WO2024192422A1 - Compositions immunogènes et leurs utilisations - Google Patents

Compositions immunogènes et leurs utilisations Download PDF

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Publication number
WO2024192422A1
WO2024192422A1 PCT/US2024/020317 US2024020317W WO2024192422A1 WO 2024192422 A1 WO2024192422 A1 WO 2024192422A1 US 2024020317 W US2024020317 W US 2024020317W WO 2024192422 A1 WO2024192422 A1 WO 2024192422A1
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WIPO (PCT)
Prior art keywords
polyribonucleotide
spacer element
sequence
circularization
circular
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PCT/US2024/020317
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English (en)
Inventor
Revital BRONSTEIN
Elissa Magdalene HOBERT
Teymur Spartakovich KAZAKOV
Ki Young PAEK
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Flagship Pioneering Innovations Vi, Llc
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Publication of WO2024192422A1 publication Critical patent/WO2024192422A1/fr

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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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular

Definitions

  • Vaccination has made an enormous contribution to both human and animal health. Since the invention of the first vaccine in 1796, vaccines have come to be considered the most successful method for preventing many infectious diseases by provoking an immune response in a subject. According to the World Health Organization, immunization currently prevents 2-3 million deaths every year across all age groups. Today, vaccines have been developed to prevent and control the spread of more than 20 infectious diseases, including diphtheria, tetanus, pertussis, influenza, and measles, and have led to the complete eradication of smallpox. There remains a need to develop new and improved immunogenic compositions and uses thereof.
  • the disclosure provides a circular polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) one or more expression augmenting element; (c) a polyribonucleotide cargo encoding an immunogen; and (d) a second postcircularization element; wherein the first post-circularization element and the second postcircularization element together form a circularization junction.
  • the disclosure provides circular polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a polyribonucleotide cargo encoding an immunogen; (c) one or more expression augmenting element; and (d) a second postcircularization element; wherein the first post-circularization element and the second postcircularization element together form a circularization junction.
  • the one or more expression augmenting element comprises a fusion of a translation enhancer and a spacer element comprising a polyA region.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the one or more expression augmenting element comprises a translation enhancer.
  • the translation enhancer is from a plant virus.
  • the translation enhancer is from a mammalian virus.
  • the translation enhancer is a human translation enhancer.
  • the translation enhancer is selected from the group comprising a BYDV like-element (BTE), a translation enhancer element (TED), a PMV/PEMV-like translation enhancer (PTE), an l-shaped structure (ISS), a Y- shaped structure (YSS), a t-shaped structure (TSS), dumbbell shaped structure, viral RNA UTRs (including Dengue, West Nile, Zika, Rotavirus), EMCV, CVB3, hepatitis B virus posttranscriptional regulatory element, human genomic fragments, a histone mRNA sequence, a cyclin D mRNA sequence, or an elF4g aptamer sequence.
  • BTE BYDV like-element
  • TED translation enhancer element
  • PTE PMV/PEMV-like translation enhancer
  • ISS l-shaped structure
  • YSS Y- shaped structure
  • TSS t-shaped structure
  • dumbbell shaped structure including Dengue, West Nile, Zika, Rotavirus
  • the translation enhancer binds to elF4g.
  • the translation enhancer comprises between 50 and 2000 (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500) ribonucleotides.
  • the translation enhancer comprises between 60 and 800 (e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500) ribonucleotides.
  • 60 and 800 e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500
  • the circular polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without a translation enhancer. In some embodiments, the circular polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without a translation enhancer after the circular polyribonucleotide is administered to a subject. In some embodiments, the circular polyribonucleotide comprises a polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 4-fold greater expression in comparison to a polyribonucleotide without a translation enhancer.
  • the one or more expression augmenting element comprises a fusion of a stability element and a spacer element comprising a polyA region.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the expression augmenting element comprises a stability element.
  • the stability element is an untranslated region (UTR).
  • the stability element is a 3’ UTR.
  • the stability element is a 5’ UTR.
  • the UTR is from a gene encoding a translocation associated membrane protein 1 (TRAM1 ), a transmembrane p24 trafficking protein 2 (TMED2), a vesicle associated membrane protein 3 (VAMP3), a CXXC repeat containing interactor of PDZ3 domain (CRIP), an adaptor related protein complex 2 subunit alpha 2 (AP2A2), a proteasome 26S subunit, a non-ATPase 5 (PSMD5), glutathione peroxidase 4 (GPX4), or a protein kinase AMP-activated non-catalytic subunit beta 1 (PRKAB1 ).
