleotides in order to improve stability and / or translation efficiency of the modified mRNA in cells and thereby enhance the production of encoded gene products, such as proteins. Also provided are methods of making the modified mRNAs described herein by adding to the 5’ end of an mRNA a 5’ cap region comprising one or more modified nucleotides at position +3 or higher. Also provided are methods of screening for altered mRNA stability and / or translation efficiency conferred by one or more modified nucleotides in a 5’ cap. Furthermore, provided herein are methods of producing a composition of RNA transcripts wherein at least 95% of the RNA transcripts in the composition comprise a 5’ cap. Finally, provided are methods of treating or preventing a disease in a subject comprising administering to a subject a composition comprising an RNA molecule of the present disclosure. In particular, provided are methods of treating or preventing an infection, such as a viral infection, such as a SARS-CoV-2 infection.Equivalents

[0026] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0027] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.Definitions

[0028] In the claims, as well as in the specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0029] In the claims, as well as in the specification, recitation of the phrase “between X and Y”, wherein X and Y are two separate values, it should be appreciated that these ranges include the use of these end values. For example, if a claim recites a range of between 1 and 10, this includes the values of 1, 10, and any value in between (e g., 2, 3, 4, 5, 6, 7, 8, 9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, etc.).

[0030] A “messenger RNA” (“mRNA”) as used herein refers to a nucleic acid comprising an open reading frame (ORF) encoding a gene product, such as a protein. An mRNA may comprise a poly-A region that is 3’ to the ORF. An mRNA may also comprise a 5’ untranslated region (5’ UTR) that is 5’ to (upstream of) the ORF, and a 3’ untranslated region (3’ UTR) that is 3’ to (downstream of) the ORF. A mRNA may also comprise a 5’ cap at the 5’ end of the mRNA.

[0031] An “open reading frame” (“ORF”), such as an ORF encoding a protein, as used herein refers to a nucleic acid sequence comprising a coding sequence that leads to the production of the protein when the ORF is translated. The nucleic acid sequence may be an RNA sequence, in which case translation of the RNA sequence produces a polypeptide with the amino acid sequence of the protein. The nucleic acid sequence may be a DNA sequence, in which case the protein is produced when an RNA polymerase uses the DNA sequence to transcribe an RNA molecule comprising an RNA sequence that is complementary to the DNA sequence, and translation of the RNA sequence produces a polypeptide with the amino acid sequence of the protein. An ORF typically begins with a START codon, such as AUG in the RNA sequence (ATG in the DNA sequence), and ends with a STOP codon, such as UAG, UAA, or UGA in the RNA sequence (TAG, TAA, or TGA in the DNA sequence), with the number of bases between the G of the start codon and the T or U of the STOP codon being a multiple of 3 (e.g., 3, 6, 9, 12, etc.).

[0032] With reference to numbering of the nucleotide positions within a nucleic acid molecule, a position of +1 refers to the first nucleotide of the nucleic acid molecule (e.g., of the RNA molecule), +2 is the second nucleotide, +3 is the third nucleotide, and so on.

[0033] In some embodiments of the modified mRNAs provided herein, the mRNA comprises a 5' untranslated region (5' UTR) and a 3' untranslated region (3' UTR). 5' and 3' UTRs are sequences within an mRNA that do not encode amino acids of the protein encoded by the mRNA, and are thus not part of the open reading frame. The 5' UTR is 5' to (upstream of) the open reading frame. The 3' UTR is 3' to (downstream of) the open reading frame. In some embodiments, the 3' UTR comprises one or more nucleotides that are 3' to the open reading frame and 5' to (upstream of) the poly-A region of the mRNA.

[0034] In some embodiments of the modified mRNAs provided herein, the mRNA comprises, in 5’-to-3’ order: 1) a 5’ cap, optionally modified; 2) a modified 5’ UTR; 3) an open reading frame (ORF); 4) a 3’ UTR; and 5) a poly-A region. In some embodiments, the first nucleotide of the 5’UTR is 3’ to (downstream of) the 5’ cap, and the last nucleotide of the 5’ UTR is 5’ to (upstream of) the first nucleotide of the ORF. In some embodiments, the first nucleotide of the ORF is 3’ to (downstream of) the last nucleotide of the 5’ UTR, and the last nucleotide of the ORF is 5’ to (upstream of) the first nucleotide of the 3’ UTR. In some embodiments, the ORF is between the last nucleotide of the 5’ UTR and the first nucleotide of the 3’ UTR. In some embodiments, the first nucleotide of the 3’ UTR is 3’ to (downstream of) the last nucleotide of the ORF, and the last nucleotide of the 3’ UTR is 5’ to (upstream of) the first nucleotide of the poly-A region. In some embodiments, the 5’ UTR is between the 5’ cap and the first nucleotide of the ORF. In some embodiments, the 3’ UTR is between the ORF and the poly-A region. In some embodiments, the 5’ cap is 5’ to (upstream of) the first nucleotide of the 5’ UTR. In some embodiments, the first nucleotide of the poly-A region is 3’ to (downstream of) the last nucleotide of the 3’ UTR.

[0035] In some embodiments, the RNA is a linear RNA. A linear RNA is an RNA with a 5' terminal nucleotide and a 3' terminal nucleotide. The 5' terminal nucleotide of a linear RNA is covalently bonded to only one adjacent nucleotide of the RNA, with the adjacent nucleotide occurring 3' to the 5' terminal nucleotide in the nucleic acid sequence of the RNA. The 3' terminal nucleotide of a linear RNA is covalently bonded to only one adjacent nucleotide of the RNA, with the adjacent nucleotide occurring 5' to the 3' terminal nucleotide in the nucleic acid sequence of the RNA. In a nucleic acid sequence comprising every nucleotide of a linear RNA in 5'-to-3' order, the 5' terminal nucleotide is the first nucleotide in the sequence, and the 3' terminal nucleotide is the last nucleotide in the sequence. The linear RNA may be a coding RNA or a non-coding RNA.

[0036] In some embodiments, the mRNA is a circular mRNA. A circular mRNA is an mRNA with no 5' terminal nucleotide or 3' terminal nucleotide. Every nucleotide in a circular mRNA is covalently bonded to both 1) a 5' adjacent nucleotide; and 2) a 3' adjacent nucleotide. In a circular mRNA with a nucleic acid sequence comprising every nucleotide of the circular mRNA in 5 '-to-3 ' order, the last nucleotide of the nucleic acid sequence is covalently bonded to the first nucleotide of the nucleic acid sequence. In some embodiments of circular mRNAs with a 5' cap region, a 5' UTR, a 3' UTR, and a poly-A region, the poly-A region is 3' to (downstream from) the 3' UTR and 5' to (upstream of) the 5' cap region. The circular RNA may be a coding RNA or a non-coding RNA.

[0037] For the purposes of the present disclosure, the disclosed RNA may be coding (i.e., encode a gene or protein of interest) or non-coding (i.e., is not translated and / or does not encode a gene or protein of interest). An RNA molecule that can be translated is referred to as a messenger RNA, or mRNA. A DNA or RNA sequence encodes a gene through codons. A codon refers to a group of three nucleotides within a nucleic acid, such as DNA or RNA, sequence. An anticodon refers to a group of three nucleotides within a nucleic acid, such as a transfer RNA (tRNA), that are complementary to a codon, such that the codon of a first nucleic acid associates with the anticodon of a second nucleic acid through hydrogen bonding between the bases of the codon and anticodon. For example, the codon 5'-AUG-3' on an mRNA has the corresponding anticodon 3'-UAC-5' on a tRNA. During translation, a tRNA with an anticodon complementary to the codon to be translated associates with the codon on the mRNA, generally to deliver an amino acid that corresponds to the codon to be translated, or to facilitate termination of translation and release of a translated polypeptide from a ribosome.

[0038] Translation is the process in which the RNA coding sequence is used to direct the production of a polypeptide. The first step in translation is initiation, in which a ribosome associates with an mRNA, and a first transfer RNA (tRNA) carrying a first amino acid associates with the first codon, or START codon. The next phase of translation, elongation, involves three steps. First, a second tRNA with an anticodon that is complementary to codon following the START codon, or second codon, and carrying a second amino acid, associates with the mRNA. Second, the carbon atom of terminal, non-side chain carboxylic acid moiety of the first amino acid reacts with the nitrogen of the terminal, non-side chain amino moiety of the second amino acid carried, forming a peptide bond between the two amino acids, with the second amino acid being bound to the second tRNA, and the first amino acid bound to the second amino acid, but not the first tRNA. Third, the first tRNA dissociates from the mRNA, and the ribosome advances along the mRNA, such that the position at which the first tRNA associated with the ribosome is now occupied by the second tRNA, and the position previously occupied by the second tRNA is now free for an additional tRNA carrying an additional amino acid to associate with the mRNA. These three steps of 1) association of a tRNA carrying amino acid, 2) formation of a peptide bond, which adds an additional amino acid to a growing polypeptide, and 3) advancement of the ribosome along the mRNA, continue until the ribosome reaches a STOP codon, which results in termination of translation. Generally, tRNAs that associate with STOP codons do not carry anamino acid, so the association of a tRNA that does not carry an amino acid during the elongation step results in cleavage of the bond between the polypeptide and the tRNA carrying the final amino acid in the polypeptide, such that the polypeptide is released from the ribosome.Alternatively, ribosomes may dissociate from the mRNA and release the polypeptide if no tRNA associates with the STOP codon.

[0039] A “nucleic acid,” or “polynucleotide,” as used herein, refers to an organic molecule comprising two or more covalently bonded nucleotides. A “nucleotide,” as used herein, refers to an organic molecule comprising a 1) a nucleoside comprising a sugar covalently bonded to a nitrogenous base (nucleobase); and 2) a phosphate group that is covalently bonded to the sugar of the nucleoside. Nucleotides in a polynucleotide are typically joined by a phosphodiester bond, in which the 3' carbon of the sugar of a first nucleotide is linked to the 5' carbon of the sugar of a second nucleic acid by a bridging phosphate group. Typically, the bridging phosphate comprises two non-bridging oxygen atoms, which are bonded only to a phosphorus atom of the phosphate, and two bridging oxygen atoms, each of which connects the phosphorus atom to either the 3' carbon of the first nucleotide or the 5' carbon of the second nucleotide. In a nucleic acid sequence describing the order of nucleotides in a nucleic acid, a first nucleotide is said to be 5' to (upstream of) a second nucleotide if the 3' carbon of first nucleotide is connected to the 5' carbon of the second nucleotide. Similarly, a second nucleotide is said to be 3' to (downstream of) a first nucleotide if the 5' carbon of the second nucleotide is connected to the 3' carbon of the first nucleotide. Nucleic acid sequences are typically read in 5 '->3' order, starting with the 5' nucleotide and ending with the 3' nucleotide.

[0040] A “modified nucleotide,” as used herein, refers to a nucleotide with a structure that is not the canonical structure of an adenosine nucleotide, cytidine nucleotide, guanine nucleotide, or uracil nucleotide. A canonical structure of a molecule refers to a structure that is generally known in the art to be the structure referred to by the name of the molecule. A canonical structure of an adenosine nucleotide, which comprises an adenine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of adenosine monophosphate:The canonical structure of AMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0041] The canonical structure of a cytosine nucleotide which comprises a cytosine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of cytidine monophosphate:The canonical structure of CMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0042] The canonical structure of a guanine nucleotide which comprises a guanine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of guanosine monophosphate:The canonical structure of GMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0043] The canonical structure of a uracil nucleotide which comprises a uracil base, ribose sugar, and one or more phosphate groups, is shown below, in the form of uridine monophosphate:The canonical structure of UMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0044] The structure of a modified nucleotide may differ from the structure of a canonical nucleotide due to one or more modifications in the sugar, nitrogenous base, or phosphate of the nucleotide. In some embodiments, the modified nucleotide comprises a modified nucleoside that is not the canonical structure of an adenine nucleoside, cytosine nucleoside, guanine nucleoside, or uracil nucleoside.

[0045] An example of a canonical structure of adenosine, an adenine nucleoside, is reproduced below:(adenosine). The canonical structure of adenosine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0046] An example of a canonical structure of cytidine, a cytosine nucleoside, is reproduced below:(cytidine). The canonical structure of cytidine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5’ phosphate in a nucleic acidsequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0047] An example of a canonical structure of guanosine, a guanine nucleoside, is reproduced below:(guanosine). The canonical structure of guanosine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0048] An example of a canonical structure of uridine, a uracil nucleoside, is reproduced below:(uridine). The canonical structure of uridine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0049] A “ligase,” as used herein, refers to an enzyme that is capable of forming a covalent bond between two nucleotides, and the process of “ligation” refers to the formation of the covalent bond between the two nucleotides.

[0050] A “poly-A tail,” as used herein, refers to a nucleic acid sequence comprising adenosine nucleotides that is attached to the 3' end of a nucleic acid, such as an RNA. A poly-A tail or poly-A region may consist of nucleotides that are 25-100%, 30-100%, 40-100%, 50-100%, 60- 100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100% adenosine nucleotides. As used herein, the terms “poly-A tail” and “poly-A region” are usedinterchangeably. The adenosine nucleotides comprised by a poly-A tail may be canonical adenosine nucleotides or modified (non-canonical) adenosine nucleotides.

[0051] A “5' cap,” as used herein, refers to one or more nucleotides that are covalently attached to the 5' end of a nucleic acid, such as an RNA molecule. A “5' cap region,” as used herein, refers to a nucleic acid comprising a 5' nucleotide cap and one or more modified nucleotides. A 5' cap may comprise a 5' capping nucleotide that is attached to the 5' end of a mRNA by a 5' to 5' triphosphate intemucleotide linkage. In some embodiments, a nucleotide attached to a mRNA by a 5' to 5' triphosphate intemucleotide linkage is referred to as a “native” 5' capping nucleotide. In some embodiments, a native 5' capping nucleotide is a 7-methylguanosine (m7G) nucleotide. In some embodiments, a 5' cap is a modified 5' cap, comprising one or more modified nucleotides, such as the 5' capping nucleotide, or one or more modified intemucleotide modifications, such as modifications to the 5' to 5' triphosphate intemucleotide linkage. In some embodiments, a 5' cap comprises one or more nucleotides with a sugar modification, such as 2'-O-methylation.

[0052] An example of a canonical structure of 7-methylguanosine (m7G) attached to a ribonucleic acid sequence (e.g., a mRNA) by a 5' to 5' triphosphate intemucleotide linkage is reproduced below:

[0053] A “counterion” or “anionic counterion” is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. In some embodiments, an anionic counterion is monovalent (e.g., including one formal negative charge). An anionic counterion may also be multivalent (e.g., including more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F , Cl", Br", I"), NOs , CIO4 , OH , H2PO4 , HCO3 , HSO4 , sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthal ene-1 -sulfonic acid-5-sulfonate, ethan-l-sulfonic acid-2- sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate,tartrate, glycolate, gluconate, and the like), BF4 , PF 4 , PFe ", AsFe , SbFe , B[3,5-(CF3)2CeH3]4]-, B(C6Fs)4 , BPt , AI(OC(CF3 ? 4 , and carborane anions (e.g., CBi 1H12 or (HCBi iMcsBre) ). Exemplary counterions which may be multivalent include CO32, HPO42, PO43, B4O72, SO42, S2O32, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.

[0054] Use of the phrase “at least one instance” refers to 1, 2, 3, 4, or more instances, but also encompasses a range, e.g., for example, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive.

[0055] The terms “prevent,” “preventing” or “prevention” as used herein with reference to a disease, such as a bacterial or viral infection, refer to precluding or reducing the risk of the disease (e.g., the infection) from developing in a subject, such as a subject. Prevention may also refer to the prevention of a subsequent infection after an initial infection has been treated or cured or prevention of recurrence of a disease after an initial disease has been treated or cured.

[0056] The terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to any individual mammalian subject, e.g., bovine, canine, feline, equine, or human. In specific embodiments, the subject, individual, or patient is a human. In general, the individual, subject, or patient is preferably a humanModified mRNAs

[0057] In some aspects, the present disclosure provides modified mRNAs comprising a 5’ cap region, wherein the 5’ cap region comprises a 5’ nucleotide cap and one or more modified nucleotides. In some embodiments, a modified mRNA is a modified linear mRNA. In some embodiments, a modified mRNA is a modified circular mRNA.

