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WO2025231018A2 - Compositions thérapeutiques et leurs procédés de production et d'utilisation - Google Patents

Compositions thérapeutiques et leurs procédés de production et d'utilisation

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Publication number
WO2025231018A2
WO2025231018A2 PCT/US2025/026868 US2025026868W WO2025231018A2 WO 2025231018 A2 WO2025231018 A2 WO 2025231018A2 US 2025026868 W US2025026868 W US 2025026868W WO 2025231018 A2 WO2025231018 A2 WO 2025231018A2
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WO
WIPO (PCT)
Prior art keywords
composition
sequence
rna molecule
seq
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2025/026868
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English (en)
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WO2025231018A9 (fr
Inventor
Xiao Wang
Hongyu Chen
Dangliang LIU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Massachusetts Institute of Technology
Broad Institute Inc
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Massachusetts Institute of Technology
Broad Institute Inc
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Publication of WO2025231018A2 publication Critical patent/WO2025231018A2/fr
Publication of WO2025231018A9 publication Critical patent/WO2025231018A9/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • mRNA messenger RNA
  • 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)
  • VEGF-A vascular endothelial growth factor A
  • EPO erythropoietin
  • GATA4 GATA Binding Protein 4
  • MEF2C Myocyte Enhancer Factor 2C
  • T-Box Transcription Factor 5 TBX5
  • MYOCD Myocardin
  • 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.
  • the present disclosure provides a composition
  • 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.
  • the RNA molecule comprises two or more 5’ caps.
  • the two or more 5’ caps are conjugated to a 5’ UTR of the RNA molecule.
  • the two or more 5’ caps are conjugated to the RNA molecule via click chemistry.
  • the one or more modified nucleotides comprises a modified sugar.
  • 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
  • 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.
  • 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.
  • the one or more modified nucleotides comprises a modified phosphate.
  • 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.
  • PS phosphorothioate
  • thiophosphate 5 '-O-methylphosphonate
  • 3 '-O-methylphosphonate 5'- hydroxyphosphonate, hydroxyphosphanate
  • phosphoroselenoate selenophosphat
  • 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.
  • 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.
  • the one or more modified nucleotides comprises a modified nucleobase.
  • 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-brom
  • 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.
  • 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.
  • 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.
  • 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
  • the composition further comprises at least one poly-A tail.
  • the at least one poly-A tail comprises between 25 and 500 nucleotides.
  • 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.
  • the at least one poly-A tail comprises 10 or more adenosine nucleotides.
  • nucleotides of the at least one poly-A tail are adenosine nucleotides.
  • the 5’ cap is added to the RNA molecule through a chemical capping method.
  • 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.
  • the RNA molecule further comprises a 5’ untranslated region (5’ UTR).
  • the 5’ UTR comprises a promoter.
  • the RNA molecule further comprises a 3’ untranslated region (3’ UTR).
  • the 3’ UTR comprises at least one exonuclease-resistant modification.
  • 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).
  • PS phosphorothioate
  • the RNA molecule comprises two or more 5’ caps. In some embodiments, the RNA molecule comprises two or more poly-A tails.
  • the RNA molecule further comprises an open reading frame (ORF).
  • the ORF encodes a protein.
  • the protein is a therapeutic protein.
  • the protein is an antigen.
  • the antigen is a SARS-CoV-2 spike protein or fragment thereof.
  • 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.
  • 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.
  • the RNA molecule further comprises a sequence encoding a therapeutic nucleic acid.
  • the therapeutic nucleic acid is an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.
  • the RNA molecule is a circular RNA molecule.
  • 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.
  • the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.
  • 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.
  • the nanoparticle is a lipid nanoparticle.
  • 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.
  • 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 poly A tail modification having the sequence of SEQ ID NO: 6; and (b) the delivery agent comprises a lipid nanoparticle.
  • the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • the subject is a human subject.
  • the disease is a bacterial or viral infection, such as a SARS-CoV-2 infection.
  • 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.
  • 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.
  • RBD Receptor Binding Domain
  • the control mRNA contained mono-m 7 G-rG cap, and the optimized dual-capped mRNA contained two LNAm 7 G 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.
  • LNP lipid nanoparticles
  • 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 RBD mRNA was detected by RIBOmap.