  • TAM1 translocation associated membrane protein 1
  • TMED2 transmembrane p24 trafficking protein 2
  • VAMP3 vesicle associated membrane protein 3
  • CRIP CXXC repeat containing interactor of PDZ3 domain
  • A2A2 adaptor related protein complex 2 subunit alpha 2
  • PSMD5 non-
  • the UTR is a UTR from a human beta actin, DDB2, TP53I3, FcIgG, LSP1 , AES, DRB4, or a mitochondrially encoded 12S rRNA.
  • the stability element comprises between 50 and 2000 (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500) ribonucleotides.
  • the stability element comprises between 60 and 800 (e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500) ribonucleotides.
  • the circular polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without a stability element.
  • the circular polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without a stability element after the circular polyribonucleotide is administered to a subject.
  • the polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 2-fold greater expression in comparison to a polyribonucleotide without a stability element.
  • the circular polyribonucleotide further comprises one or more spacer elements comprising a polyA region.
  • the spacer element has a length of between 50 to 500 (e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 50 to 300, 50 to 200, 60 to 300, and 60 to 200) ribonucleotides.
  • 50 to 500 e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500,
  • the spacer element has a length of between 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500,
  • 100 to 500 e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500,
  • the spacer element has a length of between 110 to 500 (e.g., 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 150 to 500, 200 to
  • the spacer element has a length of between 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • 120 to 500 e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500
  • the spacer element has a length of between 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 110 to 300, 115 to 300, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, 110 to 200, and 280 to 300) ribonucleotides.
  • 100 to 300 e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 110 to 300, 115 to 300, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, 110 to 200, and 280 to 300
  • 100 to 300 e.g., 100 to 280, 100 to 260, 100 to 240, 100 to
  • the spacer has a length of between 10 and 200 (e.g., between 10 and 175, 10 and 150, 10 and 125, 10 and 100, 10 and 75, 10 and 50, 10 and 25, 25 and 200, 50 and 200, 75 and 200, 100 and 200, 150 and 200, 175 and 200, and 25 and 100) ribonucleotides.
  • the spacer element has a length of about 120 ribonucleotides. In some embodiments, the spacer element has a length of about 50 ribonucleotides.
  • the disclosure provides a circular polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first expression augmenting element; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second expression augmenting element; and (e) a second post-circularization element; wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the first expression augmenting element comprises a fusion of a translation enhancer and a spacer element.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the second expression augmenting element comprises a fusion of a translation enhancer and a spacer element.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the first expression augmenting element comprises a translation enhancer.
  • the second expression augmenting element comprises a translation enhancer.
  • the translation enhancer is from a plant virus. In some embodiments, the translation enhancer is from a mammalian virus. In some embodiments, the translation enhancer is a human translation enhancer.
  • the translation enhancer is selected from the group comprising a BTE, a TED, a PTE, an ISS, a YSS, a TSS, dumbbell shaped structure, viral RNA UTRs (including Dengue, West Nile, Zika, Rotavirus), EMCV, CVB3, hepatitis B virus posttranscriptional regulatory element, human genomic fragments, a histone mRNA sequence, a cyclin D mRNA sequence, or an elF4g aptamer sequence.
  • the translation enhancer binds to elF4g.
  • the translation enhancer comprises between 50 and 2000 (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500) ribonucleotides.
  • the translation enhancer comprises between 60 and 800 (e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500) ribonucleotides.
  • the circular polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without a translation enhancer.
  • the circular polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without a translation enhancer after the circular polyribonucleotide is administered to a subject.
  • the circular polyribonucleotide comprises a polynucleotide cargo encoding an immunogen, wherein the immunogen has at least 4-fold greater expression in comparison to a polyribonucleotide without a translation enhancer.
  • the first expression augmenting element comprises a fusion of a stability element and a spacer element comprising a polyA region.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the first expression augmenting element comprises a stability element.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the second expression augmenting element comprises a stability element.
  • the stability element is a UTR.
  • the UTR is from a gene encoding a TRAM1 , a TMED2, a VAMP3, a CRIP, an AP2A2, a proteasome 26S subunit, a PSMD5, a GPX4, or a PRKAB1 .