[0058] The “5' cap region”, as used herein, refers to a region of an mRNA that is 5' to (upstream of) the ORF. In some embodiments, the 5’ cap region comprises a 5’ untranslated region (5’ UTR). In some embodiments, the 5’ cap region comprises a 5’ cap. In eukaryotic cells, mRNAs possess a cap structure in which an N7-methylguanine (m7G) moiety is linked to the first transcribed nucleotide by a 5’-5’-triphosphate bridge. The 5' cap plays multiple roles in pre- mRNA splicing, mRNA export, RNA stability through blocking degradation by the 5 ’-3’ exoribonuclease (ExoN), escaping recognition of the cellular innate immune system, and theproduction of proteins encoded by mRNAs. The presence of a 5' cap in an mRNA facilitates the initiation of translation (see, e.g., Gallie. Genes & Dev. 1991. 5:2108-2116, and Munroe et al. Mol Cell Biol. 1990. 10(7):3441-3455). The 5' cap is added by a 5' capping enzyme, such as mRNA guanylyltransferase. Translation initiation is a rate-limiting step of mRNA translation and heavily depends on the 5’ N7-methylguanosine (m7G) cap and its interaction with eukaryotic translation initiation factors (elFs), including the cap-binding eIF4E protein. Chemical modification on or near the 5’ cap influence binding of elFs and decapping enzymes, which subsequently impact downstream mRNA translation and stability. For example, the presence of 2’ O-methyl (2’0Me) groups on the first and second transcribed nucleotides (known as Cap- 0 / 1 / 2, referring to zero, one, or two 2’0Me groups) reduces mRNA immunogenicity and increases protein expression. Additionally, N6-methyladenosine (m6A) on the first base controls mRNA stability through increased resistance to decapping by Dcp2. Furthermore, the 5' cap stabilizes the mRNA by protecting the ORF from the activity of exonucleases, such as polynucleotide phosphorylase (PNPase), which can remove 3' and 5' nucleotides from an mRNA. As an exonuclease removes nucleotides, the mRNA becomes progressively shorter, and once all the nucleotides downstream of the open reading frame are removed, the nucleotides removed by the exonuclease will be nucleotides of the ORF. Removal of nucleotides from the ORF prevents translation of the encoded protein. Additionally, the association of an exonuclease with the mRNA near the ORF can inhibit translation by sterically hindering ribosomes and tRNAs from associating with the mRNA. The composition of a 5' cap typically comprises a 5' m7G attached to the mRNA by a 5' to 5' triphosphate intemucleotide linkage.

[0059] In some embodiments of the modified mRNAs provided herein, the modified mRNA comprises one or more modified nucleotides in the 5' cap region of the mRNA. In some embodiments, the 5' cap region includes one or more nucleotides that are not canonical adenosine, cytidine, guanosine, or uridine nucleotides. In some embodiments, the 5' cap region comprises between 1 and 3, between 3 and 5, between 5 and 7, or between 7 and 10 5' caps. In some embodiments, the 5' cap region comprises between 10-500 nucleotides. In some embodiments, the 5' cap region comprises between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 50, between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 and 500 nucleotides.

[0060] In some aspects, the present disclosure provides an RNA molecule comprising (i) one or more modified nucleotides at position +3 or higher with reference to a 5’ terminus of the RNA molecule, (ii) at least one 5’ cap, (iii) and an open reading frame (ORF). In some embodiments, the RNA molecule comprises two or more 5’ caps. In some embodiments, the two or more 5’ caps are conjugated to a 5’ UTR of the RNA molecule. In some embodiments, the two or more 5’ caps are conjugated to the RNA molecule via click chemistry. In some embodiments, the one or more modified nucleotides comprises a modified sugar. In some embodiments, the one or more modified nucleotides comprises a modified phosphate. In some embodiments, the one or more modified nucleotides comprises a modified nucleobase.

[0061] In some embodiments, an RNA molecule comprises a 5’ untranslated region (5’ UTR). In some embodiments, the 5’ UTR comprises a promoter. In some embodiments, the RNA molecule further comprises a 3’ untranslated region (3’ UTR). In some embodiments, the 3’ UTR comprises at least one exonuclease-resistant modification.

[0062] In some embodiments, the RNA molecule comprises two or more 5’ caps. In some embodiments, the RNA molecule comprises two or more poly-A tails.

[0063] In some embodiments, the RNA molecule further comprises an open reading frame (ORF). In some embodiments, the ORF encodes a protein. In some embodiments, the ORF encodes a therapeutic protein. In some embodiments, the ORF encodes an antigen. In some embodiments, the antigen is a SARS-CoV-2 spike protein or fragment thereof. In some embodiments, the ORF comprises a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the ORF encodes an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

[0064] In some embodiments, the RNA molecule further comprises a sequence encoding a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

[0065] In some embodiments, the RNA molecule is a circular RNA molecule.

[0066] In some embodiments, the RNA molecule comprises a stem oligo modification having the sequence of SEQ ID NO: 1. In some embodiments, the RNA molecule comprises a branch oligo modification having the sequence of SEQ ID NO: 2. In some embodiments, the RNAmolecule comprises a 5’UTR having the sequence of SEQ ID NO: 3. In some embodiments, the RNA molecule comprises a 3’UTR having the sequence of SEQ ID NO: 5. In some embodiments, the RNA molecule comprises a polyA tail modification having the sequence of SEQ ID NO: 6. In some embodiments, the RNA molecule comprises two 5’ caps, wherein each of the two 5’ caps is LNAm7G.

[0067] In some aspects, the RNA molecule comprises two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF comprising a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6. In some aspects, the RNA molecule comprises two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF encoding an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6.Chemical synthesis of 5’ cap regions

[0068] Existing methods for preparing capped linear mRNA do not accommodate modifications that are not tolerated by RNA polymerase or capping enzymes, nor modifications that extend beyond the first two bases, creating a screening bias due to differing cap incorporation efficiencies. To overcome these challenges, the capping process was decoupled from mRNA synthesis as described herein.

[0069] In some embodiments of the methods provided herein, the method comprises first synthesizing a 5 ’-phosphorylated RNA oligonucleotide with a specific sequence and / or desired modifications. In the methods described herein, the synthesized 5 ’-phosphorylated RNA oligonucleotide defines the 5’ UTR when ligated to an RNA transcript. Thus, as used herein, the terms “5’-phosphorylated RNA oligonucleotide,” “5 ’-phosphorylated oligonucleotide,” and “5’- phosphorylated UTR” are used interchangeably. In some embodiments, the 5 ’-phosphorylatedRNA oligonucleotide comprises one or more modified nucleotides which may affect RNA translation and / or stability.

[0070] In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises a modified phosphate, resulting in a modified intemucleotide linkage. Modified phosphates used in the present invention may be, but are not limited to, 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, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. In some embodiments, more than one modified phosphate is used. In some embodiments, the 5’- phosphorylated oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more modified phosphates. In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified phosphates. In some embodiments, the modified phosphates of the 5 ’-phosphorylated oligonucleotide comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total phosphates in the 5 ’-phosphorylated oligonucleotide.

[0071] In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises a modified sugar. Modified sugars used in the present invention may be, but are not limited to, 2'-deoxy fluoro (2FA), Z-adenosine (ZA), 2 '-deoxy adenosine (dA), locked nucleic acid (LNA), 2'- methoxy (2OMe), 2 '-methoxy ethoxy (2M0E), 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-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C- methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose. Z-adenosine (ZA) refers to the enantiomer of Z>-adenosine. A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2’ and 4’ carbons. This structure effectively “locks” the ribose in the 3’-endo structural conformation. In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more modified sugars. In some embodiments, the 5 ’ -phosphorylated oligonucleotide comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified sugars. In some embodiments, the modified sugars of the 5 ’-phosphorylated oligonucleotide comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total sugars in the 5 ’-phosphorylated oligonucleotide.

[0072] In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises a modified nucleobase. Modified nucleobases used in the present invention may be, but are not limited to, inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3- Indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5- bromocytosine, 5 -carboxy cytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5 -formyluracil, 5 -hydroxy cytosine, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5- methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5- propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6- chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7- propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8- chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin- 16-aminoallylcytosine, biotin-16- aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3 -aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, Nl- methoxymethylpseudouracil, N 1 -methyladenine, N1 -methylpseudouracil, Nl- propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, 06-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3 -deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, 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-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyl adenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more modified nucleobases. In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified nucleobases. In some embodiments, the modified nucleobases of the 5 ’-phosphorylated oligonucleotide comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total nucleobases in the 5’- phosphorylated oligonucleotide.

[0073] In some embodiments, the 5 ’-phosphorylated oligonucleotide is synthesized on a solidphase support. In some embodiments, the solid support is controlled-pore glass (CPG) or polystyrene (PS). In some embodiments, the 5 ’-phosphorylated oligonucleotide is synthesized via phosphoramidite oligonucleotide synthesis. In some embodiments, the 5 ’-phosphorylated oligonucleotide is synthesized in a solvent system comprising a nonpolar counterion. In some embodiments, the nonpolar counterion used in oligonucleotide synthesis is ammonium. In some embodiments, the nonpolar counterion used in oligonucleotide synthesis is ammonium.

[0074] In some embodiments, a 5’ cap is added to the 5 ’-phosphorylated oligonucleotide to produce a 5’-capped oligonucleotide (i.e., a 5’-capped UTR). A 5’ cap can be added to an RNA oligonucleotide via enzymatic or chemical reactions. In some embodiments, the cap is added to the 5 ’-phosphorylated oligonucleotide through chemical capping methods. Chemical capping may be performed by any method known in the art. Preferably, the chemical capping reaction is performed through an anhydrous reaction between the 5 ’-phosphorylated RNA oligonucleotideand a capping nucleotide conjugated to imidazole in the presence of 1 -methylimidazole (see Abe et al., “Complete Chemical Synthesis of Minimal Messenger RNA by Efficient Chemical Capping Reaction” ACS Chem. Biol. 2022, 17: 1308-1314). In this method, the cap of interest is first conjugated to imidazole. A chemical reaction is then performed between the imidazole- conjugated capping oligonucleotide and a 5’-phoshporylated oligonucleotide under anhydrous conditions and in the presence of 1 -methylimidazole. In some embodiments, the capping reaction is performed in dimethyl sulfoxide (DMSO). The desired product of this reaction is an oligonucleotide capped on its 5’ end with the cap of interest.

[0075] In some embodiments, the 5’ cap used in the present invention may be, but is not limited to, 7-methy guanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7G- 3’m-ppp-G), N7,2’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7Gm-ppp-G), 7- benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G- LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4-chlorophenoxyethyl)-guanosine, 7- ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7- (carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2-PhEt)G], 7-(l- phenylethyl) guanosine [7-(l-PhEt)G], m7GpppBH3G (DI and D2 stereoisomers), m7GppBH3G (DI and D2 stereoisomers), m7GpBii3G (DI and D2 stereoisomers), m7GppBii3pm7G, m27’2' °GpppBH3G (DI and D2 stereoisomers), m27’2'°GppBH3pG (DI and D2 diastereomers), m27’2' °GppspG (Dl and D2 diastereomers), N-Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3, 5 -dihydroxy pentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, capl, cap2, cap3, cap4, ARC A, modified ARC A, inosine, Nl- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0076] Thus, in some embodiments, a 5’ cap region provided herein comprises a 5’-capped oligonucleotide (i.e., a 5’-capped UTR) synthesized as described above. In some embodiments,the 5’ cap region comprises a modified 5’ cap, one or more modified phosphates, one or more modified sugars, and / or one or more modified nucleobases. The 5’ cap region may comprise any combination of modifications. In some embodiments, the 5’ cap region is between 5 and 50, between 10 and 45, between 15 and 40, between 20 and 35, between 25 and 30, or more than 30 nucleotides in length. In some embodiments, the 5’ cap region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more modified nucleotides. In some embodiments, the 5’ cap region comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified nucleotides. In some embodiments, the modified nucleotides of the 5’ cap region comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total nucleotides in the 5’ cap region.

[0077] In some embodiments, a capped RNA transcript as provided herein comprises more than one 5’ cap or 5’ UTR region. In some embodiments, a capped RNA transcript as provided herein comprises more than one poly-A tail. Methods of producing multi-capped RNA strands have been described in U.S. Patent Application Number 63 / 300,602, the contents of which are incorporated herein in their entirety. These methods comprise incorporating azide handles into an RNA molecule such that it is compatible with an alkyne-containing nucleotide to undergo a click chemistry reaction. The present application builds upon these these techniques. In some embodiments, an azide handle is introduced into the RNA molecule through tRNA guanine transglycosylase (TGT) in combination with a pre-queuosine 1 (preQi) substrate (Ehret et al. “Site-specific covalent conjugation of modified mRNA by tRNA guanine transglycosylase.” Mol. Pharm. 15, 737-742 (2018)). In some embodiments, an azide handle is incorporated into an RNA molecule (e.g., 5 ’-phosphorylated RNA oligonucleotide or a capped RNA transcript) through during transcription, providing an azide-linked nucleotide as substrate for incorporation into a growing RNA strand (e.g., 5-Azido-PEG4-CTP). In some embodiments, more than one azide handle is introduced into an RNA molecule. In some embodiments, more than one azide handle is introduced into an RNA molecule using more than one introduction technique (e.g., both TGT and IVT).Producing RNA transcripts with a modified 5’ cap region

[0078] In some aspects, the methods provided herein produce a capped RNA transcript with a modified 5’ cap region. In some embodiments, the methods comprise attaching a 5’ cap region as described herein to an RNA precursor, thereby producing a capped RNA transcript comprising a modified 5’ cap and UTR.

[0079] In some aspects, the present disclosure provides methods of producing modified RNAs comprising ligating an RNA (e.g., an RNA precursor) to a 5’ cap region comprising a 5’ cap and a 5’ UTR in the presence of a ligase, whereby the ligase forms a covalent bond between the 3’ nucleotide of the 5’ cap region and the 5’ nucleotide of the RNA (e.g., the RNA precursor) to produce capped RNA transcript, (e.g., a modified capped RNA transcript). The RNAs produced by the disclosed methods may be coding or non-coding RNAs. In some embodiments, a 5’ cap region is produced as described herein. When a ligase forms a covalent bond between two linear nucleic acids, a new nucleic acid is produced, with the produced nucleic acid comprising the nucleic acid sequences of both nucleic acids. Ligation of the 3’ terminal nucleotide of a first nucleic acid to the 5’ terminal nucleotide of a second nucleic acid produces a third nucleic acid, with the third nucleic acid comprising the sequence of the first nucleic acid and the second nucleic acid, and the second nucleic acid being 3’ to (downstream of) the first nucleic acid sequence. Ligation by an RNA ligase occurs in several steps. First, an amino (-NH2) group of an amino acid (e.g., a lysine) of the ligase bonds to a phosphate group of adenosine triphosphate (ATP), such that an adenosine monophosphate (AMP) group is bound to the RNA ligase.Second, a 5' terminal phosphate of the second nucleic acid displaces the phosphate of the RNA ligase-bound AMP. Finally, an oxygen of the 3' terminal hydroxyl group of the first nucleic acid binds to the phosphorus atom of the 5' terminal phosphate of the second nucleic acid. This final step forms a phosphodiester bond between terminal nucleotides of the nucleic acids, thereby forming a single nucleic acid with a continuous sugar-phosphate backbone. In some embodiments, the ligase is T4 RNA Ligase I, T4 RNA Ligase II, or RtcB. In some embodiments, the ligation is performed using a split ribozyme (see, e.g., Gambill et al., “A split ribozyme that links detection of a native RNA to orthogonal protein outputs.” Nat Commun 14, 543 (2023)).

[0080] In some embodiments, the RNA precursor comprises an open reading frame (ORF). In some embodiments, the ORF encodes a therapeutic protein. As used herein, a “therapeutic protein” refers to a protein that prevents, reduces, or alleviates one or more signs or symptoms ofa disease or disorder when expressed in a subject, such as a human subject that has, for example, an essential enzyme, clotting factor, transcription factor, growth factor, cytokine, chemokine, antibody (or antibody fragment thereof), protein hormone, signaling protein, structural protein, or cell surface receptor encoded by a gene that is mutated in a subject. A mutation in a gene encoding such a protein may cause diminished levels of the protein to be expressed in one or more cells of the subject. For example, IPEX syndrome in humans is caused by a mutation in the F0XP3 gene, which hinders development of F0XP3+ regulatory T cells and results in increased susceptibility to autoimmune and inflammatory disorders. Expression of an essential enzyme, clotting factor, transcription factor, growth factor, cytokine, chemokine, antibody (or antibody fragment thereof), protein hormone, signaling protein, structural protein, or cell surface receptor from an RNA may therefore compensate for a mutation in the gene encoding such a protein in a subject. In some embodiments, the therapeutic protein is a protein that is expressed in one or more cells of a subject a level that is less than (e.g., significantly less than) that of a reference value, such as the level of expression of the protein that is typical in cells of one or more healthy subjects (i.e., subjects who do not have and are not at risk for developing the disease or disorder). Non-limiting examples of therapeutic proteins include base editors (e.g., adenine base editors or RNA base editors), CRISPR-associated proteins (Cast, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Casl2 [Cpfl], or Casl3 [C2c2] endonuclease), RNase proteins (e.g., RNase III), hormones (e.g., insulin, renin, parathyroid hormone, thyroid hormone), thrombin, fibrinogen, metabolic enzymes, erythropoietin (EPO), growth hormone (e.g., GSH), interferons, antibodies (e.g., monoclonal antibodies), colony-stimulating factors (CSFs, e.g., granulocyte colony-stimulating factor [G-CSF]), tissue plasminogen activator (tPA), Factor VIII, Factor IX, enzymes (e.g., for conditions such as Gaucher’s disease or Fabry disease), interleukins, bone morphogenic proteins (BMPs), relaxin, alpha-1 antitrypsin, filgrastim, oxytocin, somatostatin, calcitonin, glucagon, liraglutide, vasopressin, epigenetic modulating proteins, and growth factors.