  • 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 RBD mRNA 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).
  • S-peptides Spike peptide pool
  • DMSO negative control
  • FIG. 1G shows a graph depicting FACS analysis results showing the percentages of CD4
  • 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.
  • APCs antigen presenting cells
  • 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.
  • 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.
  • RNA transcripts wherein at least 95% of the RNA transcripts in the composition comprise a 5’ cap.
  • 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.
  • methods of treating or preventing an infection such as a viral infection, such as a SARS-CoV-2 infection.
  • a “messenger RNA” 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.
  • 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.).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ORF is between the last nucleotide of the 5’ UTR and the first nucleotide of the 3’ UTR.
  • 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.
  • the 5’ UTR is between the 5’ cap and the first nucleotide of the ORF.
  • the 3’ UTR is between the ORF and the poly-A region.
  • the 5’ cap is 5’ to (upstream of) the first nucleotide of the 5’ UTR.
  • the first nucleotide of the poly-A region is 3’ to (downstream of) the last nucleotide of the 3’ UTR.
  • 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.
  • 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.
  • 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.
  • the last nucleotide of the nucleic acid sequence is covalently bonded to the first nucleotide of the nucleic acid sequence.
  • 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.
  • 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.
  • tRNA transfer RNA
  • the codon 5'-AUG-3' on an mRNA has the corresponding anticodon 3'-UAC-5' on a tRNA.
  • 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.
  • 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.
  • tRNA first transfer RNA
  • 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.
  • 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.
  • 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.
  • ribosomes may dissociate from the mRNA and release the polypeptide if no tRNA associates with the STOP codon.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the modified nucleotide comprises a modified nucleoside that is not the canonical structure of an adenine nucleoside, cytosine nucleoside, guanine nucleoside, or uracil nucleoside.
  • adenosine 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.
  • cytidine 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the terms “poly-A tail” and “poly-A region” are used interchangeably.
  • the adenosine nucleotides comprised by a poly-A tail may be canonical adenosine nucleotides or modified (non-canonical) adenosine nucleotides.
  • 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.
  • a nucleotide attached to a mRNA by a 5' to 5' triphosphate intemucleotide linkage is referred to as a “native” 5' capping nucleotide.
  • a native 5' capping nucleotide is a 7-methylguanosine (m7G) nucleotide.
  • 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.
  • a 5' cap comprises one or more nucleotides with a sugar modification, such as 2'-O-methylation.
  • m7G 7-methylguanosine
  • a ribonucleic acid sequence e.g., a mRNA
  • a “counterion” or “anionic counterion” is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality.
  • 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 , Sb
  • carborane anions e.g., CBi 1H12 or (HCBi iMcsBre)
  • exemplary counterions which may be multivalent include CO3 2 , HPO4 2 , PO4 3 , B4O7 2 , SO4 2 , S2O3 2
  • 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
  • carboranes e.g., CBi 1H12 or (HCBi iMcsBre
  • Exemplary counterions which may be multivalent include CO3 2 , HPO4 2 , PO4 3 , B4O7 2 , SO4 2 , S2O3 2
  • carboxylate anions e.g., tartrate, cit
  • 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.
  • prevent refers 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.
  • 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 human
  • 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.
  • a modified mRNA is a modified linear mRNA.
  • a modified mRNA is a modified circular mRNA.
  • the “5' cap region”, as used herein, refers to a region of an mRNA that is 5' to (upstream of) the ORF.
  • the 5’ cap region comprises a 5’ untranslated region (5’ UTR).
  • the 5’ cap region comprises a 5’ cap.
  • 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 the production 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.
  • elFs eukaryotic translation initiation factors
  • Chemical modification on or near the 5’ cap influence binding of elFs and decapping enzymes, which subsequently impact downstream mRNA translation and stability.
  • 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.
  • N6-methyladenosine (m6A) on the first base controls mRNA stability through increased resistance to decapping by Dcp2.
  • 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.
  • PNPase polynucleotide phosphorylase
  • composition of a 5' cap typically comprises a 5' m7G attached to the mRNA by a 5' to 5' triphosphate intemucleotide linkage.
  • the modified mRNA comprises one or more modified nucleotides in the 5' cap region of the mRNA.