  • the UTR is a UTR from a human beta actin, DDB2, TP53I3, FcIgG, LSP1 , AES, DRB4, or a mitochondrially encoded 12S rRNA.
  • the stability element is a 3’ UTR. In some embodiments, the stability element is a 5’ UTR. In some embodiments, the stability element comprises between 50 and 2000 (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500) ribonucleotides.
  • the stability element comprises between 60 and 800 (e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500) ribonucleotides.
  • the circular polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without a stability element.
  • the circular polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without a stability element after the circular polyribonucleotide is administered to a subject.
  • the polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 2-fold greater expression in comparison to a polyribonucleotide without a stability element.
  • the circular polyribonucleotide further comprises one or more spacer elements comprising a polyA region.
  • the spacer element has a length of between 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the spacer element has a length of between 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides. In some embodiments, the spacer element has a length of between 100 to 300 (e.g., 100 to 280, 100 to 260,
  • 100 to 240 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to
  • the disclosure provides a circular polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a spacer element; (c) a polyribonucleotide cargo encoding an immunogen; (d) an expression augmenting element; and (e) a second post-circularization element and wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a circular polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) an expression augmenting element; (c) a polyribonucleotide cargo encoding an immunogen; (d) a spacer element; and (e) a second post-circularization element and wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a circular polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first expression augmenting element; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second expression augmenting element; and (e) a second post-circularization element and wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a circular polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first spacer element having a length of at least 100 ribonucleotides; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second spacer element; and (e) a second post-circularization element; and wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a circular polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first spacer element; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second spacer element having a length of at least 100 ribonucleotides; and (e) a second post-circularization element; and wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the first spacer element has a length of between 50 to 500 (e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 50 to 300, 50 to 200, 60 to 300, and 60 to 200) ribonucleotides.
  • 50 to 500 e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500
  • the first spacer element has a length of between 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • 100 to 500 e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500
  • the second spacer element has a length of between 50 to 500 (e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 50 to 300, 50 to 200, 60 to 300, and 60 to 200) ribonucleotides.
  • 50 to 500 e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500
  • the second spacer element has a length of between 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • 100 to 500 e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500
  • the first spacer element and the second spacer element each comprise 110 to 500 (e.g., 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the first spacer element and the second spacer element each comprise 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the first spacer element and the second spacer element each have a length of 150 to 500 (e.g., 150 to 450, 150 to 400, 150 to 350, 150 and 300, 150 and 250, 150 and 200, 200 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, or 450 and 500) ribonucleotides.
  • the first spacer element and the second spacer element each have a length of 200 to 500 (e.g., 200 to 450, 200 to 400, 200 to 350, 200 to 300, 200 to 250, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500) ribonucleotides.
  • the first spacer element and the second spacer element each have a length of 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300) ribonucleotides.
  • 100 to 300 e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300
  • 100 to 300 e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140,
  • the first spacer element and the second spacer element each have a length of 100 to 200 (e.g., 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 100 to 110, 110 to 200, 120 to 200, 130 to 200, 140 to 200, 150 to 200, 160 to 200, 170 to 200, 180 to 200, and 90 to 200) ribonucleotides.
  • 100 to 200 e.g., 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 100 to 110, 110 to 200, 120 to 200, 130 to 200, 140 to 200, 150 to 200, 160 to 200, 170 to 200, 180 to 200, and 90 to 200
  • the first spacer element and the second spacer element each have a length of 110 to 300 (e.g., 110 to 280, 110 to 260, 110 to 240, 110 to 220, 110 to 200, 110 to 180, 110 to 160, 110 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300) ribonucleotides.
  • 110 to 300 e.g., 110 to 280, 110 to 260, 110 to 240, 110 to 220, 110 to 200, 110 to 180, 110 to 160, 110 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300
  • the first spacer element and the second spacer element each have a length of 120 to 300 (e.g., 120 to 280, 120 to 260, 120 to 240, 120 to 220, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300) ribonucleotides.
  • 120 to 300 e.g., 120 to 280, 120 to 260, 120 to 240, 120 to 220, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300
  • the first spacer element and the second spacer element each have a length of 150 to 300 (e.g., 150 to 290, 150 to 280, 150 to 270, 150 to 260, 150 to 250, 150 to 240, 150 to 230, 150 to 220, 150 to 210, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 150 to 160, 160 to 300, 170 to 300, 180 to 300, 190 to 300, 200 to 300, 210 to 300, 220 to 300, 230 to 300, 240 to 300, 250 to 300, 260 to 300, 270 to 300, 280 to 300, and 290 to 300) ribonucleotides.