[0081] In some embodiments, the ORF encodes an antigen. As used herein, “antigen” refers to a molecule (e.g., a protein) that, when expressed in a subject, elicits the generation of antibodies in the subject that bind to the antigen. In some embodiments, the antigen is a protein derived from a pathogen, such as a pathogenic virus, bacterium, protozoan, or fungus. In some embodiments, the antigen is a protein derived from a virus (viral antigen) or a fragment thereof. In someembodiments, the antigen is a protein or fragment thereof derived from SARS-CoV-2. In some embodiments, the antigen is a fragment of a SARS-CoV-2 spike protein. In some embodiments, the antigen comprises a SARS-CoV-2 spike protein receptor-binding domain (RBD). In some embodiments, the ORF encodes an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the antigen is a protein derived from a bacterium (bacterial antigen) or a fragment thereof. In some embodiments, the antigen is a protein derived from a protozoan (protozoal antigen) or a fragment thereof. In some embodiments, the antigen is a protein derived from a fungus (fungal antigen) or a fragment thereof. A fragment of a full-length protein refers to a protein with an amino acid sequence that is present in, but shorter than, the amino acid sequence of the full-length protein. Thus, in some embodiments, the RNA transcripts produced by the methods provided herein may be used for prophylactic purposes, such as for vaccination of a subject.MFVFLVLLPLVSSQCVGSGSGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRK RISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQA GSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV KNKCVNFGSGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 10).

[0082] In another aspect, the methods disclosed herein provide an RNA precursor and / or a capped RNA transcript comprising one or more noncoding genes. In some embodiments, the noncoding heterologous genes are therapeutic nucleic acids. As used herein, a therapeutic nucleic acid is a nucleic acid or related compound that alters gene expression to prevent or treat diseases or disorders. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide (ASO), N-acetylgalactosamine (GalNAc) ligand-modified short interfering RNA (siRNA) conjugate, DNA aptamer, RNA aptamer, ribozyme, RNA decoy, siRNA, shRNA, miRNA, gRNA, or CRISPRi molecule.

[0083] In some embodiments, a composition provided herein (e.g., a pharmaceutical composition) further comprises one or more additional agents. In some embodiments, the additional agent is a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate. In some embodiments, the additional agent is an agent which has a therapeutic effect when administered to a subject. In some embodiments, theadditional agent is an agent that is capable of modulating expression of a gene and / or protein is a subject, such as a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an antisense oligonucleotide (ASO). In some embodiments, the additional agent is a small molecular inhibitor. In some embodiments, the additional agent is an agent that is capable of eliciting or enhancing an immune response in a subject. In some embodiments, the additional agent is an antigen (e.g., a viral antigen, a bacterial antigen). In some embodiments, the additional agent is an adjuvant, which is defined as an agent that is sufficient for enhancing an immune response in a subject when administered at an effective amount, but does not elicit an immune response in a subject when administered alone. In some embodiments, the additional agent is an enzyme, such as an enzyme that is capable of catalyzing one or more chemical reactions in a subject or in cells of a subject.

[0084] In some embodiments of the methods provided herein, an RNA precursor for which 5’ capping is desired must first be chemically prepared for capping. In vitro transcription (IVT) of RNA results in uncapped 5 ’-triphosphorylated RNA. The 5 ’-triphosphate of the in vitro transcribed RNA is incompatible with enzymatic ligation on its 5’ end, as enzymatic ligation requires 5 ’-monophosphorylated RNA. For example, T4 RNA Ligase 1 catalyzes the ligation of a 5’-phosphoryl-terminated nucleic acid donor (e.g., the RNA precursor described herein) to a 3’ hydroxyl-terminated nucleic acid acceptor (e.g., the 5’ cap region described herein) through the formation of a 3’U 5’ phosphodiester bond with hydrolysis of adenosine triphosphate (ATP) to adenosine monophosphate (AMP) and pyrophosphate (PPi).Thus, in some embodiments, an RNA precursor is prepared for capping by removing pyrophosphate from the 5’ end of the triphosphorylated RNA, leaving a 5 ’-monophosphorylated RNA to be used in a ligation reaction. In some embodiments of the methods provided herein, RNA 5’ Pyrophosphohydrolase (RppH) is used to produce 5 ’-monophosphorylated RNA from 5 ’ -triphosphorylated RNA.

[0085] Ligation of the 5’ cap region to the RNA precursor using conventional enzymatic ligation methods demonstrated the requirement of a high ratio (>200) of 5’ cap region oligo:RNA precursor (oligo:mRNA) to achieve complete labeling of the RNA precursor. The requirement for such high levels of 5’ cap region oligos is inconsistent with scalability of such methods. Thus, to address this issue, in some embodiments a short unstructured spacer region was introduced into the 5’ end of the RNA precursor such that the 5’ end was exposed, allowing enzymes (e.g., RNA ligase) better access to the 5’ end.

[0086] In some embodiments, the spacer region comprises a plurality of identical consecutive nucleotides. In some embodiments, the spacer region comprises between 5 and 10, between 10 and 20, between 15 and 30, between 20 and 50, between 50 and 100, or between 100 and 200 consecutive adenosine, cytosine, guanine, or thymine nucleotides. In some embodiments, the spacer region comprises about 15 consecutive adenosine nucleotides. In some embodiments, the spacer region comprises at least one non-canonical nucleotide (e.g., inosine).

[0087] In some embodiments, the RNA precursor to which the 5’ cap region is ligated comprises one or more exonuclease-resistant nucleotide modifications in its 3’ end. Modified nucleotides containing one or more structural modifications to the nucleobase, sugar, or phosphate linkage of the RNA can interfere with 3’ and 5’ exonuclease activity, rendering the RNA more stable. Nucleotide modifications conferring exonuclease resistance are known in the art. In some embodiments, the RNA precursor comprises one or more modified phosphates, sugars, and / or nucleobases to confer exonuclease resistance. In some embodiments, the RNA precursor comprises one or more 2’-O-Methyl (2’OMe) modifications. In some embodiments, the RNA precursor comprises one or more 2’-fluoro bases. In some embodiments, the RNA precursor comprises one or more phosphorothioate (PS) or thiophosphate (SP) linkages. In some embodiments, the 3’ end of the RNA precursor comprises a phosphate group. In some embodiments, the RNA precursor comprises a C3 spacer incorporated internally or at its 3’ end. A C3 spacer modification adds a 3-carbon spacer to the 3’ terminus of an oligonucleotide. In some embodiments, the RNA precursor comprises a 2’-O-methoxy-ethyl base (2’-MOE), a G- quadruplex, or a 2’-3’-dideoxy nucleotide (ddN). In some embodiments, the RNA precursor comprises one or a combination of any of the modifications known in the art to confer exonuclease resistance (see, e.g., Clave et al., “Modified internucleoside linkages for nuclease- resistant oligonucleotides.” RSC Chem. Biol. (2021) 2:94-150)Capped-circular mRNA (QRNA)

[0088] As described herein, a “capped-circular mRNA” is a circular mRNA characterized by one or more covalent linkages to one or more cap structures (or a derivative thereof). The circular mRNA can contain all the canonical elements of a linear mRNA: (1) Cap, (2) 5’ UTR (untranslated region), (3) protein-coding regions (CDS), (4) 3’ UTR, and (5) polyA tail. By circularizing these features into a capped-circular RNA, it is intended to enhance half-life(increased nuclease resistance) of a canonical circular mRNA, while retaining the benefits of efficient cap-dependent translation, such as in linear mRNA.

[0089] The RNA embodiments and methods disclosed herein take advantage of the exonucleaseresistant feature of circRNA while utilizing the strong m7G-cap dependent translation initiation machinery. Such features can be achieved via chemical conjugation of a capped oligonucleotide with a circRNA through click chemistries such as copper catalyzed azide-alkyne cycloaddition (CuAAC) or tetrazine-trans cyclooctene inverse electron demand Diels- Alder reaction (IEDDA). In some embodiments, the invention contemplates two generic structures of capped circular messenger RNAs (QRNAs): Type 1 QRNA and Type 2 QRNA. In Type 1 QRNA, a circular poly-phosphodiester backbone is present while capping is achieved via chemical ligation of a short, capped oligonucleotide to an internal handle on the circular mRNA through click chemistry. The 5’ cap may comprise of a 7-methylguanylate that enables efficient translation of an mRNA or alternative common mRNA cap structures, as shown, for example, in Mccaffreyanton, 2019, Genetic Engineering & Biotechnology News. 39. In Type 2 QRNA, a continuous mRNA poly-phosphodiester backbone is present; circularization is achieved via chemical conjugation between the 3’-end and 5’-UTR of the mRNA through click chemistry.

[0090] Enzymes capable of catalyzing the reaction of linking a cap molecule to the mRNA include, but are not limited to, Vaccinia capping system including 2’-O-Methyl Transferase, tRNA guanine transglycosylase (TGT), Faustovirus capping enzyme, and T4-RNA ligase. Capping can also occur during the synthesis of mRNA called co-transcriptional capping.

[0091] As used herein, the term “molecular handle” or “handle” refers to a chemical group that is attached to a nucleotide on mRNA and can form a covalent bond to another molecule that is separate from the mRNA to link this other molecule to the mRNA. The covalent bond can be formed via various appropriate functional crosslinking reactions. In some embodiments described herein, the crosslinking reaction is click chemistry. As used herein, the term “click handle” refers to a molecule on mRNA that can covalently bind to another molecule via click chemistry reaction. Examples of a handle include, but are not limited to, alkyne or azide (when CuAAC is used in click chemistry), or trans-cyclotene or tetrazine (when IEDDA is used in click chemistry), or hydrozone or oxime, or any equivalent structures thereof. Other crosslinking chemistries including thio-ene and tiol-yne reactions (Escorihuela et al., 2014, Bioconjug. Chem. 25:618-627), a phosphate-amine based reaction (El-Sagheer and Brown, 2017, Chem. Commun.53: 10700-10702; Kalinowski et al., 2016, Chembiochem. \ . 1150-1155), thiol-yne, amino-yne, and hydroxyl-yne reactions (Worch et al., 2021, Chem Rev. 121(12): 6744-6776), and other bioconjugation reactions (Gassensmith, chem . libretexts . org / Bookshel ves / Organi c_Chemi stry / Supplemental_Modules_(Organic_Chemi st ry) / Reactions / Introduction_to_Bioconjugation, accessed June 23, 2023) have also been contemplated.

[0092] As used herein, the term “hairpin” or “hairpin oligonucleotide” refers to a single-stranded oligonucleotide that has a sequence of complementary base pairs at both ends capable of forming a “stem-and-loop” structure.

[0093] As used herein and understood in the art, the term “click chemistry” is intended to encompass chemical methods for linking chemical components together, including but not limited to nucleotides into polynucleotides and amino acids into peptides and polypeptides, that are “simple to perform, have high yields, require no or minimal purification, and are versatile in joining diverse structures without the prerequisite of protection steps” (see, for example, Hein et al., 2006, Pharm. Res. 10: 2216-2230). In current chemical synthetic practice four primary reactions are employed: 1) cycloadditions (including for example monovalent copper-catalyzed Huisgen 1,3-dipolar cycloadditions of azides and alkynes, the most widely used); 2) nucleophilic ring openings (including ring systems comprising strained heterocyclic electrophiles); 3) non- Aldol carbonyl chemistry (including for example hydrazone / oxime ether formation); and 4) carbon multiple bond additions (including for example certain Michael additions and formation of various three-membered rings by inter alia epoxidations). Click chemistry has been found to be particularly useful for polymeric substances such as proteins and nucleic acids as illustrated herein.

[0094] As used herein, the term “equivalent structure” means any molecule that are sufficiently structurally similar and perform the same function in a chemical reaction.

[0095] As used herein, the terms “derivatized” or “functionalized” means modification of a nucleotide that leads to some functional consequences in its chemical properties or reactivity or both. Both terms shall be understood to be equivalent to the extent that particular embodiments of the capped, circular RNA molecules have by benefit of derivatization thereof a function, particularly with regard to crosslink-dependent circularization embodiments provided herein. Insome embodiments, a derivatized nucleotide is a nucleotide that is modified to comprise a chemical group / handle can participate in a cross-linking reaction.

[0096] As used herein, the term “QRNA” is intended as a generic term meaning capped circular messenger RNAs. Particularly encompassed by this term are the various species of circularized RNA molecules and in particular circularized mRNA molecules disclosed herein, but these examples are not intended to be limiting.

[0097] In some embodiments, the synthesis pathway of Type 1 and Type 3 QRNA enables multiple oligonucleotides containing 5’ cap binding to the circular RNA. For example, circular RNA can include multiple derivatized nucleotides that can covalently bind to multiple oligonucleotides containing 5’ cap. Alternatively, a single circular RNA backbone can encode multiple TGT sites to enable binding of multiple oligonucleotides containing 5’ cap onto the circular RNA simultaneously.

[0098] In some embodiments, the capped, circular RNA molecule comprises an mRNA region encoding one or a plurality of peptides or polypeptides.

[0099] As provided herein, the cap used in the capped, circularized RNA molecules of the invention can include 7-methylguanine (m7G) but in addition cap analogues as set forth, inter alia, in U.S. patent application No. 2020 / 0055891 to Walczak et al.; Holstein et al., 2016, Agnew Chem. Int. Ed. Engl. 55: 10899-10903; Walczak et al., 2017, Chem. Sci. 8: 260-267; Muttach et al., 2017, J. Org. Chem. 13: 2819-2832) can be incorporated into the circular RNA molecule precursors to create the capped, circularized RNA molecules provided herein.Cap modifications for QRNA

[0100] Several variations of the cap structure have been contemplated here to optimize translation efficiency of QRNA. These variations include: including multiple cap structures (cap 0, 1, and 2; Shanmugasundaram t al., 2022, Chem Rec. 22(8): e202200005); including N6, 2’-O- dimethyladenosine (m6Am) as a terminal modification adjacent to the mRNA cap (Sun et al., 2021, Nat Commnn. 12(1): 4778); using cap structures with modified triphosphate bridges (Sun et al., 2021, Nat Cornmun. 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; Nowakowskaet al., 2014, Org. biomol. Chem. 12: 4841-4847); hydrophobic cap analogs (WO 2017066782 Al); 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).

[0101] In some embodiments, the methyl group in 7-methylguanosine (m7G) cap structure can be modified to produce 7-benzylguanosine (Bn7G), 7-chlorobenzylguanosine (ClBn7G), and chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G). Introduction of one or more Locked Nucleic Acid (LNA), 2’ -methoxy (20Me), and 2-m ethoxy ethoxy (2M0E) into m7G structure significantly increase mRNA translation. In some embodiments, the cap structures include, but are not limited to, m7G-LNA, LNAm7G-LNA, LNAm7G-LNAx6, LNAm7G- 2OMex6. In some embodiments, the cap structure is m7G diphosphate imidazolide (m7GDP-Im).Nucleotide modifications

[0102] In some embodiments, as disclosed and recognized herein it is beneficial to alter the type of nucleotide / nucleotide identity, specifically incorporation of adenosine (A), guanosine (G), 6-methyladenosine (m6A), or the non-canonical inosine (I) in the mRNA, preferably, at the +1 position, increases translation efficiency. In some embodiments, substitution of some or all uridine residues to A7-methylpseudouridine (m1T) in the mRNA also boosts the translation. The nucleotides are numbered according to their position immediately downstream of the cap structure. For example, the cap structure found at the 5' end of eukaryotic mRNAs consists of a 7-methylguanosine (m7G) moiety linked to the first nucleotide (+1 position) of the transcript via a 5'-5' triphosphate bridge.

[0103] Other modified nucleotides include, but are not limited to, pseudouridine, 5- methylcytidine, 2-thiouridine, 5-methoxyuridine, 4-acetylcytidine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5 -methyluracil, 4-thiothymidine, 4-thiouracil, 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-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin- 16-7-deaza-7-propargylaminoguanine, biotin- 16- aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3 -aminoallylcytosine, cyanine 3 -aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, Nl- 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- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, 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-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A) have also been contemplated at +1 and other positions.