  • the 5' cap region includes one or more nucleotides that are not canonical adenosine, cytidine, guanosine, or uridine nucleotides.
  • the 5' cap region comprises between 1 and 3, between 3 and 5, between 5 and 7, or between 7 and 10 5' caps.
  • the 5' cap region comprises between 10-500 nucleotides.
  • 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.
  • 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).
  • the RNA molecule comprises two or more 5’ caps.
  • the two or more 5’ caps are conjugated to a 5’ UTR of the RNA molecule.
  • the two or more 5’ caps are conjugated to the RNA molecule via click chemistry.
  • the one or more modified nucleotides comprises a modified sugar.
  • the one or more modified nucleotides comprises a modified phosphate.
  • the one or more modified nucleotides comprises a modified nucleobase.
  • an RNA molecule comprises a 5’ untranslated region (5’ UTR).
  • the 5’ UTR comprises a promoter.
  • the RNA molecule further comprises a 3’ untranslated region (3’ UTR).
  • the 3’ UTR comprises at least one exonuclease-resistant modification.
  • the RNA molecule comprises two or more 5’ caps. In some embodiments, the RNA molecule comprises two or more poly-A tails.
  • the RNA molecule further comprises an open reading frame (ORF).
  • the ORF encodes a protein.
  • the ORF encodes a therapeutic protein.
  • the ORF encodes an antigen.
  • the antigen is a SARS-CoV-2 spike protein or fragment thereof.
  • 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.
  • 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.
  • the RNA molecule further comprises a sequence encoding a therapeutic nucleic acid.
  • the therapeutic nucleic acid is an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.
  • ASO antisense oligonucleotide
  • the RNA molecule is a circular RNA molecule.
  • 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.
  • 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.
  • 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.
  • the method comprises first synthesizing a 5 ’-phosphorylated RNA oligonucleotide with a specific sequence and/or desired modifications.
  • the synthesized 5 ’-phosphorylated RNA oligonucleotide defines the 5’ UTR when ligated to an RNA transcript.
  • the terms “5’-phosphorylated RNA oligonucleotide,” “5 ’-phosphorylated oligonucleotide,” and “5’- phosphorylated UTR” are used interchangeably.
  • the 5 ’-phosphorylated RNA oligonucleotide comprises one or more modified nucleotides which may affect RNA translation and/or stability.
  • 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.
  • PS phosphorothioate
  • thiophosphate 5'-O- methylphosphonate
  • 3'-O-methylphosphonate 5'-
  • 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.
  • 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.
  • 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'-a
  • Z-adenosine 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.
  • 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.
  • 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.
  • 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.
  • 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-aminoally
  • 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.
  • 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.
  • the 5 ’-phosphorylated oligonucleotide is synthesized on a solidphase support.
  • the solid support is controlled-pore glass (CPG) or polystyrene (PS).
  • the 5 ’-phosphorylated oligonucleotide is synthesized via phosphoramidite oligonucleotide synthesis.
  • the 5 ’-phosphorylated oligonucleotide is synthesized in a solvent system comprising a nonpolar counterion.
  • the nonpolar counterion used in oligonucleotide synthesis is ammonium.
  • the nonpolar counterion used in oligonucleotide synthesis is ammonium.
  • 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.
  • 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.
  • the chemical capping reaction is performed through an anhydrous reaction between the 5 ’-phosphorylated RNA oligonucleotide and 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).
  • 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.
  • the capping reaction is performed in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the desired product of this reaction is an oligonucleotide capped on its 5’ end with the cap of interest.
  • 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-
  • a 5’ cap region provided herein comprises a 5’-capped oligonucleotide (i.e., a 5’-capped UTR) synthesized as described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • TGT tRNA guanine transglycosylase
  • preQi pre-queuosine 1
  • 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).
  • more than one azide handle is introduced into an RNA molecule.
  • 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
  • the methods provided herein produce a capped RNA transcript with a modified 5’ cap region.
  • 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.
  • 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.
  • a 5’ cap region is produced as described herein.
  • 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.
  • 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.
  • an amino acid e.g., a lysine
  • ATP adenosine triphosphate
  • AMP adenosine monophosphate
  • a 5' terminal phosphate of the second nucleic acid displaces the phosphate of the RNA ligase-bound AMP.
  • 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.