  • 150 to 300 e.g., 150 to 290, 150 to 280, 150 to 270, 150 to 260, 150 to 250, 150 to 240, 150 to 230, 150 to 220, 150 to 210, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 150 to 160, 160 to 300, 170 to 300,
  • the difference between the length of the first spacer element and the length of the second spacer element is 0 to 100 (e.g., 0 and 10, 0 and 20, 0 and 30, 0, and 40, 0 and 50, 0 to 60, 0 to 70, 0 to 80, 0 to 90, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 5 to 10, 5 to 20, 10 to 30 or 5 to 50) nucleotides.
  • 0 to 100 e.g., 0 and 10, 0 and 20, 0 and 30, 0, and 40, 0 and 50, 0 to 60, 0 to 70, 0 to 80, 0 to 90, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 5 to 10, 5 to 20, 10 to 30 or 5 to 50
  • the difference between the length of the first spacer element and the length of the second spacer element is 0 to 50 (e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, and 50) nucleotides.
  • the first spacer element and the second spacer element are the same number of ribonucleotides in length.
  • the first spacer or the second spacer element consists of: (i) a polyA region comprising 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine residues; (ii) a polyAC region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine or cytosine residues; (iii) a polyAU region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
  • the circular polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without the first spacer element or the second spacer element. In some embodiments, the circular polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without the first spacer element or the second spacer element after the circular polyribonucleotide is administered to a subject.
  • the polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 4-fold greater expression in comparison to a polyribonucleotide without the first spacer element or the second spacer element.
  • the polyribonucleotide cargo encoding an immunogen comprises an expression sequence. In some embodiments, the polyribonucleotide cargo encoding an immunogen comprises an IRES operably linked to an expression sequence. In some embodiments, the expression sequence further comprises a 3’ untranslated region or a 5’ untranslated region. In some embodiments, the expression sequence encodes an immunogen.
  • the polyribonucleotide comprises between 500 and 20,000 (e.g., between 500 to 15,000, 500 to 10,000, 500 to 5,000, 500 to 1 ,000, 1 ,000 to 20,000, 5,000 to 20,000, 10,000 to 20,000, and 15,000 to 20,000) ribonucleotides. In some embodiments, the polyribonucleotide comprises between 2,000 and 20,000 (e.g., 2,000 to 5,000, 2,000 to 7,500, 2,000 to 10,000, 2,000 to 12,500, 2,000 to 15,000, 2,000 to 17,500, 5,000 to 20,000, 7,500 to 20,000, 10,000 to 20,000, 12,500 to 20,000, 15,000 to 20,000, and 17,500 to 20,000) ribonucleotides.
  • the circularization junction comprises a splice junction.
  • the first post-circularization element comprises a first exon fragment and the second post-circularization element comprises a second exon fragment, and wherein the first exon fragment and the second exon fragment are joined by the splice junction.
  • the circularization junction comprises an oligonucleotide splint that is hybridized to the first postcircularization element and to the second post-circularization element.
  • the oligonucleotide splint is a DNA splint or an RNA splint.
  • the first postcircularization element comprises a region that is capable of hybridizing to a first region of the oligonucleotide splint and the second post-circularization element comprises a region that is capable of hybridizing to a second region of the oligonucleotide splint.
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) one or more expression augmenting element; (c) a polyribonucleotide cargo encoding an immunogen; and (d) a second postcircularization element; wherein the first post-circularization element and the second postcircularization element together form a circularization junction.
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a polyribonucleotide cargo encoding an immunogen; (c) one or more expression augmenting element; and (d) a second post-circularization element; wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the one or more expression augmenting element comprises a fusion of a translation enhancer and a spacer element comprising a polyA region.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the one or more expression augmenting element comprises a translation enhancer.
  • the translation enhancer is from a plant virus.
  • the translation enhancer is from a mammalian virus.
  • the translation enhancer is a human translation enhancer.
  • the translation enhancer is selected from the group comprising a BTE, a TED, a PTE, an ISS, a YSS, a TSS, dumbbell shaped structure, viral RNA UTRs (including Dengue, West Nile, Zika, Rotavirus), EMCV, CVB3, hepatitis B virus posttranscriptional regulatory element, human genomic fragments, a histone mRNA sequence, a cyclin D mRNA sequence, or an elF4g aptamer sequence.