[0104] In some embodiments, the modified phosphate backbone can be phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5- hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphorami date, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, or guani di nopropyl phosphoramidate.

[0105] In some embodiments, introduction of locked nucleic acid (LNA), 2’- methoxyribose (2-OMe), and 2-methoxyethoxy (2-MOE) into the ribose sugar backboneincreases mRNA translation. Addition of multiple 2-OMe and 2-MOE modified bases increases translation further. LNA specifically increased expression at the +1 position.

[0106] 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 ’-di deoxyribose, 3’- azido-2, 3 -dideoxyribose, 3 ’-deoxyribose, 3’-O-(2-nitrobenzyl)-2’-deoxyribose, 3’-O- methylribose, 5 ’-aminoribose, 5 ’-thioribose, 5-nitro-l-indolyl-2’-deoxyribose, 5’-biotin-ribose, 2’-O,4’-C-methylene-linked, 2’-O,4’-C-amino-linked ribose, or 2’-O,4’-C-thio-linked ribose.

[0107] In these backbone modifications, stereoisomer structures are also considered since they have been shown to impact the RNA’s nuclease-resistance properties (Iwamoto et al., 2017, Nat. Biotech. 35: 845-851; Jahns etal., 2022, Nucleic Acids Res. 50(3): 1221-1240).

[0108] Modification of nucleotides on traditional circRNA is limited because not all of them are compatible with the internal ribosome entry site (IRES). QRNA translation does not require an IRES; thus, is tolerable to more modified nucleotides in a wide range of percentage. These modifications could be spiked into the circular backbone in varying percentages (m6A is typically spiked in at 5%). And the “stem” oligo containing the cap, or the 573’ UTR and tails could likely tolerate a higher percentage of modifications. Alternatively, these modifications can be present in different percentages along different regions of the circular RNA backbone (e.g. in the 5’ UTR, or 3’ UTR, or CDS, or close to the cap structure, or combinations thereof).Furthermore, the “stem” oligo of a Type 1 QRNA (the oligonucleotide containing the cap) is chemically synthesized and could potentially tolerate more complex structures that are difficult to enzymatically incorporate, such as locked nucleic acids (LNAs), 2’ O-methyl nucleotides, peptide nucleic acids (PNAs), morpholinos, and various internal chemical linkers as provided herein.Peptides and polypeptides encoded by QRNA

[0109] Polypeptides encoded by the capped, circularized RNA molecules provided by the invention include any therapeutically useful polypeptide for treatment or intervention of any disease process associated with or dependent on polymorphic or mutant polypeptide species, heritable or acquired as a result of environmental insult or injury. QRNA can encode multiple polypeptides, for example, self-amplifying mRNA cassettes, or multiple therapeutic peptides orpolypeptides. In some embodiments, the capped, circular RNA molecule comprises an mRNA region encoding one or a plurality of peptides or polypeptides. A plurality of polypeptides include multiple copies of the same polypeptide or multiple copies of different polypeptides.

[0110] An IRES, or self-cleaving peptide such as T2A sequence, can exist between the multiple polypeptide coding sequences on the QRNA. Alternatively, an RNA oligonucleotide containing cap residue site is located before each polypeptide coding sequence, which ultimately will result in a QRNA with multiple cap residue-containing RNA oligonucleotides and ensure that all coding sequences are translated efficiently.

[0111] Peptides encoded by capped, circularized RNA molecules of the 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 cells) or cellular (T cells) immune system cells. In certain embodiments these antigenic peptides are adapted to and effective for use as vaccines. In other embodiments the antigenic peptides are adapted to or effective in suppressing immune responses, for example in autoimmune diseases or transplant patients. In additional embodiments the antigenic peptides are adapted to and effective for eliciting specific antitumor immune responses in tumor cells or in attracting cytotoxic native (natural killer cells) or engineered (e.g., CAR-T) cells. Therapeutic peptides encoded by capped, circularized RNA molecules of the invention can include but are not limited to human parathyroid hormone, filgrastim, oxytocin, somatostatin, calcitonin, glucagon, insulin, liraglutide, vasopressin, and the like (see, Fosgerau & Hoffman, 2015, Drug Discovery Today 20:122-128; al Musaimi etal., 2021, Pharmaceuticals (Basil) 14: 145; Wang et al., 2022, Signal Transduct, and Targeted Therap. 7: 1-27).

[0112] In some embodiments, peptides encoded by the capped, circular RNA molecules of the 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 delivery of genome or epigenome editing therapies.

[0113] In some embodiments, 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, biologies, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, or targeting moieties.Synthesis of QRNA

[0114] Type 2: The invention also provides methods for producing a type 2 capped, circularized RNA molecules of this aspect of the invention, the methods 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 the covalently linked capped circular RNA molecule.

[0115] Type I: The invention also provides methods for producing a type 1 capped, circularized RNA molecules of this aspect of the invention, the methods comprising the steps of: producing a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide, and a derivatized nucleotide outside the mRNA region; synthesizing an RNA oligonucleotide comprising a 5’ end containing cap structure and a 3’ end containing a moiety reactive with the derivatized nucleotide; and reacting the derivatized nucleotide with the 3’ end moiety of the RNA oligonucleotide form a covalently link between the RNA oligonucleotide and the circular RNA. In certain embodiments, the derivatized nucleotide comprises a moiety that can react with the 3’ end moiety by bioconjugation chemistry, wherein in the bioconjugation chemistry is click chemistry. In addition, the circularized RNA is produced by ribozyme- mediated splicing, enzymatic ligation, or click chemistry-mediated circularization.

[0116] Type 3: The invention also provides methods for producing a type 3 capped, circularized RNA molecules of this aspect of the invention, the methods comprising the steps of: producing a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide, and a derivatized nucleotide outside the mRNA region; synthesizing an RNA oligonucleotide comprising a 5’ end containing cap structure and a 3’ end containing a moiety reactive with the derivatized nucleotide; and reacting the derivatized nucleotide with the 3’ end moiety of the RNA oligonucleotide form a covalently link 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 the mRNA region encoding a peptide or polypeptide, a hairpin structure containing an enzyme-recognition site, and a twister ribozyme sequence on both 5’ and 3’ ends; reacting the RNA oligonucleotide with the enzyme toproduce the derivatized nucleotide within the hairpin structure; circularizing the RNA oligonucleotide using the twister ribozyme sequence.

[0117] The derivatized nucleotide in these 3 types of QRNA can be generated using numerous strategies. Namely, the derivatized nucleotide can be specifically targeted by having a hairpin structure containing a specific enzyme-recognition site. The enzyme described in some examples in tRNA guanine transferases (TGT). In other examples, the derivatized nucleotide is generated by replacement of a single cytidine with azide-cytidine.

[0118] Circularization of RNA molecule 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’ end and 3’ end of the linear oligonucleotide comprise the appropriate moiety to participate in the enzymatic reaction to form the circular RNA. Alternatively, the click chemistry moiety has also been contemplated for the circularization. In some embodiments, additional splint probe containing complementary sequences to the 5’ and 3’ ends of the linear oligonucleotide can be used to bring the two ends in proximity and facilitate circularization.Purification of capped RNA

[0119] In some embodiments, the nucleic acids described herein are purified by any method known in the art to remove undesired components from IVT or associated reactions (including unincorporated rNTPs, protein enzymes, salts, metal ions, etc.). Techniques for the isolation of RNA molecules are well known in the art. Well-known procedures include phenol / chloroform extraction and or precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, et al. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012). Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).Techniques to remove contaminants, such as dsDNA, have been developed and are known in theart including but not limited to scalable HPLC purification (see, e.g., Kariko, et al., 2011, Nucl Acid Res, v. 39 el42; Weissman, et al., 2012, Synthetic Messenger RNA and Cell Metabolism Modulation v.969 (Rabinovich, P.H. Ed)). In a preferred embodiment, the capped RNA described herein is purified through HPLC, as HPLC-purified RNA has been reported to be translated at much greater levels compared to other purification methods, particularly in primary cells and in vivo.

[0120] In some aspects, the capped RNA oligonucleotides provided herein (e.g., comprising one or more modified oligonucleotides) are purified by high-performance liquid chromatography (HPLC). In some embodiments, the capped RNA oligonucleotide is purified by reverse-phase HPLC (RP-HPLC). The addition of a counterion to the mobile phase of a HPLC setup can improve separation of the desired product from unwanted products. In a preferred embodiment, HPLC gradients used to isolate the capped RNA oligonucleotides comprise hydrophobic hexylammonium ions. In some embodiments, the gradient is chosen from ethyl ammonium, diethyl ammonium, triethyl ammonium, propyl ammonium, dipropyl ammonium, hexyl ammonium, dihexyl ammonium, octyl ammonium, dioctyl ammonium, etc. The number and lengths of carbon chains may be altered based on the lengths of the oligonucleotide to be captured and the desired feature for separation. In some embodiments, the concentration of hydrophobic ions (e.g., hexylammonium ions) used for HPLC purification of RNA oligonucleotides is between 10 mM to 200 mM. In some embodiments, the concentration of hydrophobic ions is between 10 mM and 20 mM, between 20 mM and 30 mM, between 30 mM and 40 mM, between 40 mM and 50 mM, between 50 mM and 60 mM, between 60 mM and 70 mM, between 70 mM and 80 mM, between 80 mM and 90 mM, between 90 mM and 100 mM, between 100 mM and 110 mM, between 110 mM and 120 mM, between 120 mM and 130 mM, between 130 mM and 140 mM, between 140 mM and 150 mM, between 150 mM and 160 mM, between 160 mM and 170 mM, between 170 mM and 180 mM, between 180 mM and 190 mM, or between 190 mM and 200 mM. In some embodiments, the concentration of hydrophobic ions is greater than 200 mM.Compositions comprising capped RNA transcripts

[0121] In some aspects, the present disclosure provides a delivery reagent comprising any of the capped RNA molecules provided herein. In some embodiments, any of the cappedRNA molecules provided herein are conjugated to a delivery agent. Any of the capped RNA molecules provided herein may be conjugated to a delivery agent that includes, for example, to a lipid, a peptide, a protein, an antibody, or a carbohydrate. Lipids used in the conjugation and delivery of modified mRNAs are generally known in the art, and include, for example, cholesterol. Peptides, proteins, antibodies, and carbohydrates used in the conjugation and delivery of modified mRNAs are generally known in the art and include, for example, any peptide, protein, antibody, or carbohydrate known to bind specifically to a moiety (e.g., a protein) on the surface of a target cell type. Methods for conjugating a lipid, peptide, protein, antibody, or carbohydrate to a capped RNA molecule include, for example, methods of conjugating a lipid, peptide, protein, antibody, or carbohydrate to a capped RNA molecule at a 5’ or 3’ terminus, and are generally known in the art.

[0122] In some embodiments, any of the capped RNA molecules provided herein are conjugated to or encapsulated by a delivery agent that includes, for example, a nanoparticle, a microparticle, or an exosome. A nanoparticle refers to a particle having a diameter between approximately 10 nm and 1000 nm. A microparticle is defines as a particle having a diameter greater than 1000 nm (1 pm), such as a particle having a diameter between approximately 1 pm and 100 pm. In some embodiments, a nanoparticle or microparticle is approximately spherical. In some embodiments, a nanoparticle or microparticle is hollow, comprising an internal core. In some embodiments, a nanoparticle or microparticle is a lipid nanoparticle or lipid microparticle, respectively. A lipid nanoparticle or lipid microparticle refers to a composition comprising one or more lipids that form an aggregate of lipids, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a lipid nanoparticle or lipid microparticle comprises a lipid bilayer that encloses an aqueous core. Lipids used in the formulation of lipid nanoparticles and lipid microparticles for delivering RNAs are generally known in the art, and include, but are not limited to, ionizable amino lipids, non-cationic lipids, sterols, and polyethylene glycol-modified lipids. See, e.g., Buschmann et al. Vaccines. 2021. 9(1):65. In some embodiments, the capped RNA molecule is surrounded by the lipids of the lipid nanoparticle or the lipid microparticle and are present in the interior of the lipid nanoparticle or lipid microparticle. In some embodiments, the capped RNA molecule is dispersed throughout the lipids of the lipid nanoparticle or lipid microparticle. In some embodiments, the lipid nanoparticle or lipid microparticle comprises an ionizable amino lipid, a non-cationic lipid, asterol, and / or a polyethylene glycol (PEG)-modified lipid. Lipid nanoparticles and lipid microparticles comprising modified mRNAs may be prepared by any means generally known in the art, such as, for example, detergent dialysis, emulsion, centrifugation, evaporation, thin film hydration, or ethanol dilution. See, e.g., Barba et al. Pharmaceutics. 2019. 11 (8) :360. An exosome refers to a type of lipid nanoparticle produced by eukaryotic cells as a result of the inward budding of vesicles within multivesicular bodies and are generally between 30 nm and 150 nm in diameter. Exosomes comprise a heterogenous mixture of endogenous lipids, such as phospholipids, membrane-anchored proteins, and carbohydrates present in eukaryotic cells, and enclose an aqueous core. Exosomes may have beneficial features that are difficult to achieve with synthetically produced lipid nanoparticles, such as, for example, the ability to pass through the blood brain barrier and deliver capped RNA molecules to tissues within the brain. Exosomes comprising capped RNA molecules may be produced by any means generally known in the art, such as, for example, by sonicating or electroporating isolated exosomes in the presence of a capped RNA molecule, or mixing exosomes with a lipid-conjugated capped RNA molecule, such as, for example, a capped RNA molecule that has been conjugated to cholesterol. See, e.g., Roberts et al. Nat Rev Drug Discov. 2020. 19(10):673-694.

[0123] In some embodiments, a nanoparticle or microparticle is a polymeric nanoparticle or polymeric microparticle, respectively. A polymeric nanoparticle or polymeric microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more polymers that form an aggregate of polymers, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a polymeric nanoparticle or polymeric microparticle comprises a polymeric layer that encloses an aqueous core. Polymers used in the formulation of polymeric nanoparticles and polymeric microparticles for delivering RNA are generally known in the art, and include cationic polymers such as, but are not limited to, polyethylenimine (PEI), poly-amido-amine (PAA), poly-beta amino-esters (PBAEs), polylysine (PLL), spermine, chitosan, polyurethane, and derivatives thereof (e.g., PEI stearic acid (PSA) copolymer). See, e.g., Liu et al. Front Bioeng Biotechnol. 2021. 9:718753. In some embodiments, the capped RNA molecule is surrounded by the polymers of the polymeric nanoparticle or the polymeric microparticle and are present in the interior of the polymeric nanoparticle or polymeric microparticle. In some embodiments, the capped RNA molecule is dispersed throughout the polymers of the polymeric nanoparticle or polymeric microparticle.

[0124] In some embodiments, a nanoparticle or microparticle is a protein nanoparticle or protein microparticle, respectively. A protein nanoparticle or protein microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more proteins that form an aggregate of proteins, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a protein nanoparticle or protein microparticle comprises a protein layer that encloses an aqueous core. Proteins used in the formulation of protein nanoparticles and protein microparticles for delivering RNA are generally known in the art, and include but are not limited to, viral coat proteins and ferritin. See, e.g., Wang et al. Nat Nanotechnol. 2020. 15(5):406-416. In some embodiments, the capped RNA molecule is surrounded by the proteins of the protein nanoparticle or the protein microparticle and are present in the interior of the protein nanoparticle or protein microparticle. In some embodiments, the capped RNA molecule is external to the proteins of the protein nanoparticle or the protein microparticle and are attached to the exterior surface of the protein nanoparticle or protein microparticle. In some embodiments, the capped RNA molecule is conjugated to proteins of the protein nanoparticle or protein microparticle through a covalent linkage, such as, for example, that formed by a click chemistry reaction, or by fusing the capped RNA molecule and protein each to a protein or peptide of a protein / peptide pair known to react to form a covalent linkage.

[0125] In some embodiments, a nanoparticle or microparticle is a solid nanoparticle or solid microparticle. A solid nanoparticle or solid microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more materials that form a solid structure, which has an external surface and may or may not comprise an internal surface. A solid nanoparticle or solid microparticle may comprise any suitable material that is generally known in the art, such as, for example, gold, silver, or silicon dioxide (silica). In some embodiments, a capped RNA molecule is conjugated to the external surface of a solid nanoparticle or solid microparticle. Solid nanoparticles and solid microparticles comprising capped RNA molecules may be produced by any means generally known in the art, such as, for example, by linking the capped RNA molecules to the surface of the solid nanoparticle or solid microparticle through thiol linkages (e.g., modifying the DNA to comprise cyclic disulfide- anchoring groups), or by modifying the external surface of the solid nanoparticle or solid microparticle with one or more cationic materials (e.g., PEI) within which capped RNA molecules are present. See, e.g., Roberts et al. Nat Rev Drug Discov. 2020. 19(10):673-694, Leeet al. Nano Lett. 2007, 7(7):2112-21 15, and Paris and Vallet-Regi. Pharmaceutics. 2020, 12(6):526.