  • the ligase is T4 RNA Ligase I, T4 RNA Ligase II, or RtcB.
  • 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)).
  • the RNA precursor comprises an open reading frame (ORF).
  • the ORF encodes a therapeutic protein.
  • a “therapeutic protein” refers to a protein that prevents, reduces, or alleviates one or more signs or symptoms of a 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.
  • 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.
  • 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
  • the ORF encodes an antigen.
  • 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.
  • the antigen is a protein derived from a pathogen, such as a pathogenic virus, bacterium, protozoan, or fungus.
  • the antigen is a protein derived from a virus (viral antigen) or a fragment thereof.
  • the antigen is a protein or fragment thereof derived from SARS-CoV-2.
  • the antigen is a fragment of a SARS-CoV-2 spike protein.
  • the antigen comprises a SARS-CoV-2 spike protein receptor-binding domain (RBD).
  • 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.
  • the antigen is a protein derived from a bacterium (bacterial antigen) or a fragment thereof.
  • the antigen is a protein derived from a protozoan (protozoal antigen) or a fragment thereof.
  • 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.
  • the RNA transcripts produced by the methods provided herein may be used for prophylactic purposes, such as for vaccination of a subject.
  • the methods disclosed herein provide an RNA precursor and/or a capped RNA transcript comprising one or more noncoding genes.
  • the noncoding heterologous genes are therapeutic nucleic acids.
  • a therapeutic nucleic acid is a nucleic acid or related compound that alters gene expression to prevent or treat diseases or disorders.
  • 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.
  • ASO antisense oligonucleotide
  • GaNAc N-acetylgalactosamine
  • siRNA short interfering RNA
  • a composition provided herein further comprises one or more additional agents.
  • 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.
  • the additional agent is an agent which has a therapeutic effect when administered to a subject.
  • the additional 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).
  • the additional agent is a small molecular inhibitor.
  • the additional agent is an agent that is capable of eliciting or enhancing an immune response in a subject.
  • the additional agent is an antigen (e.g., a viral antigen, a bacterial antigen).
  • 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.
  • 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.
  • 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.
  • 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).
  • ATP adenosine triphosphate
  • AMP adenosine monophosphate
  • PPi pyrophosphate
  • 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.
  • RNA 5’ Pyrophosphohydrolase RppH is used to produce 5 ’-mono
  • the 5’ cap region 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.
  • 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.
  • the spacer region comprises a plurality of identical consecutive nucleotides.
  • 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).
  • non-canonical nucleotide e.g., inosine
  • 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.
  • the RNA precursor comprises one or more modified phosphates, sugars, and/or nucleobases to confer exonuclease resistance.
  • 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.
  • PS phosphorothioate
  • SP thiophosphate
  • the RNA precursor comprises a 2’-O-methoxy-ethyl base (2’-MOE), a G- quadruplex, or a 2’-3’-dideoxy nucleotide (ddN).
  • 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)
  • QRNA Capped-circular mRNA
  • 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.
  • 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).
  • CuAAC copper catalyzed azide-alkyne cycloaddition
  • IEDDA tetrazine-trans cyclooctene inverse electron demand Diels- Alder reaction
  • the invention contemplates two generic structures of capped circular messenger RNAs (QRNAs): Type 1 QRNA and Type 2 QRNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • the crosslinking reaction is click chemistry.
  • 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. ⁇ .
  • 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.
  • 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).
  • equivalent structure means any molecule that are sufficiently structurally similar and perform the same function in a chemical reaction.
  • a derivatized nucleotide is a nucleotide that is modified to comprise a chemical group/handle can participate in a cross-linking reaction.
  • RNA 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.
  • the synthesis pathway of Type 1 and Type 3 QRNA enables multiple oligonucleotides containing 5’ cap binding to the circular RNA.
  • circular RNA can include multiple derivatized nucleotides that can covalently bind to multiple oligonucleotides containing 5’ cap.
  • a single circular RNA backbone can encode multiple TGT sites to enable binding of multiple oligonucleotides containing 5’ cap onto the circular RNA simultaneously.
  • the capped, circular RNA molecule comprises an mRNA region encoding one or a plurality of peptides or polypeptides.
  • 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.
  • m7G 7-methylguanine
  • cap structure [00100] 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 N 6 , 2’-O- dimethyladenosine (m 6 Am) 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.