  • the translation enhancer binds to elF4g.
  • the translation enhancer comprises between 50 and 2000 (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500) ribonucleotides.
  • the translation enhancer comprises between 60 and 800 (e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500) ribonucleotides.
  • 60 and 800 e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500
  • the linear polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without a translation enhancer. In some embodiments, the linear polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without a translation enhancer after the linear polyribonucleotide is administered to a subject. In some embodiments, the linear polyribonucleotide is polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 4-fold greater expression in comparison to a polyribonucleotide without a translation enhancer.
  • the UTR is from a gene encoding TRAM1 , a TMED2, a VAMP3, a CRIP, an AP2A2, a proteasome 26S subunit, a PSMD5, a GPX4, or a PRKAB1 .
  • the UTR is a UTR from a human beta actin, DDB2, TP53I3, FcIgG, LSP1 , AES, DRB4, or a mitochondrially encoded 12S rRNA.
  • the stability element is a 3’ UTR. In some embodiments, the stability element is a 5’ UTR.
  • the stability element comprises between 50 and 2000 (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500) ribonucleotides.
  • 50 and 2000 e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500
  • the stability element comprises between 60 and 800 (e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500) ribonucleotides.
  • 60 and 800 e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500
  • the linear polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without a stability element. In some embodiments, the linear polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without a stability element after the linear polyribonucleotide is administered to a subject. In some embodiments, the polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 2-fold greater expression in comparison to a polyribonucleotide without a stability element.
  • the linear polyribonucleotide further comprises one or more spacer elements comprising a polyA region.
  • the spacer element has a length of between 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the spacer element has a length of between 110 to 500 (e.g., 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the spacer element has a length of between 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the spacer element has a length of between 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 110 to 300, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300) ribonucleotides.
  • 100 to 300 e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 110 to 300, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300
  • 100 to 300 e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first expression augmenting element; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second expression augmenting element; and (e) a second post-circularization element; wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the first expression augmenting element comprises a fusion of a translation enhancer and a spacer element.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the second expression augmenting element comprises a fusion of a translation enhancer and a spacer element.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the first expression augmenting element comprises a translation enhancer.
  • the second expression augmenting element comprises a translation enhancer.
  • the translation enhancer is from a plant virus. In some embodiments, the translation enhancer is from a mammalian virus. In some embodiments, the translation enhancer is a human translation enhancer.
  • the translation enhancer is selected from the group comprising a BTE, a TED, a PTE, an ISS, a YSS, a TSS, dumbbell shaped structure, viral RNA UTRs (including Dengue, West Nile, Zika, Rotavirus), EMCV, CVB3, hepatitis B virus posttranscriptional regulatory element, human genomic fragments, a histone mRNA sequence, a cyclin D mRNA sequence, or an elF4g aptamer sequence.
  • the translation enhancer binds to elF4g.
  • the translation enhancer comprises between 50 and 2000 (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500) ribonucleotides.
  • the translation enhancer comprises between 60 and 800 (e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500) ribonucleotides.
  • the linear polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without a translation enhancer.
  • the linear polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without a translation enhancer after the linear polyribonucleotide is administered to a subject.
  • the linear polyribonucleotide is polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 4-fold greater expression in comparison to a polyribonucleotide without a translation enhancer.
  • the first expression augmenting element comprises a fusion of a stability element and a spacer element comprising a polyA region.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the first expression augmenting element comprises a stability element.
  • the second expression augmenting element comprises a fusion of a stability element and a spacer element.
  • the spacer element comprises from 5 to 200 ribonucleotides in length (e.g., from 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150 ribonucleotides in length).
  • the second expression augmenting element comprises a stability element.
  • the stability element is a UTR.
  • the UTR is from a gene encoding a TRAM1 , a TMED2, a VAMP3, a CRIP, an AP2A2, a proteasome 26S subunit, a PSMD5, a GPX4, or a PRKAB1 .
  • the UTR is a UTR from a human beta actin, DDB2, TP53I3, FcIgG, LSP1 , AES, DRB4, or a mitochondrially encoded 12S rRNA.
  • the stability element is a 3’ UTR. In some embodiments, the stability element is a 5’ UTR.
  • the stability element comprises between 50 and 2000 (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500) ribonucleotides.