[0126] In some aspects, the present disclosure provides cells comprising any of the capped RNA molecules provided herein. In some embodiments, the cell is a human cell comprising any one of the capped RNA molecules provided herein. A “cell” is the basic structural and functional unit of all known independently living organisms. It is the smallest unit of life that is classified as a living thing. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as plants, fungi, and animals, including cattle, horses, chickens, turkeys, sheep, swine, dogs, cats, and humans, are multicellular. In some embodiments, the half-life of the capped RNA molecule in the cell is 15-900 minutes. In some embodiments, the half-life of the capped RNA molecule in the cell is 30-600 minutes. In some embodiments, the half-life of the capped RNA molecule in the cell is 60-300 minutes. In some embodiments, the half-life of the capped RNA molecule is at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 minutes. In some embodiments, the half-life of the capped RNA molecule in the cell is at least 30, at least 60, at least 90, at least 120, at least 150, at least 180, at least 210, at least 240, at least 270, at least 300, at least 330, at least 360, at least 390, at least 420, at least 450, at least 480, at least 510, at least 540, at least 570, at least 600, at least 630, at least 660, at least 690, at least 720, at least 750, at least 780, at least 810, at least 840, or at least 870 minutes. In some aspects, the present disclosure provides compositions comprising any of the modified mRNAs, delivery agents, or cells provided herein. In some embodiments, the composition further comprises one or more additional agents, such as a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate. In some embodiments, the additional agent has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is an agent for use in modulating the expression and / or activity of one or more gene products (e.g., proteins) in a subject. In some embodiments, the additional agent is a nucleic acid for use in decreasing the expression and / or activity of one or more gene products (e.g., proteins), such as a short hairpin RNA (shRNA), small interfering RNA (siRNA), or an antisense oligonucleotide (ASO). In some embodiments, the additional agent is an inhibitor for decreasing the activity of one or more gene products (e.g., proteins). In some embodiments, the agent is a small molecular inhibitor. In some embodiments, the additional agent is an agent for enhancing an immuneresponse in a subject. In some embodiments, the additional agent is an antigen, such as a nucleic acid antigen, a protein antigen, or a phospholipid antigen. In some embodiments, the additional agent is an adjuvant, such as, for example, aluminum hydroxide or potassium aluminum sulfate (alum), monophosphoryl lipid A (MPL), an oil-in-water emulsion (e.g., a squalene emulsion), a cytosine phosphoguanine (CpG) oligodeoxynucleotide, or another adjuvant that is known in the art. See, e.g., Di Pasquale, A et al. Vaccines. 2015. 3(2):320-343. In some embodiments, the composition is a pharmaceutical composition comprising any one of the capped RNA molecules, delivery agents, or cells provided herein, and a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients, carriers, buffers, stabilizers, isotonicising agents, preservatives or antioxidants, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g., parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal.

[0127] In some aspects, the present disclosure provides a comprising an RNA molecule and a delivery agent. In some embodiments, the composition comprises (a) an RNA molecule comprising (i) one or more modified nucleotides at position +3 or higher with reference to a 5’ terminus of the RNA molecule, (ii) at least one 5’ cap, (iii) and an open reading frame (ORF), and (b) a delivery agent. In some embodiments, the RNA molecule of the composition comprises two or more 5’ caps. In some embodiments, the two or more 5’ caps are conjugated to a 5’ UTR of the RNA molecule of the composition. In some embodiments, the two or more 5’ caps are conjugated to the RNA molecule of the composition via click chemistry. In some embodiments, the one or more modified nucleotides comprises a modified sugar. In some embodiments, the one or more modified nucleotides comprises a modified phosphate. In some embodiments, the one or more modified nucleotides comprises a modified nucleobase.

[0128] In some embodiments, a composition comprises an RNA molecule comprising a 5’ untranslated region (5’ UTR). In some embodiments, the 5’ UTR comprises a promoter. In some embodiments, the RNA molecule of the composition further comprises a 3’ untranslated region (3’ UTR). In some embodiments, the 3’ UTR comprises at least one exonuclease-resistant modification.

[0129] In some embodiments, the RNA molecule of the composition comprises two or more 5’ caps. In some embodiments, the RNA molecule of the composition comprises two or more poly- A tails.

[0130] In some embodiments, the RNA molecule of the composition further comprises an open reading frame (ORF). In some embodiments, the ORF encodes a protein. In some embodiments, the ORF encodes a therapeutic protein. In some embodiments, the ORF encodes an antigen. In some embodiments, the antigen is a SARS-CoV-2 spike protein or fragment thereof. In some embodiments, the ORF comprises a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the ORF encodes an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

[0131] In some embodiments, the RNA molecule of the composition further comprises a sequence encoding a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

[0132] In some embodiments, the RNA molecule of the composition is a circular RNA molecule.

[0133] In some embodiments, the RNA molecule of the composition comprises a stem oligo modification having the sequence of SEQ ID NO: 1. In some embodiments, the RNA molecule of the composition comprises a branch oligo modification having the sequence of SEQ ID NO: 2. In some embodiments, the RNA molecule of the composition comprises a 5’UTR having the sequence of SEQ ID NO: 3. In some embodiments, the RNA molecule of the composition comprises a 3’UTR having the sequence of SEQ ID NO: 5. In some embodiments, the RNA molecule of the composition comprises a polyA tail modification having the sequence of SEQ ID NO: 6. In some embodiments, the RNA molecule of the composition comprises two 5’ caps, wherein each of the two 5’ caps is LNAm7G.

[0134] In some embodiments, the delivery agent of the composition comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle. In some embodiments, the nanoparticle or microparticle is a lipid nanoparticle or a lipid microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a proteinmicroparticle, or a solid nanoparticle or a solid microparticle. In some embodiments, the delivery agent of the composition comprises a lipid nanoparticle.

[0135] In some aspects, the composition comprises an RNA molecule and a delivery agent, wherein the RNA molecule comprises two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF comprising a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6; and the delivery agent comprises a lipid nanoparticle. In some aspects, the composition comprises an RNA molecule and a delivery agent, wherein the RNA molecule comprises two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF encoding an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6; and the delivery agent comprises a lipid nanoparticle.

[0136] In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

[0137] In some aspects, the present disclosure provides a method of administering to a subject any of the capped RNA molecules, delivery agents, cells, compositions, or pharmaceutical compositions provided herein. In some embodiments, the subject is a human. In some embodiments, the administration is parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal. In some embodiments, the composition is to be stored below 50°C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60°C, below -70 °C, or below -80 °C, such that the nucleic acids are relatively stable over time. In some embodiments, the capped RNA molecule is introduced into a cell in a subject by in vivo electroporation. In vivo electroporation is the process of introducing nucleic acids or other molecules into a cell of a subject using a pulse of electricity, whichpromote passage of the nucleic acids or other molecules through the cell membrane and / or cell wall. See, e.g., Somiari et al. Molecular Therapy., 2000. 2(3): 178-187. The capped RNA molecule to be delivered is administered to the subject, such as by injection, and a pulse of electricity is applied to the injection site, whereby the electricity promotes entry of the nucleic acid into cells at the site of administration. In some embodiments, the capped RNA molecule is delivered to and taken up by cells of the subject (e.g., cells local to the site of administration or throughout the subject) via a delivery agent that is associated with (e.g., conjugated to) the capped RNA molecule. In some embodiments, the capped RNA molecule is administered with other elements, such as buffers and / or excipients, that increase the efficiency of electroporation.

[0138] In some aspects, the present disclosure provides a kit comprising any of the capped RNA oligonucleotides, RNA precursors, or capped RNA molecules provided herein. The capped RNA oligonucleotide and RNA precursor can be combined in the presence of an RNA ligase to produce a capped RNA molecule, such as one of the capped RNA molecules provided herein. In some embodiments, the kit comprises a ligase. In some embodiments, the kit comprises an RNA ligase. In some embodiments, the kit comprises a T4 RNA ligase. In some embodiments, a kit comprises a T4 RNA ligase 1. In some embodiments, a kit comprises a T4 RNA ligase 2. In some embodiments, the kit comprises an RtcB RNA ligase. In some embodiments, the kit further comprises a buffer for carrying out the ligation. In some embodiments, the kit further comprises a nucleotide triphosphate, such as ATP, to provide energy required by the ligase. In some embodiments, the kit is to be stored below 50 °C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60°C, below -70 °C, or below -80 °C, such that the nucleic acids are relatively stable over time.

[0139] In some aspects, the present disclosure provides a kit comprising any of the pharmaceutical compositions provided herein and a delivery device. A delivery device refers to machine or apparatus suitable for administering a composition to a subject, such as a syringe or needle. In some embodiments, the kit is to be stored below 50 °C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60°C, below -70 °C, or below -80 °C, such that the nucleic acids of the pharmaceutical composition are relatively stable over time. In some embodiments, the kit furthercomprises instructions for administering any of the pharmaceutical compositions provided herein to a subject.Pharmaceutical compositions for delivery and methods therefore

[0140] This invention provides pharmaceutical compositions comprising capped RNA molecules of the disclosure, particularly linear and circularized mRNA molecules. In certain embodiments pharmaceutical compositions of the invention further comprise pharmaceutically acceptable excipients and in certain other embodiments comprise one or more additional therapeutics agents.

[0141] In some embodiments, the compositions are suitable to be administered to a human subject in need thereof. In the context of the present disclosure, “active ingredient” refers generally to the capped RNA molecules described herein, particularly linear and circularized mRNA molecules as well as any additional therapeutic agents provided therewith.

[0142] It is generally understood by a person of ordinary skill in the art that the compositions described herein are also suitable for administration to any non-human subjects as well. A person of ordinary skill in the veterinary arts will understand that pharmaceutical compositions described herein can 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. A person of ordinary skill in the veterinary arts also will understand that pharmaceutical compositions described herein can be suitable for administration to birds including by not limited to chickens, ducks, geese, turkey, and other domesticated birds, as well as wild birds particularly endangered species of such birds. Additionally, a person of ordinary skill in the veterinary arts will understand that pharmaceutical compositions described herein can be suitable for administration to a wide variety of fish including commercial or wild salmon, tuna, cod, sardine, zebra fish, shark, or the like.

[0143] Pharmacological compositions described herein can be prepared by any method known or developed in the art of pharmacology, immunology, virology, or in biotechnology in general.

[0144] In some embodiments, the formulations of a pharmacological composition described herein can comprise a unit dose of at least one RNA, in addition to at least one other pharmaceutically acceptable excipient. Such excipients can include but are not limited to, solvents,dispersions, buffers, diluents, surfactants, emulsifiers, isotonic agents, preservatives, thickeners, lubricating agents, oils, or the like.

[0145] In some embodiments, the pharmacological composition can comprise a delivery mechanism further comprising a lipid nanoparticle. The size of the lipid nanoparticle can be altered to counteract immunogenic response from the subject, or to allow for increased potency and pharmacological activity.

[0146] In other embodiments, the pharmacological composition can comprise a delivery mechanism further comprising a lipidoid as previously described in the art. See Akinc etal., 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 etal., 2011, Nat Biotechnol. 29: 1005-1010, all of which is incorporated herein in their entirety. Lipidoids refers broadly to lipid nanoparticles, liposomes, lipid emulsions, lipid micelles and the like. Lipidoids containing the pharmacological composition comprising the derivatized RNA can be administered parenterally by means including but not limited to, intravenous injection, intramuscular injection, subcutaneous injection, via dialysate, intrathecal injection, or intracranial injection.

[0147] A person of ordinary skill in the art would also recognize that other nucleotide delivery mechanisms exist such as the use of viral like, or viral derived particles. See Rohovie et al., 2016, Bioengineering & Translational Med. 2(1): 43-57. Virus like particles can include coat proteins or viral capsids of a virus. Such particles can be PEGylated or further annealed to compounds that avoid phagocytotic clearance. Additionally, the surface of the virus like particle can be further functionalized to provide cellular specific targeting, facilitate extravasation, facilitate radio labeling, improve permeability across cellular boundaries, or to transcytose the blood-brain barrier. The virus like particles can be derived for animal viruses, bacteriophages, or plant viruses. Examples of suitable virus for derivation of a virus like particle delivery mechanism include but are not limited to cowpea chlorotic mottle virus, cowpea mosaic virus, hepatitis B virus (core), enterobacteria phage MS2, Salmonella typhimiirium P22, enterobacteria phage Q amongst other suitable viruses. Derivatized RNA payloads can be loaded into the virus like particles by electrostatic adsorption or any other suitable method known to a person of ordinary skill in the art.

[0148] In some aspects, provided herein are methods of preventing or treating a disease in a subject. In some embodiments, provided herein are methods of preventing or treating a diseasein a subject, comprising introducing an effective amount of an RNA molecule or composition comprising the RNA molecule described herein to the subject. In some embodiments, the subject is a human subject. In some embodiments, provided are methods of vaccinating a subject against a disease. In some embodiments, provided are methods of vaccinating a subject against a disease, comprising introducing an effective amount of an RNA molecule or composition comprising the RNA molecule described herein to the subject. In some embodiments, the disease is SARS-CoV- 2.

[0149] In some embodiments of a method of preventing or treating a disease in a subject, a single dose of an effective amount of an RNA molecule or a composition comprising the RNA molecule is administered to the subject. In some embodiments of a method of preventing or treating a disease in a subject, at least doses of an effective amount of an RNA molecule or a composition comprising the RNA molecule are administered to the subject. Dose administrations me be separated by at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 7 days, at least 14 days, at least 21 days, at least 28 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, or more.

[0150] For the purposes of the present disclosure, “preventing” a disease can include reducing the risk of a given disease, such as a viral or bacterial infection. Thus, the present disclosure provides methods of reducing the risk of a disease (e.g., a bacterial or viral infection) comprising administering to a subject an effective amount of an RNA molecule or composition described herein. For example, the disclosed RNA molecules and compositions can be administered in an effective amount to a subject in need thereof to reduce circulating levels of a virus (e.g., SARS-COV-2) or bacterial, reduce viral load or bacterial titer, and / or reduce, ameliorate, or eliminate one or more signs or symptoms of an infection (e.g., a SARS-COV-2 infection). In some embodiments, the subject may be at risk of exposure to a bacteria or virus, previously exposed to a bacteria or virus, or exposed to a bacteria or virus. In some embodiments, the administration of the antigen prevents the subject from developing a HIV infection and / or AIDS. In some embodiments, the administration of the disclosed RNA molecules and compositions reduces the risk the subject will develop an infection, generally, or may reduce the risk of developing a severe infection (e.g., reducing the risk of infection requiring hospitalization). In some embodiments, the administration of the disclosed RNA molecules and compositions reduces the risk of transmission of a bacteria or virus. In some embodiments, o theadministration of the disclosed RNA molecules and compositions reduces the need for additional treatment or prophylaxis to treat or prevent a given infection. The effective amount of a binding protein is sufficient to reduce circulating viral load or bacterial titer, and / or to reduce, ameliorate, or eliminate one or more symptoms or effects of a viral (e.g., SARS-COV-2) or bacterial infection. In embodiments pertaining to treatment or prevention of SARS-COV-2, the effective amount of a binding protein is effective to reduce or prevent binding of a SARS-COV-2 spike protein to a host cell. The specific amount of a given RNA molecule or composition administered may depend on one or more of the age and / or weight of the subject and / or the stage or severity of the disease and / or the dosage form and route of administration, and can be determined by the skilled practitioner.

[0151] Various exemplary embodiments of compositions and methods according to this invention are now described in the following non-limiting Examples. The Examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.EXAMPLES OF EMBODIMENTS

[0152] The following examples are given to illustrate the present disclosure. It should be understood, however, that the disclosure is not to be limited to the specific conditions or details described in these examples.

[0153] Embodiment 1: A composition comprising: (a) an RNA molecule comprising (i) one or more modified nucleotides at position +3 or higher with reference to a 5’ terminus of the RNA molecule, (ii) at least one 5’ cap, (iii) and an open reading frame (ORF), and (b) a delivery agent or a composition comprising: (a) an RNA molecule comprising (i) one or more modified nucleotides at position +3 or higher with reference to a 5’ terminus of the RNA molecule, and (ii) at least one 5’ cap, and (b) a delivery agent.

[0154] Embodiment 2: The composition of embodiment 1, wherein the RNA molecule comprises two or more 5’ caps.

[0155] Embodiment 3: The composition of embodiment 2, wherein the two or more 5’ caps are conjugated to a 5’ UTR of the RNA molecule.