  • the methyl group in 7-methylguanosine (m 7 G) cap structure can be modified to produce 7-benzylguanosine (Bn 7 G), 7-chlorobenzylguanosine (ClBn 7 G), and chlorobenzyl-O-ethoxyguanosine (ClBnOEt 7 G).
  • LNA Locked Nucleic Acid
  • 2Me 2’ -methoxy (20Me)
  • 2-m ethoxy ethoxy (2M0E) significantly increase mRNA translation.
  • the cap structures include, but are not limited to, m 7 G-LNA, LNAm 7 G-LNA, LNAm 7 G-LNAx6, LNAm 7 G- 2OMex6.
  • the cap structure is m 7 G diphosphate imidazolide (m 7 GDP-Im).
  • nucleotide/nucleotide identity specifically incorporation of adenosine (A), guanosine (G), 6-methyladenosine (m 6 A), or the non-canonical inosine (I) in the mRNA, preferably, at the +1 position, increases translation efficiency.
  • substitution of some or all uridine residues to A 7 -methylpseudouridine (m 1 T) in the mRNA also boosts the translation.
  • the nucleotides are numbered according to their position immediately downstream of the cap structure.
  • the cap structure found at the 5' end of eukaryotic mRNAs consists of a 7-methylguanosine (m 7 G) moiety linked to the first nucleotide (+1 position) of the transcript via a 5'-5' triphosphate bridge.
  • m 7 G 7-methylguanosine
  • 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,
  • 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.
  • PS phosphorothioate
  • thiophosphate 5'-O-methylphosphonate
  • 3'-O-methylphosphonate 5- hydroxyphosphonate, hydroxyphosphanate
  • phosphoroselenoate selenophosphate
  • phosphorami date carbophosphonate, methylphosphonate, phenylphosphonate, ethy
  • LNA locked nucleic acid
  • 2-OMe 2’- methoxyribose
  • 2-MOE 2-methoxyethoxy
  • 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’
  • RNA nuclease-resistance properties
  • 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).
  • 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.
  • LNAs locked nucleic acids
  • PNAs peptide nucleic acids
  • morpholinos morpholinos
  • various internal chemical linkers as provided herein.
  • 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 or polypeptides.
  • 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.
  • An IRES or self-cleaving peptide such as T2A sequence, can exist between the multiple polypeptide coding sequences on the QRNA.
  • 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.
  • 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.
  • these antigenic peptides are adapted to and effective for use as vaccines.
  • the antigenic peptides are adapted to or effective in suppressing immune responses, for example in autoimmune diseases or transplant patients.
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the circularized RNA is produced by ribozyme- mediated splicing, enzymatic ligation, or click chemistry-mediated circularization.
  • 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 polypeptid
  • 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.
  • TGT tRNA guanine transferases
  • Circularization of RNA molecule in type 1 and type 3 can be achieved by ligation with T4 ligase, RtcB ligase, or ribozyme-mediated splicing.
  • 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.
  • the click chemistry moiety has also been contemplated for the circularization.
  • 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.
  • 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.
  • RNA v.10, 889-893 silica-based affinity chromatography and polyacrylamide gel electrophoresis
  • 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).
  • 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.
  • the capped RNA oligonucleotides provided herein are purified by high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • the capped RNA oligonucleotide is purified by reverse-phase HPLC (RP-HPLC).
  • RP-HPLC reverse-phase HPLC
  • HPLC gradients used to isolate the capped RNA oligonucleotides comprise hydrophobic hexylammonium ions.
  • 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.
  • the concentration of hydrophobic ions e.g., hexylammonium ions
  • used for HPLC purification of RNA oligonucleotides is between 10 mM to 200 mM.
  • 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
  • the present disclosure provides a delivery reagent comprising any of the capped RNA molecules provided herein.
  • any of the capped RNA 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.
  • 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.
  • a nanoparticle or microparticle is approximately spherical.
  • a nanoparticle or microparticle is hollow, comprising an internal core.
  • 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.
  • 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.
  • 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.
  • the capped RNA molecule is dispersed throughout the lipids of the lipid nanoparticle or lipid microparticle.
  • the lipid nanoparticle or lipid microparticle comprises an ionizable amino lipid, a non-cationic lipid, a sterol, 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.