  • 50 and 2000 e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500
  • the stability element comprises between 60 and 800 (e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500) ribonucleotides.
  • 60 and 800 e.g., 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 400, 60 to 300, 60 to 300, 60 to 200, 60 to 100, 100 to 800, 200 to 800, 300 to 800, 400 to 800, 500 to 800, 600 to 800, 700 to 800, and 200 to 500
  • the linear polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without a stability element. In some embodiments, the linear polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without a stability element after the linear polyribonucleotide is administered to a subject. In some embodiments, the polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 2-fold greater expression in comparison to a polyribonucleotide without a stability element.
  • the linear polyribonucleotide further comprises one or more spacer elements comprising a polyA region.
  • the spacer element has a length of between 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the spacer element has a length of between 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • 120 to 500 e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500
  • the spacer element has a length of between 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300) ribonucleotides.
  • 100 to 300 e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300
  • 100 to 300 e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a spacer element; (c) a polyribonucleotide cargo encoding an immunogen; (d) an expression augmenting element and (e) a second post-circularization element; and wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) an expression augmenting element; (c) a polyribonucleotide cargo encoding an immunogen; (d) a spacer element; and (e) a second post-circularization element, and wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first expression augmenting element; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second expression augmenting element; and (e) a second post-circularization element, and wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first spacer element having a length of at least 100 ribonucleotides; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second spacer element; and (e) a second post-circularization element; and wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first spacer element having a length of at least 100 ribonucleotides; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second spacer element and (e) a second post-circularization element; and wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the disclosure provides a linear polyribonucleotide comprising, in the following order from 5’ to 3’: (a) a first post-circularization element; (b) a first spacer element; (c) a polyribonucleotide cargo encoding an immunogen; (d) a second spacer element having a length of at least 100 ribonucleotides and (e) a second post-circularization element; and wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the first spacer element has a length of between 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the first spacer element has a length of between 100 and 300 (e.g., 100 to 250, 100 to 200, 100 to 150, 110 to 300, 120 to 300, 150 to 300, 200 to 300, and 250 to 300).
  • the second spacer element has a length of between 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • 100 to 500 e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500
  • the first spacer element and the second spacer element each comprise 110 to 500 (e.g., 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the first spacer element and the second spacer element each comprise 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides.
  • the first spacer element and the second spacer element each have a length of 150 to 500 (e.g., 150 to 450, 150 to 400, 150 to 350, 150 and 300, 150 and 250, 150 and 200, 200 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, or 450 and 500) ribonucleotides.
  • the first spacer element and the second spacer element each have a length of 200 to 500 (e.g., 200 to 450, 200 to 400, 200 to 350, 200 to 300, 200 to 250, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500) ribonucleotides.
  • the first spacer element and the second spacer element each have a length of 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to
  • the first spacer element and the second spacer element each have a length of 100 to 200 (e.g., 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to
  • the first spacer element and the second spacer element each have a length of 110 to 300 (e.g., 110 to 280, 110 to 260, 110 to 240, 110 to 220, 110 to 200, 110 to 180, 110 to 160, 110 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300) ribonucleotides.
  • 110 to 300 e.g., 110 to 280, 110 to 260, 110 to 240, 110 to 220, 110 to 200, 110 to 180, 110 to 160, 110 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300
  • the first spacer element and the second spacer element each have a length of 120 to 300 (e.g., 120 to 280, 120 to 260, 120 to 240, 120 to 220, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300) ribonucleotides.
  • 120 to 300 e.g., 120 to 280, 120 to 260, 120 to 240, 120 to 220, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300
  • the first spacer element and the second spacer element each have a length of 150 to 300 (e.g., 150 to 290, 150 to 280, 150 to 270, 150 to 260, 150 to 250, 150 to 240, 150 to 230, 150 to 220, 150 to 210, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 150 to 160, 160 to 300, 170 to 300, 180 to 300, 190 to 300, 200 to 300, 210 to 300, 220 to 300, 230 to 300, 240 to 300, 250 to 300, 260 to 300, 270 to 300, 280 to 300, and 290 to 300) ribonucleotides.
  • 150 to 300 e.g., 150 to 290, 150 to 280, 150 to 270, 150 to 260, 150 to 250, 150 to 240, 150 to 230, 150 to 220, 150 to 210, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 150 to 160, 160 to 300, 170 to 300,
  • the difference between the length of the first spacer element and the length of the second spacer element is 0 to 100 (e.g., 0 and 10, 0 and 20, 0 and 30, 0, and 40, 0 and 50, 0 to 60, 0 to 70, 0 to 80, 0 to 90, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 5 to 10, 5 to 20, 10 to 30 or 5 to 50) nucleotides.