[0156] Embodiment 4: The composition of embodiment 2 or 3, wherein the two or more 5’ caps are conjugated to the RNA molecule via click chemistry.

[0157] Embodiment 5: The composition of any one of embodiments 1-4, wherein the one or more modified nucleotides comprises a modified sugar.

[0158] Embodiment 6: The composition of embodiment 5, wherein the modified sugar is selected from the group consisting of 2'-deoxy fluoro (2FA), Z-adenosine (LA), 2'- deoxyadenosine (dA), locked nucleic acid (LNA), 2'-methoxy (2OMe), 2 '-methoxy ethoxy (2M0E), 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-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio- linked ribose.

[0159] Embodiment 7: The composition of embodiment 5 or 6, comprising between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified sugars.

[0160] Embodiment 8: The composition of embodiment 5 or 6, comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified sugars.

[0161] Embodiment 9: The composition of any one of embodiments 1-8, wherein the one or more modified nucleotides comprises a modified phosphate.

[0162] Embodiment 10: The composition of embodiment 9, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O- methylphosphonate, 3 '-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxy phosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0163] Embodiment 11 : The composition of embodiment 9 or 10, comprising between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified phosphates.

[0164] Embodiment 12: The composition of embodiment 9 or 10, comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified phosphates.

[0165] Embodiment 13: The composition of any one of embodiment 1-12, wherein the one or more modified nucleotides comprises a modified nucleobase.

[0166] Embodiment 14: The composition of embodiment 13, wherein the modified nucleobase is selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 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-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin- 16-7-deaza-7-propargylaminoguanine, biotin- 16- aminoallylcytosine, biotin- 16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3 -aminoallylcytosine, cyanine 3 -aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, Nl-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- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, 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-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0167] Embodiment 15: The composition of embodiment 13 or 14, comprising between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified nucleobases.

[0168] Embodiment 16: The composition of embodiment 13 or 14, comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified nucleobases.

[0169] Embodiment 17: The composition of any one of embodiments 1-16, wherein the one or more modified nucleotides comprise one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof.

[0170] Embodiment 18: The composition of any one of embodiments 1-17, wherein the 5’ cap is selected from the group consisting of 7-methyguanosine (m7G), N7,3’-O-dimethyl- guanosine-5 ’ -triphosphate-5 ’ -guanosine (m7G-3 ’m-ppp-G), N7,2’ -O-dimethyl-guanosine-5 ’ - triphosphate-5 ’-guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4-chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2-PhEt)G], 7-(l -phenyl ethyl) guanosine [7-(l-PhEt)G], m7GpppBH3G (DI and D2 stereoisomers), m7GppBH3G (DI and D2 stereoisomers), m7GpBH3G (DI and D2 stereoisomers), m7GppBH3pm7G, m27’2'°GpppBH3G (DI and D2 stereoisomers), m27’2’’ ^GppsmpG (DI and D2 diastereomers), m27,2'^GppspG (DI and D2 diastereomers), N- Arylmethyl analogs, glyceryl, 4', 5'-methylene nucleotide, l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L- nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3, 5 -dihydroxy pentyl nucleotide, 3 '-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3 '-phosphate, 3'-phosphorothioate, phosphorodi thioate, capl, cap2, cap3, cap4, ARCA, modified ARC A, inosine, N1 -methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0171] Embodiment 19: The composition of any one of embodiments 1-18, further comprising at least one poly-A tail.

[0172] Embodiment 20: The composition of embodiment 19, wherein the at least one poly-A tail comprises between 25 and 500 nucleotides.

[0173] Embodiment 21 : The composition of embodiment 20, wherein the at least one poly-A tail comprises between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 and 500 nucleotides.

[0174] Embodiment 22: The composition of any one of embodiments 19-21, wherein the at least one poly-A tail comprises 10 or more adenosine nucleotides.

[0175] Embodiment 23: The composition of any one of embodiments 19-21, wherein 25- 100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96- 100%, 97-100%, 98-100%, or 99-100% of nucleotides of the at least one poly-A tail are adenosine nucleotides.

[0176] Embodiment 24: The composition of any one of embodiments 1-23, wherein the 5’ cap is added to the RNA molecule through a chemical capping method.

[0177] Embodiment 25: The composition of embodiment 24, wherein the chemical capping method is an anhydrous reaction between a 5 ’-phosphorylated RNA molecule and a capping nucleotide conjugated to imidazole in the presence of 1 -methylimidazole.

[0178] Embodiment 26: The composition of any one of embodiments 1-25, wherein the RNA molecule further comprises a 5’ untranslated region (5’ UTR).

[0179] Embodiment 27: The composition of embodiment 26, wherein the 5’ UTR comprises a promoter.

[0180] Embodiment 28: The composition of any one of embodiments 1-27, wherein the RNA molecule further comprises a 3’ untranslated region (3’ UTR).

[0181] Embodiment 29: The composition of embodiment 28, wherein the 3’ UTR comprises at least one exonuclease-resistant modification.

[0182] Embodiment 30: The composition of embodiment 29, wherein the exonucleaseresistant modification is selected from the group consisting of phosphorothioate (PS) linkage, 2’- O-methyl (2OMe), 2’ Fluoro, inverted deoxythymidine (dT), inverted dideoxythymidine (ddT), 3’ phosphorylation, C3 spacer, 2'-O-methoxy-ethyl (2'-MOE), G-quadruplex, and 2'-3'-dideoxy nucleotide (ddN).

[0183] Embodiment 31 : The composition of any one of embodiments 1-30, wherein the RNA molecule comprises two or more 5’ caps.

[0184] Embodiment 32: The composition of any one of embodiments 1-32, wherein the RNA molecule comprises two or more poly-A tails.

[0185] Embodiment 33: The composition of any one of embodiments 1-32, wherein the RNA molecule further comprises an open reading frame (ORF).

[0186] Embodiment 34: The composition of embodiment 33, wherein the ORF encodes a protein.

[0187] Embodiment 35: The composition of embodiment 34, wherein the protein is a therapeutic protein.

[0188] Embodiment 36: The composition of embodiment 33, wherein the protein is an antigen.

[0189] Embodiment 37: The composition of embodiment 36, wherein the antigen is a SARS-CoV-2 spike protein or fragment thereof.

[0190] Embodiment 38: The composition of embodiment 37, wherein the ORF comprises a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4.

[0191] Embodiment 39: The composition of embodiment 37, wherein the ORF encodes an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

[0192] Embodiment 40: The composition of any one of embodiments 31-39, wherein the RNA molecule further comprises a sequence encoding a therapeutic nucleic acid.

[0193] Embodiment 41 : The composition of embodiment 40, wherein the therapeutic nucleic acid is an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

[0194] Embodiment 42: The composition of any one of embodiments 1-41, wherein the RNA molecule is a circular RNA molecule.

[0195] Embodiment 43: The composition of any one of embodiments 1-42, wherein the RNA molecule comprises a stem oligo modification having the sequence of SEQ ID NO: 1.

[0196] Embodiment 44: The composition of any one of embodiments 1-43, wherein the RNA molecule comprises a branch oligo modification having the sequence of SEQ ID NO: 2.

[0197] Embodiment 45: The composition of any one of embodiments 1-44, wherein the RNA molecule comprises a 5’UTR having the sequence of SEQ ID NO: 3.

[0198] Embodiment 46: The composition of any one of embodiments 1-45, wherein the RNA molecule comprises a 3’UTR having the sequence of SEQ ID NO: 5.

[0199] Embodiment 47: The composition of any one of embodiments 1-46, wherein the RNA molecule comprises a polyA tail modification having the sequence of SEQ ID NO: 6.

[0200] Embodiment 48: The composition of any one of embodiments 1-47, wherein the RNA molecule comprises two 5’ caps, wherein each of the two 5’ caps is LNAm7G.

[0201] Embodiment 49: The composition of any one of embodiments 1-48, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

[0202] Embodiment 50: The composition of embodiment 49, wherein the nanoparticle or microparticle is a lipid nanoparticle or a lipid microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

[0203] Embodiment 51 : The composition of embodiment 50, wherein the nanoparticle is a lipid nanoparticle.

[0204] Embodiment 52: The composition of any one of embodiments 1-51, wherein: (a) the RNA molecule comprises two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF comprising a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; a 3’UTR having the sequence of SEQ ID NO: 5; and a poly A tail modification having the sequence of SEQ ID NO: 6; and (b) the delivery agent comprises a lipid nanoparticle.

[0205] Embodiment 53: The composition of any one of embodiments 1-52, wherein: (a) the RNA molecule comprises two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF encoding an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6; and (b) the delivery agent comprises a lipid nanoparticle.

[0206] Embodiment 54: The composition of any one of embodiments 1-53, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

[0207] Embodiment 55: An RNA molecule comprising two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQ ID NO: 3; an ORF comprising a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; a 3’UTR having the sequence of SEQ ID NO: 5; and a polyA tail modification having the sequence of SEQ ID NO: 6.

[0208] Embodiment 56: An RNA molecule comprising two 5’ caps, wherein each of the caps is LNAm7G; a stem oligo modification having the sequence of SEQ ID NO: 1; a branch oligo modification having the sequence of SEQ ID NO: 2; a 5’UTR having the sequence of SEQID NO: 3; an ORF encoding an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; a 3’UTR having the sequence of SEQ ID NO: 5; and a poly A tail modification having the sequence of SEQ ID NO: 6.

[0209] Embodiment 57: A vector comprising the RNA molecule of embodiment 55 or 56.

[0210] Embodiment 58: A cell comprising the RNA molecule of embodiment 55 or 56 or the vector of embodiment 57.

[0211] Embodiment 59: The cell of embodiment 58, wherein the cell is a mammalian cell.

[0212] Embodiment 60: A method of preventing or treating a disease in a subject, comprising introducing an effective amount of the RNA molecule of embodiment 55 or 56, the vector of embodiment 57, or the composition of any one of embodiments 1-54 to the subject.

[0213] Embodiment 61 : The method of embodiment 60, wherein the subject is a human subject.

[0214] Embodiment 62: The method of embodiment 60 or 61, wherein the disease is SARS-CoV-2.

[0215] Embodiment 63: The RNA molecule of embodiment 55 or 56, the vector of embodiment 57, or the composition of any one of embodiments 1-54 for use in preventing or treating a disease in a subject.

[0216] Embodiment 64: The RNA molecule of embodiment 63, wherein the disease is SARS-CoV-2.

[0217] Embodiment 65: The RNA molecule of 63 or 64, wherein the subject is a human subject.

[0218] Embodiment 66: A kit comprising the composition of any one of embodiments 1- 54, a device for administering the composition to a subject, and / or instructions for administering the composition to the subject.EXAMPLESExample 1: Modified dual-capped mRNA for enhanced mRNA vaccine against SARS-CoV- 2

[0219] Applicant evaluated the effects of multi-capped RNAs on SARS-CoV-2 vaccination outcomes. Applicant used an optimized dual-LNAm7G-LNA+5xOMe-6*PS+2MOE mRNA and regular mono-m7G-rG control mRNA encoding the receptor binding domain (RBD) of the Spike glycoprotein from SARS-CoV-2 with a trimerization domain. Applicant adopted a conventional prime-boost scheme, separated by 2 weeks between each dose and with serum drawn every week for antibody titering (FIG. 1A). The results from ELISA for anti-spike IgG antibody quantification showed that m7G-rG capped control mRNA induced the production of anti-spike IgG to 221±122 pg / mL three weeks post-injection, which is comparable with previous reports. In contrast, the optimized dual-capped mRNA induced a detectable level of IgG antibody even at 7 days post-injection of the prime dose, and showed 17. l-fold / 3.7-fold higher antibody level than the control mRNA at Day 14 / Day21 (FIG. IB). This result indicated that modified mRNA with increased expression capacity of antigen is able to stimulate a stronger RBD-specific humoral immune response even with only a single dosage at earlier stages of vaccination.

[0220] To gain immunological insights into the effectiveness of the optimized dualcapped mRNA vaccine, Applicant extracted the mouse inguinal lymph nodes 24 hrs after booster injection and performed in situ sequencing to immunotype the local splenocytes / lymphocytes. Specifically, Applicant used STARmap to target endogenous transcripts for cell typing, whereas actively translating synthetic RBD mRNAs were detected by RIBOmap (FIG. 1C). Cells in the lymph node samples were spatially clustered into regions (B-cell zone, T-cell zone, marginal zone at B / T zone boundary, and capsule) by SPIN and subtyped by k-means clustering (FIG. 2A-B). Significantly higher actively translating RBD mRNAs was observed in the B-cell zone with all classes of potential antigen-presenting cells (APCs), including B cells, dendritic cells (DCs), and macrophages (mph.) by the optimized dual-capped mRNA group 24 hrs post booster injection, in comparison with mono-m7G-rG control and polyC control groups (FIG. 1D-E). It is unexpected to see higher RBD translation within B cells than DCs and macrophages, but this result was consistent with recent reports about the distribution of mRNA-LNP within the immune organ where significant uptake of LNP by B cells was observed. In addition to observing higher translation of modified dual-cap mRNA, Applicant found significant upregulation of the MHC-I molecule encoding gene H2-K1 and the chemokine receptor Ccr7 responsible for immune cell migratory events in all APC subtypes, indicating overall higheractivation of the adaptive immune response by the optimized dual -capped mRNA. Spatially, activation of B cell-APCs in both T / B zones and activation of cDC2-, other DC-, and mph.-APCs are equally efficient in vaccinated conditions. However, the optimized dual-capped mRNA resulted in greater activation of other (non-cDCl / 2) DC- and mph.-APCs within the T zone, and more significant activation of cDCl-, activated mph.-APCs in both T / B zones, which potentially explains our observed higher T / B cell responses (FIG. 2C-H).

[0221] Beyond the B-cell mediated humoral response as evidenced by antibody titers and spatial transcriptomic profiling, Applicant next investigated the T-cell immune response, which is the other major component in mediating immunity against SARS-CoV-2 in patients. To compare memory CD4+ and CD8+ T cells mediated effector functions induced by either the m7G-rG or optimized dual-capped mRNA, the splenocytes of the immunized mice were collected and then stimulated in culture with SARS-CoV-2 Spike-RBD pooled peptides (S- peptides), or DMSO as a negative control (FIG. IF). The cytokine-producing T cells were quantified by intracellular cytokine staining among effector memory T cells (Tern, CD44+CD62L-) (FIG. 1G-L, FIG. 3A-C) or by ELISA for cytokines secreted into the cell culture medium (FIG. 3D-H). Quantification results from flow cytometry showed that the percentage of CD8+ T cells producing IFN-y were significantly increased by 3.9-fold (FIG. 1G), indicating a stronger RBD-specific CD8+ T cell response elicited by the optimized dual-capped mRNA compared to the mono-m7G-rG mRNA mRNA. ELISA results from secreted cytokines indicated that both control and optimized dual-capped mRNA induced the secretion of cytokines IFN-y, TNF-a, IL2, IL4 and IL 13, demonstrating the activation of both the Thl and Th2 CD4+ T cell immune response. In particular, the percentage of CD4+ T cells producing IFN-y, a Thl- marker, was significantly increased by 4.7-fold for the mice immunized with optimized dualcapped mRNA than the mono-m7G-rG mRNA (FIG. 1G, FIG. 1 J), aligning with the observed higher cDCl-APC activation in the T zone observed by STARmap (FIG. 2D). Together, these results indicated that optimized dual-capped mRNA vaccines induced significantly stronger Thl T cell immune responses and comparable Th2 T cell responses to the control mono-m7G-rG mRNA.Example 2. Materials and MethodsCloning, characterization, and purification of plasmids

[0222] Briefly, the proteins of interest coding sequences (CDS) were inserted into an optimized backbone containing (from 5' to 3'): a T7 “CleanCap AG” promoter sequence (TAATACGACTCACTATAAG) (SEQ ID NO: 11) a 5' human alpha-globin UTR; a variable CDS region; a 3' UTR derived from AES mRNA and mitochondrial encoded 12S rRNA; a 100xA template-encoded split polyA tail; and an Esp3I linearization site 3' to the end of the poly A sequence. The CDS-containing plasmid / gene blocks were PCR amplified, gel -purified, and assembled into the optimized backbone using NEBuilder HiFi DNA Assembly Master Mix [NEB, E2621S], transformed into NEB Stable cells, miniprepped with ZymoPURE Plasmid Miniprep kits [Zymo Research, D4015], and sequence-verified with whole plasmid and Sanger sequencing. Short inserted sequences or deletions, for example, the 3' 15* A linker or 3' truncated polyA variant constructs, were generated by site-directed mutagenesis using Q5 Site-Directed Mutagenesis Kit [NEB, E0554S], Linear mRNA synthesis and characterization