  • 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.
  • 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.
  • 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.
  • PEI polyethylenimine
  • PAA poly-amido-amine
  • PBAEs poly-beta amino-esters
  • PLL polylysine
  • spermine e.g., chitosan, polyurethane, and derivatives thereof (e.g., PEI stearic acid (PSA) copo
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • thiol linkages e.g., modifying the DNA to comprise cyclic disulfide- anchoring groups
  • PEI cationic materials
  • the present disclosure provides cells comprising any of the capped RNA molecules provided herein.
  • 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.
  • 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.
  • 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.
  • the present disclosure provides compositions comprising any of the modified mRNAs, delivery agents, or cells provided herein.
  • 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.
  • the additional agent has a therapeutic effect when administered to a subject.
  • 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.
  • 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).
  • the additional agent is an inhibitor for decreasing the activity of one or more gene products (e.g., proteins).
  • the agent is a small molecular inhibitor.
  • the additional agent is an agent for enhancing an immune response in a subject.
  • the additional agent is an antigen, such as a nucleic acid antigen, a protein antigen, or a phospholipid antigen.
  • 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.
  • 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.
  • compositions for example, 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.
  • the present disclosure provides a comprising an RNA molecule and a delivery agent.
  • 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.
  • 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.
  • the two or more 5’ caps are conjugated to the RNA molecule of the composition via click chemistry.
  • the one or more modified nucleotides comprises a modified sugar.
  • the one or more modified nucleotides comprises a modified phosphate.
  • the one or more modified nucleotides comprises a modified nucleobase.
  • a composition comprises an RNA molecule comprising a 5’ untranslated region (5’ UTR).
  • the 5’ UTR comprises a promoter.
  • the RNA molecule of the composition further comprises a 3’ untranslated region (3’ UTR).
  • the 3’ UTR comprises at least one exonuclease-resistant modification.
  • the RNA molecule of the composition comprises two or more 5’ caps.
  • the RNA molecule of the composition comprises two or more poly- A tails.
  • the RNA molecule of the composition further comprises an open reading frame (ORF).
  • the ORF encodes a protein.
  • the ORF encodes a therapeutic protein.
  • the ORF encodes an antigen.
  • the antigen is a SARS-CoV-2 spike protein or fragment thereof.
  • 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.
  • 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.
  • the RNA molecule of the composition further comprises a sequence encoding a therapeutic nucleic acid.
  • the therapeutic nucleic acid is an antisense oligonucleotide (ASO), an aptamer, an RNA decoy, an siRNA, a shRNA, a miRNA, or a gRNA.
  • the RNA molecule of the composition is a circular RNA molecule.
  • 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.
  • the delivery agent of the composition comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.
  • 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.
  • the delivery agent of the composition comprises a lipid nanoparticle.
  • 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.
  • 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.
  • the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • 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.
  • the subject is a human.
  • the administration is parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal.
  • 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.
  • 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, which promote 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.
  • 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.
  • the capped RNA molecule is administered with other elements, such as buffers and/or excipients, that increase the efficiency of electroporation.
  • the present disclosure provides a kit comprising any of the capped RNA oligonucleotides, RNA precursors, or capped RNA molecules provided herein.
  • the kit comprises a ligase.
  • the kit comprises an RNA ligase.
  • the kit comprises a T4 RNA ligase.
  • a kit comprises a T4 RNA ligase 1.
  • a kit comprises a T4 RNA ligase 2.
  • the kit comprises an RtcB RNA ligase.
  • the kit further comprises a buffer for carrying out the ligation.
  • the kit further comprises a nucleotide triphosphate, such as ATP, to provide energy required by the ligase.
  • 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.
  • 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.
  • 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.
  • the kit further comprises instructions for administering any of the pharmaceutical compositions provided herein to a subject.
  • compositions for delivery and methods therefore, are provided.
  • compositions comprising capped RNA molecules of the disclosure, particularly linear and circularized mRNA molecules.
  • pharmaceutical compositions of the invention further comprise pharmaceutically acceptable excipients and in certain other embodiments comprise one or more additional therapeutics agents.
  • compositions are suitable to be administered to a human subject in need thereof.
  • 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.