  • 0 to 100 e.g., 0 and 10, 0 and 20, 0 and 30, 0, and 40, 0 and 50, 0 to 60, 0 to 70, 0 to 80, 0 to 90, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 5 to 10, 5 to 20, 10 to 30 or 5 to 50
  • the difference between the length of the first spacer element and the length of the second spacer element is 0 to 50 (e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, and 50) nucleotides.
  • the first spacer element and the second spacer element are the same number of ribonucleotides in length.
  • the first spacer or the second spacer element consists of: (i) a polyA region comprising 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine residues; (ii) a polyAC region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine or cytosine residues; (iii) a polyAU region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
  • the polyribonucleotide exhibits at least 2-fold greater stability in comparison to a polyribonucleotide without the first spacer element or the second spacer element. In some embodiments, the polyribonucleotide may be detected for at least 2-fold longer in comparison to a polyribonucleotide without the first spacer element or the second spacer element after the polyribonucleotide is administered to a subject. In some embodiments, the polynucleotide cargo encodes an immunogen, wherein the immunogen has at least 4-fold greater expression in comparison to the polyribonucleotide without the first spacer element or the second spacer element.
  • the polyribonucleotide cargo encoding an immunogen comprises an expression sequence. In some embodiments, the polyribonucleotide cargo encoding an immunogen comprises an IRES operably linked to an expression sequence. In some embodiments, the expression sequence further comprises a 3’ untranslated region or a 5’ untranslated region. In some embodiments, the expression sequence encodes an immunogen.
  • the polyribonucleotide comprises between 500 and 20,000 (e.g., between 500 to 15,000, 500 to 10,000, 500 to 5,000, 500 to 1 ,000, 1 ,000 to 20,000, 5,000 to 20,000, 10,000 to 20,000, and 15,000 to 20,000) ribonucleotides. In some embodiments, the polyribonucleotide comprises between 2,000 and 20,000 (e.g., 2,000 to 5,000, 2,000 to 7,500, 2,000 to 10,000, 2,000 to 12,500, 2,000 to 15,000, 2,000 to 17,500, 5,000 to 20,000, 7,500 to 20,000, 10,000 to 20,000, 12,500 to 20,000, 15,000 to 20,000, and 17,500 to 20,000) ribonucleotides.
  • the circularization junction comprises a splice junction.
  • the first post-circularization element comprises a first exon fragment and the second post-circularization element comprises a second exon fragment, and wherein the first exon fragment and the second exon fragment are joined by the splice junction.
  • the circularization junction comprises an oligonucleotide splint that is hybridized to the first postcircularization element and to the second post-circularization element.
  • the oligonucleotide splint is a DNA splint or an RNA splint.
  • the first postcircularization element comprises a region that is capable of hybridizing to a first region of the oligonucleotide splint and the second post-circularization element comprises a region that is capable of hybridizing to a second region of the oligonucleotide splint.
  • the first circularization element comprises a first catalytic intron fragment, a first splice site dinucleotide, and a first exon fragment; and the second circularization element comprises a second catalytic intron fragment, a second splice site dinucleotide, and a second exon fragment.
  • the first catalytic intron fragment and the second catalytic intron fragment are capable of selfsplicing thereby covalently joining the first exon region and the second exon region to produce a circular polyribonucleotide.
  • the first catalytic intron fragment and the second catalytic intron fragment are from a cyanobacterium Anabaena pre-tRNA-Leu gene or a Tetrahymena pre-rRNA.
  • the first circularization element comprises a region that is capable of hybridizing to a first region of an oligonucleotide splint and the second circularization element comprises a region that is capable of hybridizing to a second region of the oligonucleotide splint.
  • the disclosure provides a DNA vector comprising an RNA polymerase promoter operably linked to a sequence that encodes the any one of the linear polyribonucleotides described herein.
  • the disclosure provides a circular polyribonucleotide produced from any one of the linear polyribonucleotides or from the DNA vector described herein.
  • the disclosure provides a pharmaceutical composition comprising any one of the linear polyribonucleotides, circular polyribonucleotides, or the DNA vector described herein and a pharmaceutically acceptable excipient.