[0223] DNA plasmids were obtained as aforementioned and linearized by Esp3I [NEB, R0734S], or another Type IIS restriction enzyme as specified if Esp3I was already present in the CDS. Linearized plasmids are purified with the DNA Clean & Concentrator-25 kit [Zymo Research, D4033] and characterized for complete linearization with agarose gel electrophoresis. mRNA constructs were synthesized by IVT using HiScribe T7 High Yield RNA Synthesis Kit [NEB, E2040S] per manufacturer’s protocol except with 100% replacement of UTP with N1- methyl pseudouridine-5 '-triphosphate [Trilink, N-1081-1] and addition of 1 :50 SUPERase-In RNase inhibitor [ThermoFisher Scientific, AM2694], Following IVT reaction, DNA templates were digested by TURBO DNase [ThermoFisher Scientific, AM2238] and purified using Monarch RNA cleanup kit [NEB, T2040L], mRNA concentrations were quantified using the Qubit RNA HS Assay [ThermoFisher Scientific, Q32852] or the Qubit RNA BR Assay [ThermoFisher Scientific, QI 0210], Unless otherwise specified, mRNA products are suspended in 1 :50 (v / v) RNase inhibitor-containing RNase-free water (subsequently referred to as RNase- free water) and stored at -80 °C.Synthesis of the capping reagents for cap analogs

[0224] m7G-Im, Bn7G-Im, ClBn7G-Im, ClBnOEt7G-Im and LNAm7G-Im capping reagents were synthesized following previously reported protocol. Briefly, m7GDP, Bn7GDP,ClBn7GDP, ClBnOEt7GDP were synthesized by treating the GDP sodium salt with dimethyl sulfoxide or corresponding alkylation reagents, followed by HPLC purification. LNAm7GDP was synthesized by introducing the phosphate to the 5' hydroxyl group of LNA guanosine. Characterization data of all GDP derivatives were in agreement with previous reports: m7GDP, ClBn7GDP, LNAm7GDP. Data for Bn7GDP: LC-MS (ESI-): calculated 532.32, found 532.40.NMR (400 MHz, D2O) 8 9.44 (s, 1H), 7.68-7.53 (m, 2H), 7.51-7.39 (m, 3H), 6.08 (d, J = 4.0, 1H), 5.69 (s, 2H), 4.78-4.73 (m, 1H), 4.58-4.51 (m, 1H), 4.47-4.40 (m, 1H), 4.39-4.21 (m, 2H).31P NMR (162 MHz, D2O) 5 -10.75 (d, J =21.06), -11.31 (d, >19.90). Data for ClBnOEt7GDP: LC-MS (ESI-): calculated 596.79, found 596.37. *HNMR (400 MHz, D2O) 5 7.20 (m, 1H), 7.08 (d, >9.2, 2H), 6.75 (d, >8.8, 2H), 5.81 (d, >3.2, 1H), 4.80-4.72 (m, 2H), 4.47-4.37 (m, 4H), 4.26-4.21 (m, 2H), 4.21-4.14 (m, 2H).31P NMR (162 MHz, D2O) 8 -10.69 (d, J =21.07), -11.28 (d, >19.44).

[0225] Then, GDP-Imidazole derivatives were prepared according to the general protocol and purified through acetone precipitation to give the capping reagents that can be used directly in the capping reaction without further purification.General conditions for RNA RP-HPLC purification

[0226] All purification was conducted on an Agilent 1260 Infinity II HPLC. Acetonitrile (solvent A) [Sigma Aldrich, 34851], 100 mM hexylamine / acetic acid in water (pH 7.0, with 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 phases and PLRP-S column as the stationary phase.

[0227] Method 1 : 100 A pore size was used, 0% A + 100% B (0~5 mins, hold); 10% A + 90% B (5~10 mins, linear increase); 25% A + 75% B (10-55 mins, linear increase).

[0228] Method 2: 300 A pore size was used, 0% A + 100% B (0-5 mins, hold); 15% A + 85% B (5-10 mins, linear increase); 50% A + 50% B (10-45 mins, linear increase).

[0229] Method 3 : 4000 A pore size was used, 0% A + 100% B (0 mins); 20% A + 80% B (0-2 mins, linear increase); 70% A + 30% B (2-30 mins, linear increase).

[0230] Method 4: 4000 A pore size was used, 0% A + 100% C (0 mins); 25% A + 75% B (0-25 mins, linear increase).Capped oligonucleotide synthesis

[0231] The oligonucleotides used in this study were ordered from IDT with final quality control. 12 nmol of solid phase synthesized oligonucleotide (with ammonium as counterion) was dissolved in a solution of 40 mM m7GDP-Im (or corresponding cap analogue) in 42 pL of anhydrous DMSO, and 8 pL of 1-methyl-imidazole was added. The reaction was mixed well and heated at 55 °C for 3 hrs. The reaction was then quenched by addition of 50 pL of water and directly subjected to HPLC purification using method 1. Fractions containing the capped products were pooled, lyophilized, and resuspended in RNase-free water and stored at -80 °C until being used. Concentrations of capped oligos were quantified using Qubit microRNA assay kit [Invitrogen, Q32880] and nanodrop.Oligonucleotide conjugation using CuAAC

[0232] Azide / alkyne-labeled oligonucleotides at a final concentration of -200 pM were mixed with modified 1.5x click chemistry buffer ([Lumiprobe, 61150] containing additive 5% SUPERase Inhibitor, 5% DMSO, and 5% 10 mM dNTP mix [ThermoFisher Scientific, 18427089]) that was degassed by argon purging. For a typical 100 uL reaction, 33 uL of oligonucleotide solution was mixed with 66 uL of click chemistry buffer and 4 uL of 100 mM freshly prepared ascorbic acid solution [Sigma Aldrich, A5960] was added immediately prior to the reaction. The mixture was incubated at 37 °C for 1 hr and quenched by addition of 1 uL of 500 mM EDTA (pH 8.0). The reaction was first purified using Monarch RNA Cleanup Kit [NEB, T2040] and then subjected to RNase-free HPLC purification using method 2.Enzymatic ligation of modified oligonucleotides to mRNAs

[0233] 5 '-triphosphorylated mRNA generated by IVT was first treated with RppH [NEB,M0356S] per manufacturer’s protocol to generate 5P-mRNA and purified with the Monarch RNA cleanup kit. Synthetic oligo and 5P-mRNA were mixed at a molar ratio of 25: 1, and diluted in 2x 50% PEG-8000, 10x T4 RNA ligase buffer, 10x T4 RNA ligase [Promega, M1051], and RNase-free water. The reaction was incubated at 37 °C for 30 mins and inactivated by the addition of 50x 500 mM EDTA (pH 8.0). Products were purified first by the Monarch RNA cleanup kit and then by RNase-free HPLC (method 3). Purified fractions were pooled and desalted using the Monarch RNA cleanup kit and ligation efficiency was characterized usingRNase H assay as described before. In case of incomplete ligation, a second round obligation was performed. mRNA profding in transfected cell culture / tissue with STARmap / RIBOmap

[0234] For the cell culture experiments, dual-capped / mono-capped mRNA encoding FLuc and unmodified mRNA encoding RLuc (transfection control) were cotransfected to HeLa cells seeded in 24-well plates as described in previous sections. After a 6-hrs incubation, the transfection mixture was removed, and cells were rinsed with DPBS and trypsinized to reseed into two glass-bottom 96-well plates ([MatTek, PBK96G-1.5-5-F] poly-D-lysine [Sigma- Aldrich, A-003-M] coated). Cell culture based STARmap / RIBOmap was performed and quantified as previously described, where the FLuc mRNA was profiled by STARmap or RIBOmap and the RLuc mRNA was profiled by STARmap. The following laser settings were used: DAPI, Diode 405 nm / ~[420-489] nm; Alexa546, white light laser 557 nm / ~[569-612] nm; Alexa647, white light laser 653 nm / ~[668-738] nm. MATLAB 2021a and CellProfiler 4.0.7 were used for the amplicon count quantifications.

[0235] For STARmap / RIBOmap experiments in mouse lymph nodes, 24 hrs after the booster injection, the mouse inguinal lymph nodes were harvested and frozen by liquid nitrogen in optimal cutting temperature (OCT) compound. STARmap / RIBOmap procedures were adopted as previously described where the RIBOmap probes were designed to target the synthetic RBD mRNA and STARmap probes were designed to target endogenous mRNA. Probe sequences are listed in Supplementary Table 1.

[0236] The analysis for STARmap / RIBOmap data in mouse tissue was performed similarly to Wang et al. and Shi et al. Briefly, image deconvolution was achieved with Huygens Essential version 21.04 (Scientific Volume Imaging, The Netherlands, http: / / svi.nl), using the CMLE algorithm, with SNR: 10 and 10 iterations. Image registration, spot calling, and barcode filtering were performed as previously described. For 3D cell segmentation, enhanced contrast synthetic images were created by multiplying the inverted Flamingo staining image with the DAPI staining image after contrast enhancement using Fiji for each field of view (FOV). A StarDist3 3D segmentation model was trained using a manually labeled training dataset derived from the synthetic data. The model was then used to predict segmentation for each FOV. For tissue region identification, the SPIN4 method was used to identify low-frequency transcriptionalpatterns across the tissue. Specifically, Laplacian smoothing was applied to a spatial Delaunay triangulation (nearest neighbor mesh of cells) using a heat kernel filter with time t=l 0. Subsequently, PCA was conducted on the filtered cell-by-gene matrix to detect region features. Clustering in PC space was then performed to assign categorical region labels. K-means clustering was utilized to prevent spatial autocorrelation artifacts induced by smoothing.

[0237] For quality control and cell type classification, cells with less than 2 reads and expressed fewer than 2 genes were excluded. Gene expression profiles were normalized and scaled using standard Scanpy5 procedures. Samples from multiple batches were corrected with Combat implemented in Scanpy. A hierarchical clustering approach was then utilized to create a two-level cell-type annotation. Initially, 34 clusters were identified through k-means clustering of the preprocessed gene expression profile containing 26 genes, which were further categorized into six level 1 cell types (T cells, B cells, Macrophages, Dendritic cells, NK cells, and Endothelial cells). Cells lacking expression of any of the selected 26 gene markers were removed from subsequent analysis. Each level 1 cell type underwent additional k-means clustering to establish level 2 annotations. Specifically, T cells were subdivided into CD4+ T cells (Cd4+) and CD8+ T cells (Cd8a+). Macrophages were classified as Activated Macrophages (Cd68+) and Monocytes (Csflr+, Lyz2+), while Dendritic cells were differentiated into cDCl (Irf8+) and cDC2 (Irf4+). Antigen-presenting cells (APCs) were further classified from the B cell, Macrophage, and Dendritic cell populations with four gene markers (Cd40, Cd86, Ccr7, H2-K1).Synthesis of preQl -azide

[0238] 100 mg of preQl-HCl [Princeton Bio, PBMR232165-0.5 g] was suspended in 0.5 mb anhydrous DMF, to which 179 pL DBU was added dropwisely at 0 °C which allowed the salt to dissolve, and 85 mg of bromo-PEGl -azide [Broadpharm, BP-24132] was then added. The reaction was allowed to stir overnight under argon protection. Solvents were removed in vacuo, and preQi -azide was isolated by flash chromatography as a light brown solid (1 : 1 :0.1 DCM / MeOH / NH3). LC-MS (ESI-): calculated 291.30, found 291.26. 'H NMR (400 MHz, CD3OD) 5 6.62 (s, 1H), 3.84 (s, 2H), 3.61 (m, 2H), 3.36 (m, 2H), 2.80 (m, 2H).13C NMR (126 MHz, CD3OD) 8 160.91, 152.94, 151.91, 115.77, 115.70, 98.80, 69.43, 69.33, 50.32, 45.90, 44.48.QRNA containing wild-type uridine: synthesis and characterization

[0239] CircRNA containing wild-type uridine and bearing a TGT hairpin was synthesized as described in previous sections. TGT enzyme was expressed in E. colt as described in the literature. To label the circRNA with preQi -azide, 1 pM of circRNA, 100 pM of preQi - azide, 10 pM of TGT, 10 pL of SUPERase-In RNase inhibitor were incubated in lx TGT reaction buffer (100 mM HEPES, pH 7.3, 5 mM DTT, and 20 mM MgCh) in a total of 100 pL reaction at 37 °C for 2 hours. The labeled circRNA was purified and subjected to click reaction with Bn7G-capped alkyne labeled oligo using the general condition for click reaction for 30 mins. It is noteworthy that the hydrophobicity of Bn7G cap allowed effective purification of the QRNA product from the circRNA precursor. The reaction mixture was then subjected to RP- HPLC purification to remove the linearized portions (method 3), pooled and desalted, and subjected to another round of RP-HPLC purification to isolate the QRNA product (method 4). QRNA product was characterized by RNase H assay with 2 primers upstream / downstream the TGT site.QRNA containing N1-methylpseudouridine: synthesis and characterization

[0240] DNA plasmid templates for Nkmethylpseudouridine-modified QRNA were cloned as described previously. These templates contained (from 5' to 3'): a T7 “CleanCap AG” promoter sequence (TAATACGACTCACTATAAG) (SEQ ID NO: 11); a 15xA linker followed by 5' human alpha globin UTR; a NanoLuc CDS region; a 3' UTR derived from AES mRNA and mitochondrial encoded 12S rRNA; a 6xA linker; and a 3’ Esp3I plasmid linearization site. In this case, there was sufficient homology between the 5' and 3' UTRs to enable efficient circularization via ligation, without the need for additional 5' and 3' hybridization motifs. Applicant generated mRNA as previously described, by IVT using Hi Scribe T7 High Yield RNA Synthesis Kit [NEB, E2040S] per manufacturer's protocol except with 100% replacement of UTP with N1-methyl pseudouridine-5'-triphosphate [Trilink, N-1081-1] and addition of 1 :50 SUPERase-In RNase inhibitor [ThermoFisher Scientific, AM2694],

[0241] For site-specific circRNA modification by LEGO, the 573 '-phosphate oligo (with or without a branched cap) was prepared as described in previous sections. 5' Triphosphorylated IVT mRNA was subjected to first CIAP [Promega, M2825] dephosphorylation, T4 RNA ligase 1,and RctB ligase [NEB, M0458S], The crude product was desalted and subjected to RP-HPLC purification to isolate the circular products.RNA extraction and cDNA preparation

[0242] For the evaluation of innate immune response induced by the 5' modified mRNA constructs, the cell culture media was removed post transfection for 24 hours. Then, 300 pL of Trizol reagent was added to each well, followed by extracting the total mRNA from cell lysate with RNA Miniprep Kit [Zymo Research: R2050] according to the manufacturer’s protocol. The optional DNase digestion was performed, also according to the manufacturer’s protocol. The isolated RNA was then quantified using Nanodrop. Reverse transcription of total RNA was performed using the LunaScript RT SuperMix Kit [New England Biolab, Inc., E3010L] according to the manufacturer's protocol with 1 pg of total RNA.RT-qPCR

[0243] qPCR was performed using the Luna Universal qPCR Master Mix [New England Biolabs, Inc., M3OO3] according to the manufacturer's protocol. Briefly, the reactions were set up with 250 nM primers for GAPDH, Mxl and ISG15 in 20 pL. The amplifications were conducted with the following protocol: 95 °C for 5 min, 40 cycles of 95 °C for 15s, 60 °C for 30s. The specificity of primer pairs was tested with melting curves at the end of the 40thamplification cycle. All the gene expressions were calculated and normalized to GAPDH.Mice

[0244] All animal procedures followed animal care guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute of MIT and Harvard under animal protocol # 0255-08-19. Animal experiments were conducted in compliance with IACUC policies and NIH guidelines. The BALB / cj (male, 6~8 weeks old) mice used for in vivo NanoLuc assay in this study were purchased from The lackson Laboratory (JAX). The BALB / cj (female, 3~4 weeks old) mice used for COVID vaccine evaluation in this study were purchased from The Jackson Laboratory (JAX). The C57BL / 6 mice (female, 7 weeks old), used for hEPO assay in this study were purchased from The Jackson Laboratory (JAX). Mouse were housed 4 animal per cage on a 12-h light-dark cycle with ad libitum food and water at 18-23 °C temperature and 40-60% humidity.In vivo delivery of mRNA[002451 mRNA diluted in 50 mM citrate buffer (pH 4) and lipid mix (37.5 mM in ethanol) containing 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% Cholesterol, and 1.5 mol% DMG- PEG2000 were assembled into LNP using a NanoAssemblr Spark instrument [Precision Nanosystems]. Upon formulation, mRNA-LNPs were diluted in 12 mL IxPBS solution and the buffer was exchanged by concentrating with a 30 kDa spin fdter [MilliporeSigma, UFC901008] to remove residual ethanol. Concentrations and encapsulation efficiency of mRNA were determined using Quant-it RiboGreen RNA Assay Kit [ThermoFisher, R11490], mRNA-LNP (equal molar, normalized to the control RNA) in a total volume of 100 pL was injected through intramuscular injection (for SARS-CoV-2 (COVID-19) Vaccination) into each mouse. Four mice were used for each condition of each experiment (SARS-CoV-2 (COVID- 19) Vaccination). For SARS-CoV-2 (COVID-19) Vaccination, poly(C)-LNP complex was used as negative control.In vivo SARS-CoV-2 (COVID-19) vaccination and ELIS As

[0246] DNA plasmid templates were generated as previously described, with an IVT cassette containing a T7 “CleanCap AG” promoter sequence (TAATACGACTCACTATAAG) (SEQ ID NO: 11); a 15*A linker followed by 5’ human alpha globin UTR; a CDS region; a 3' human alpha globin UTR derived from AES mRNA and mitochondrial encoded 12S rRNA; a 100xA template-encoded polyA tail; and a 3’ Esp3I plasmid linearization site. The CDS is a modified design based on BNT162bl, as it encodes the antigenic receptor binding domain (RBD) from the SARS-CoV-2 (2019 Wuhan variant) fused to a trimerization motif (foldon). Specifically, the CDS contains: (1) Spike signal sequence (AAs: 1-16); (2) (GS)a linker; (3) RBD domain (AAs: 319-541); (4) (GS)3 linker; and (5) a codon optimized T4 fibritin derived trimerization domain (foldon). IVT RNA substrates contained 100% replacement of uridine with N 1 -methylpseudouridine.