  • compositions described herein are also suitable for administration to any non-human subjects as well.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • excipients can include but are not limited to, solvents, dispersions, buffers, diluents, surfactants, emulsifiers, isotonic agents, preservatives, thickeners, lubricating agents, oils, or the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • provided herein are methods of preventing or treating a disease in a subject, comprising introducing an effective amount of an RNA molecule or composition comprising the RNA molecule described herein to the subject.
  • the subject is a human subject.
  • the disease is SARS-CoV- 2.
  • a single dose of an effective amount of an RNA molecule or a composition comprising the RNA molecule is administered to the 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.
  • preventing” a disease can include reducing the risk of a given disease, such as a viral or bacterial infection.
  • a disease e.g., a bacterial or viral infection
  • 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.
  • 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).
  • 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.
  • the administration of the antigen prevents the subject from developing a HIV infection and/or AIDS.
  • 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).
  • the administration of the disclosed RNA molecules and compositions reduces the risk of transmission of a bacteria or virus.
  • o the administration 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.
  • 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.
  • 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.
  • Embodiment 2 The composition of embodiment 1, wherein the RNA molecule comprises two or more 5’ caps.
  • Embodiment 3 The composition of embodiment 2, wherein the two or more 5’ caps are conjugated to a 5’ UTR of the RNA molecule.
  • 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.
  • Embodiment 5 The composition of any one of embodiments 1-4, wherein the one or more modified nucleotides comprises a modified sugar.
  • 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 '-azido
  • 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.
  • 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.
  • Embodiment 9 The composition of any one of embodiments 1-8, wherein the one or more modified nucleotides comprises a modified phosphate.
  • 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.
  • PS phosphorothioate
  • thiophosphate 5'-O- methylphosphonate
  • 3 '-O-methylphosphonate 5'-hydroxyphosphonate, hydroxy phosphanate
  • phosphoroselenoate selenophosphate
  • phosphoramidate
  • 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.
  • 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.
  • Embodiment 13 The composition of any one of embodiment 1-12, wherein the one or more modified nucleotides comprises a modified nucleobase.
  • 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-car
  • 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.
  • 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.
  • 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.
  • 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-prop
  • Embodiment 19 The composition of any one of embodiments 1-18, further comprising at least one poly-A tail.
  • Embodiment 20 The composition of embodiment 19, wherein the at least one poly-A tail comprises between 25 and 500 nucleotides.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 26 The composition of any one of embodiments 1-25, wherein the RNA molecule further comprises a 5’ untranslated region (5’ UTR).
  • Embodiment 27 The composition of embodiment 26, wherein the 5’ UTR comprises a promoter.
  • Embodiment 28 The composition of any one of embodiments 1-27, wherein the RNA molecule further comprises a 3’ untranslated region (3’ UTR).
  • Embodiment 29 The composition of embodiment 28, wherein the 3’ UTR comprises at least one exonuclease-resistant modification.
  • 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).
  • PS phosphorothioate
  • Embodiment 31 The composition of any one of embodiments 1-30, wherein the RNA molecule comprises two or more 5’ caps.
  • Embodiment 32 The composition of any one of embodiments 1-32, wherein the RNA molecule comprises two or more poly-A tails.
  • Embodiment 33 The composition of any one of embodiments 1-32, wherein the RNA molecule further comprises an open reading frame (ORF).
  • ORF open reading frame
  • Embodiment 34 The composition of embodiment 33, wherein the ORF encodes a protein.
  • Embodiment 35 The composition of embodiment 34, wherein the protein is a therapeutic protein.
  • Embodiment 36 The composition of embodiment 33, wherein the protein is an antigen.
  • Embodiment 37 The composition of embodiment 36, wherein the antigen is a SARS-CoV-2 spike protein or fragment thereof.
  • 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.
  • 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.
  • Embodiment 40 The composition of any one of embodiments 31-39, wherein the RNA molecule further comprises a sequence encoding a therapeutic nucleic acid.
  • 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.
  • ASO antisense oligonucleotide
  • RNA decoy an siRNA
  • shRNA a shRNA
  • miRNA a miRNA
  • gRNA gRNA
  • Embodiment 42 The composition of any one of embodiments 1-41, wherein the RNA molecule is a circular RNA molecule.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 51 The composition of embodiment 50, wherein the nanoparticle is a lipid nanoparticle.