[0247] During the vaccination study, at the indicated time points post-vaccination, approximately 100 pL of blood was collected with EDTA-coated capillary tubes from each mouse and then transferred to an EDTA-coated tube. The collected blood samples were centrifuged at 2000 g for 10 min, followed by transferring the resulting plasma into another tube. Anti-Sl Spike mouse IgG antibodies were measured via ELISA using a Mouse SARS-CoV-2Spike SI Antibody Quantitative ELISA kit [Eagle Biosciences, KBVH015-14], according to the manufacturer’s protocol. Serum samples were first titered to determine optimal dilution factors and ensure measured values for each sample fell within the quantitative range of the kit’s provided standards. For example, 1 :25 dilutions were used for Day 7 and 14 IgG measurements, while a 1 :2000 serum dilution was used for Day 21 (post-boost dose) measurements. All samples were processed in technical duplicates, with averaged values of technical duplicates being reported for each biological replicate. All samples were background subtracted using the average of the background of the 0 pg / mL standard (buffer only condition), and the standard curve was fit to a logistic regression that was used for the back-calculation of antibody titers.

[0248] For T-cell stimulation experiments in culture, culture media was collected at 48 hours post COVID-19 peptide stimulation [JPT Peptide technology, PM-WCPV-S-RBD-1] at 1.5 pg / ml for each peptide, with DMSO used as the negative control. These media samples were diluted and characterized via ELISA for each cytokine measured, as described above. The following kits were used: Mouse IL-2 ELISA Kit [Proteintech, KE10004], Mouse IL-13 ELISA Kit [Proteintech, KE10021], LEGEND MAX Mouse IFN-y ELISA Kit [BioLegend, 430807], LEGEND MAX Mouse TNF-a ELISA Kit [BioLegend, 430907], and LEGEND MAX Mouse IL-4 ELISA Kit [BioLegend, 431107],T cell flow cytometry analysis

[0249] At the indicated time point mentioned above, mice spleen single-cell suspensions were prepared in RPMI 1640 medium by mashing tissue against the surface of a 70-pm cell strainer [BD Falcon, 64752-00], Then, the single-cell suspension was centrifuge at 200 g for 5 minutes and the supernatant was removed. The red blood cells were lysed by adding 3 ml of RBC lysis buffer [BioLegend, 420301] at 4 °C for 1.5 minutes, followed by centrifugation and removal of the supernatant. The cells were washed once with RPMI 1640 medium and then resuspended with RPMI 1640 medium (10% FBS and 1% Pen-Strep antibiotic). 4* 106of splenocytes from each mouse were cultured in RPMI medium and stimulated with RBD peptide pools [JPT Peptide technology, PM-WCPV-S-RBD-1] at a final concentration of 1.5 pg / ml for each peptide. The GolgiStop transport inhibitor cocktail [BD, 554724] was added according to the manufacturer's instruction 18 hours later. Then, 6 hours later, the cells were collected and washed with FACS buffer (PBS with 2% FBS) prior to staining with LIVE / DEAD [Thermo,L34963] for 20 minutes at room temperature. Cells were then washed with FACS buffer and suspended in Fc block [BD, 553141] for 5 minutes, followed by staining with a surface staining with the antibody cocktail on ice: CD3 [Biolegend, 100229]; CD4 [Biolegend, 100552]; CD8 [Biolegend, 100714]; CD44 [Biolegend, 103006]; CD62L [Biolegend, 161203], After 20 minutes, the cells were washed with a FACS buffer and then fixed and permeabilized using a BD Cytoperm fixation / permeabilization solution kit [BD, 554714] according to the manufacturer’s instructions. Cells were washed in perm / wash solution, followed by intracellular staining (30 min, room temperature) using a cocktail of the following antibodies: IFN-y [Biolegend, 505810]; IL-2 [Biolegend, 503818]; TNF-a [Biolegend, 506324], Finally, the cells were washed in perm / wash solution and suspended in a staining buffer. Samples were washed and acquired on a Beckman CytoFLEX LX Flow Cytometer. Analysis was performed using FlowJo software.SEQUENCES

[0250] The following tables contain sequences of oligonucleotides used in the experiments shown in each referenced figure. Nucleotide modifications are denoted as follows: r = ribose sugar m = 2OMe-modified sugar* = phosphorothioate (PS) linkage+ = locked nucleic acid (LNA) i2FA = internal 2 '-deoxy fluoro iN6Me = internal N6-methyladenosine ibetaL = internal T-adenosine i5OCTdU = internal3AzideN = 3' 6-diazynyl-JV-heptylhexanamideTable 1. Sequences used in FIG. 1Table 2. Construct Cap Details for FIGs.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising:(a) an RNA molecule comprising (i) one or more modified nucleotides at position +3 or higher with reference to a 5’ terminus of the RNA molecule, (ii) at least one 5’ cap, (iii) and an open reading frame (ORF), and(b) a delivery agent.

2. The composition of claim 1, wherein the RNA molecule comprises two or more 5’ caps.

3. The composition of claim 2, wherein the two or more 5’ caps are conjugated to a 5’ UTR of the RNA molecule.

4. The composition of claim 2 or 3, wherein the two or more 5’ caps are conjugated to the RNA molecule via click chemistry.

5. The composition of any one of claims 1-4, wherein the one or more modified nucleotides comprises a modified sugar.

6. The composition of claim 5, wherein the modified sugar is selected from the group consisting of 2'-deoxy fluoro (2FA), L-adenosine (LA), 2 '-deoxy adenosine (dA), locked nucleic acid (LNA), 2'-methoxy (20Me), 2 '-methoxy ethoxy (2M0E), 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 '-di deoxyribose, 3'-azido-2',3'- dideoxyribose, 3 ’-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'- aminoribose, 5 '-thioribose, 5-nitro-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C- methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose.

7. The composition of claim 5 or 6, comprising between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified sugars.

8. The composition of claim 5 or 6, comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified sugars.

9. The composition of any one of claims 1-8, wherein the one or more modified nucleotides comprises a modified phosphate.

10. The composition of claim 9, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5 '-O-methylphosphonate, 3'-O- methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphorosel enoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

11. The composition of claim 9 or 10, comprising between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified phosphates.

12. The composition of claim 9 or 10, comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified phosphates.

13. The composition of any one of claim 1-12, wherein the one or more modified nucleotides comprises a modified nucleobase.

14. The composition of claim 13, wherein the modified nucleobase is selected from the group consisting of inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2- thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-di hydrouracil, 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-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin- 16-7-deaza-7-propargylaminoguanine, biotin- 16- aminoallylcytosine, biotin- 16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3 -aminoallylcytosine, cyanine 3 -aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, Nl- 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- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, 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-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

15. The composition of claim 13 or 14, comprising between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 400 and 500, between 600 and 700, between 800 and 900, or between 900 and 1000 modified nucleobases.

16. The composition of claim 13 or 14, comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, at least 1000, or more modified nucleobases.

17. The composition of any one of claims 1-16, wherein the one or more modified nucleotides comprise one or more modified sugars, one or more modified phosphates, one or more modified nucleobases, or any combination thereof.

18. The composition of any one of claims 1-17, wherein the 5’ cap is selected from the group consisting of 7-methy guanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’- guanosine (m7G-3 ’ m-ppp-G), N7,2 ’ -O-dimethyl-guanosine-5 ’ -triphosphate-5 ’ -guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G), 7-(4- chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2-phenylethyl) guanosine [7-(2- PhEt)G], 7-(l-phenylethyl) guanosine [7-(l-PhEt)G], m7GpppBH3G (DI and D2 stereoisomers), m7GppBH3G (DI and D2 stereoisomers), m7GpBH3G (DI and D2 stereoisomers), m7GppBH3pm7G, m272’°GpppBH3G (DI and D2 stereoisomers), ni27-2'^GppBiiipG (DI and D2 diastereomers), m27-2°GppspG (DI and D2 diastereomers), N-Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2 '-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, capl, cap2, cap3, cap4, ARC A, modified ARC A, inosine, Nl- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

19. The composition of any one of claims 1-18, further comprising at least one poly-A tail.

20. The composition of claim 19, wherein the at least one poly-A tail comprises between 25 and 500 nucleotides.

21. The composition of claim 20, wherein the at least one poly-A tail comprises between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 and 500 nucleotides.

22. The composition of any one of claims 19-21, wherein the at least one poly-A tail comprises 10 or more adenosine nucleotides.

23. The composition of any one of claims 19-21, wherein 25-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99- 100% of nucleotides of the at least one poly-A tail are adenosine nucleotides.

24. The composition of any one of claims 1-23, wherein the 5’ cap is added to the RNA molecule through a chemical capping method.

25. The composition of claim 24, wherein the chemical capping method is an anhydrous reaction between a 5 ’-phosphorylated RNA molecule and a capping nucleotide conjugated to imidazole in the presence of 1 -methylimidazole.

26. The composition of any one of claims 1-25, wherein the RNA molecule further comprises a 5’ untranslated region (5’ UTR).

27. The composition of claim 26, wherein the 5’ UTR comprises a promoter.

28. The composition of any one of claims 1-27, wherein the RNA molecule further comprises a 3’ untranslated region (3’ UTR).

29. The composition of claim 28, wherein the 3’ UTR comprises at least one exonucleaseresistant modification.

30. The composition of claim 29, wherein the exonuclease-resistant modification is selected from the group consisting of phosphorothioate (PS) linkage, 2’-O-methyl (2OMe), 2’ Fluoro,inverted deoxythymidine (dT), inverted dideoxythymidine (ddT), 3’ phosphorylation, C3 spacer, 2'-O-methoxy-ethyl (2'-M0E), G-quadruplex, and 2'-3'-dideoxy nucleotide (ddN).

31. The composition of any one of claims 1-30, wherein the RNA molecule comprises two or more 5’ caps.

32. The composition of any one of claims 1-31, wherein the RNA molecule comprises two or more poly-A tails.

33. The composition of any one of claims 1-32, wherein the RNA molecule further comprises an open reading frame (ORF).

34. The composition of claim 33, wherein the ORF encodes a protein.

35. The composition of claim 34, wherein the protein is a therapeutic protein.

36. The composition of claim 33, wherein the protein is an antigen.

37. The composition of claim 36, wherein the antigen is a SARS-CoV-2 spike protein or fragment thereof.

38. The composition of claim 37, wherein the ORF comprises a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4.

39. The composition of claim 37, wherein the ORF encodes an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

40. The composition of any one of claims 31-39, wherein the RNA molecule further comprises a sequence encoding a therapeutic nucleic acid.

41. The composition of claim 40, wherein the therapeutic nucleic acid is an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.

42. The composition of any one of claims 1-41, wherein the RNA molecule is a circular RNA molecule.

43. The composition of any one of claims 1-42, wherein the RNA molecule comprises a stem oligo modification having the sequence of SEQ ID NO: 1.

44. The composition of any one of claims 1-43, wherein the RNA molecule comprises a branch oligo modification having the sequence of SEQ ID NO: 2.

45. The composition of any one of claims 1-44, wherein the RNA molecule comprises a 5’UTR having the sequence of SEQ ID NO: 3.

46. The composition of any one of claims 1-45, wherein the RNA molecule comprises a 3’UTR having the sequence of SEQ ID NO: 5.

47. The composition of any one of claims 1-46, wherein the RNA molecule comprises a poly A tail modification having the sequence of SEQ ID NO: 6.

48. The composition of any one of claims 1-47, wherein the RNA molecule comprises two 5’ caps, wherein each of the two 5’ caps is LNAm7G.

49. The composition of any one of claims 1-48, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

50. The composition of claim 49, wherein the nanoparticle or microparticle is a lipid nanoparticle or a lipid microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

51. The composition of claim 50, wherein the nanoparticle is a lipid nanoparticle.

52. The composition of any one of claims 1-51, wherein:(a) the RNA molecule comprises i. two 5’ caps, wherein each of the caps is LNAm7G; ii. a stem oligo modification having the sequence of SEQ ID NO: 1; iii. a branch oligo modification having the sequence of SEQ ID NO: 2;iv. a 5’UTR having the sequence of SEQ ID NO: 3; v. an ORF comprising a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; vi. a 3’UTR having the sequence of SEQ ID NO: 5; and vii. a polyA tail modification having the sequence of SEQ ID NO: 6; and(b) the delivery agent comprises a lipid nanoparticle.

53. The composition of any one of claims 1-52, wherein:(a) the RNA molecule comprises i. two 5’ caps, wherein each of the caps is LNAm7G; ii. a stem oligo modification having the sequence of SEQ ID NO: 1; iii. a branch oligo modification having the sequence of SEQ ID NO: 2; iv. a 5’UTR having the sequence of SEQ ID NO: 3; v. an ORF encoding an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; vi. a 3’UTR having the sequence of SEQ ID NO: 5; and vii. a polyA tail modification having the sequence of SEQ ID NO: 6; and(b) the delivery agent comprises a lipid nanoparticle.

54. The composition of any one of claims 1-53, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

55. An RNA molecule comprising i. two 5’ caps, wherein each of the caps is LNAm7G; ii. a stem oligo modification having the sequence of SEQ ID NO: 1; iii. a branch oligo modification having the sequence of SEQ ID NO: 2; iv. a 5’UTR having the sequence of SEQ ID NO: 3; v. an ORF comprising a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4;vi. a 3’UTR having the sequence of SEQ ID NO: 5; and vii. a polyA tail modification having the sequence of SEQ ID NO: 6.

56. An RNA molecule comprising i. two 5’ caps, wherein each of the caps is LNAm7G; ii. a stem oligo modification having the sequence of SEQ ID NO: 1; iii. a branch oligo modification having the sequence of SEQ ID NO: 2; iv. a 5’UTR having the sequence of SEQ ID NO: 3; v. an ORF encoding an antigen comprising an amino acid sequencing having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; vi. a 3’UTR having the sequence of SEQ ID NO: 5; and vii. a polyA tail modification having the sequence of SEQ ID NO: 6.

57. A vector comprising the RNA molecule of claim 55 or 56.

58. A cell comprising the RNA molecule of claim 55 or 56 or the vector of claim 57.

59. The cell of claim 58, wherein the cell is a mammalian cell.

60. A method of preventing or treating a disease in a subject, comprising administering to a subject an effective amount of the composition of any one of claims 1-54, the RNA molecule of claim 55 or 56, or the vector of claim 57.

61. A method of reducing the risk of a disease in a subject, comprising administering to the subject an effective amount of the composition of any one of claims 1-54, the RNA molecule of claim 55 or 56, or the vector of claim 57.

62. The method of claim 60 or 61, wherein the subject is a human subject.

63. The method of any one of claims 60-62, wherein the disease is a SARS-CoV-2 infection.

64. The RNA molecule of claim 55 or 56, the vector of claim 57, or the composition of any one of claims 1-54 for use in preventing, treating, or reducing the risk of developing a disease in a subject.

65. The RNA molecule of claim 64, wherein the disease is SARS-CoV-2 infection.

66. The RNA molecule of 64 or 65, wherein the subject is a human subject.

67. A kit comprising the composition of any one of claims 1-54, a device for administering the composition to a subject, and / or instructions for administering the composition to the subject.