  • 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.
  • 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.
  • Embodiment 54 The composition of any one of embodiments 1-53, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • 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.
  • 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 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 poly A tail modification having the sequence of SEQ ID NO: 6.
  • Embodiment 57 A vector comprising the RNA molecule of embodiment 55 or 56.
  • Embodiment 58 A cell comprising the RNA molecule of embodiment 55 or 56 or the vector of embodiment 57.
  • Embodiment 59 The cell of embodiment 58, wherein the cell is a mammalian cell.
  • 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.
  • Embodiment 61 The method of embodiment 60, wherein the subject is a human subject.
  • Embodiment 62 The method of embodiment 60 or 61, wherein the disease is SARS-CoV-2.
  • 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.
  • Embodiment 64 The RNA molecule of embodiment 63, wherein the disease is SARS-CoV-2.
  • Embodiment 65 The RNA molecule of 63 or 64, wherein the subject is a human subject.
  • 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.
  • Example 1 Modified dual-capped mRNA for enhanced mRNA vaccine against SARS-CoV- 2
  • Applicant evaluated the effects of multi-capped RNAs on SARS-CoV-2 vaccination outcomes.
  • Applicant used an optimized dual-LNAm 7 G-LNA+5xOMe-6*PS+2MOE mRNA and regular mono-m 7 G-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).
  • 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).
  • T-cell immune response is the other major component in mediating immunity against SARS-CoV-2 in patients.
  • S- peptides SARS-CoV-2 Spike-RBD pooled peptides
  • DMSO DMSO
  • 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-m 7 G-rG mRNA mRNA.
  • CDS proteins of interest coding sequences
  • 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
  • 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 N 1 - 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)
  • m 7 G-Im, Bn 7 G-Im, ClBn 7 G-Im, ClBnOEt 7 G-Im and LNAm 7 G-Im capping reagents were synthesized following previously reported protocol. Briefly, m 7 GDP, Bn 7 GDP, ClBn 7 GDP, ClBnOEt 7 GDP were synthesized by treating the GDP sodium salt with dimethyl sulfoxide or corresponding alkylation reagents, followed by HPLC purification. LNAm 7 GDP 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: m 7 GDP, ClBn 7 GDP, LNAm 7 GDP.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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
  • 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.
  • 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.
  • modified 1.5x click chemistry buffer [Lumiprobe, 61150] containing additive 5% SUPERase Inhibitor, 5% DMSO, and 5% 10 mM dNTP mix [ThermoFisher Scientific, 18427089]
  • 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.
  • 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.
  • the model was then used to predict segmentation for each FOV.
  • 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).
  • 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.
  • TGT enzyme was expressed in E. colt as described in the literature.
  • 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 Bn 7 G-capped alkyne labeled oligo using the general condition for click reaction for 30 mins. It is noteworthy that the hydrophobicity of Bn 7 G 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.
  • 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.
  • TAATACGACTCACTATAAG T7 “CleanCap AG” promoter sequence
  • 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
  • a 3’ Esp3I plasmid linearization site
  • 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 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.
  • 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 40 th amplification cycle. All the gene expressions were calculated and normalized to GAPDH.
  • 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.
  • 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].
  • 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.
  • mRNA-LNP concentration 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.
  • 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.
  • T7 “CleanCap AG” promoter sequence TAATACGACTCACTATAAG
  • 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).
  • 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.
  • 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.
  • 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]
  • LEGEND MAX Mouse IL-4 ELISA Kit [BioLegend, 431107]
  • 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).

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Abstract

L'invention concerne des molécules d'ARN coiffées comprenant un ou plusieurs nucléotides modifiés en position +3 ou supérieure par rapport à à une extrémité 5' de la molécule d'ARN, et leurs procédés de fabrication. Les molécules d'ARN coiffées peuvent être fabriquées par ligature d'un oligonucléotide d'ARN modifié coiffé en 5' à l'extrémité 3' d'une molécule d'ARN. L'invention concerne également des compositions comprenant une ou plusieurs des molécules d'ARN coiffées selon l'invention, et des procédés d'utilisation desdites compositions pour des applications thérapeutiques, telles que dans le traitement ou la prévention d'une maladie chez un sujet, tel que le SARS-CoV-2.
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