  • the disclosure provides a method of expressing an immunogen in a cell or a subject, the method comprising providing to the cell or the subject any one of the linear polyribonucleotides, the circular polyribonucleotides, the DNA vector, or the pharmaceutical composition described herein.
  • the disclosure provides a method of producing a circular polyribonucleotide from any one of the linear polyribonucleotides described herein, the method comprising providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to produce a circular polyribonucleotide.
  • the term “about” refers to a value that is within ⁇ 10% of a recited value.
  • adaptive immune response means either a humoral or cell- mediated immune response.
  • a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells.
  • adjuvant refers to a composition (e.g., a compound, polypeptide, nucleic acid, or lipid) that increases an immune response, for example, increases a specific immune response against an immunogen.
  • Increasing an immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses.
  • any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.
  • the term “between” refers to all values that are greater than or equal to the initial value and less than or equal to the endpoint, such that the range of values between two values includes the endpoints of the range.
  • between 1 and 5 refers to all values >1 and ⁇ 5 such that the endpoints of 1 and 5 are included in the contemplated range.
  • carrier is a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., a circular polyribonucleotide) into a cell by a covalent modification of the circular polyribonucleotide, via a partially or completely encapsulating agent, or a combination thereof.
  • a composition e.g., a circular polyribonucleotide
  • Non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride-modified phytoglycogen or glycogen-type material), nanoparticles (e.g., a nanoparticle that encapsulates or is covalently linked binds to the circular polyribonucleotide), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., a protein covalently linked to the circular polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or transfection reagent).
  • carbohydrate carriers e.g., an anhydride-modified phytoglycogen or glycogen-type material
  • nanoparticles e.g., a nanoparticle that encapsulates or is covalently linked binds to the circular polyribonucleotide
  • liposomes e.g., fusosomes, ex vivo
  • RNA circular polyribonucleotide
  • RNA circular RNA
  • molecule a polyribonucleotide molecule that has a structure having no free ends (i.e. , no free 3’ and/or 5’ ends), for example a polyribonucleotide molecule that forms a circular or end-less structure through covalent (e.g., covalently closed) or non-covalent bonds.
  • the circular polyribonucleotide may be e.g., a covalently closed polyribonucleotide.
  • circularization efficiency is a measurement of resultant circular polyribonucleotide versus its non-circular starting material.
  • the terms “disease,” “disorder,” and “condition” each refer to a state of sub- optimal health, for example, a state that is or would typically be diagnosed or treated by a medical professional.
  • epitope refers to a portion or the whole of an immunogen that is recognized, targeted, or bound by an antibody or T cell receptor.
  • An epitope can be a linear epitope, for example, a contiguous sequence of nucleic acids or amino acids.
  • An epitope can be a conformational epitope, for example, an epitope that contains amino acids that form an epitope in the folded conformation of the protein.
  • a conformational epitope can contain non-contiguous amino acids from a primary amino acid sequence.
  • a conformational epitope includes nucleic acids that form an epitope in the folded conformation of an immunogenic sequence based on its secondary structure or tertiary structure.
  • the term “expression augmenting element” refers to a component of a polyribonucleotide construct which increases the expression of an immunogen cargo encoded by the polyribonucleotide in comparison to a polyribonucleotide construct lacking the expression element.
  • the expression augmenting element is a translation enhancer or a stability element.
  • the expression augmenting element increases expression of the immunogen cargo by increasing the length of time circRNA persists before it is degraded, or it increases the concentration of the immunogen cargo that is expressed over a fixed time period.
  • expression sequence is a nucleic acid sequence that encodes a product, e.g., an immunogen.
  • An exemplary expression sequence that codes for an immunogen may include a plurality of nucleotide triads, each of which can code for an amino acid and is termed as a “codon.”
  • fragment refers to a continuous, less than a whole portion of a sequence of the immunogen or the nucleic acid.
  • a fragment of an immunogen for instance, refers to continuous, less than a whole fraction (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the entire length) of the sequence such as a sequence disclosed herein. It is understood that all the present disclosure contemplates fragments of all polypeptides disclosed herein.
  • the term “fusion” refers a single, contiguous molecule containing two or more different elements.
  • the fusion elements described herein may include a translation enhancer (e.g., joined or connected) fused to a stability element, a translation enhancer fused (e.g., joined or connected) to a spacer element, or a stability element fused (e.g., joined or connected) to a spacer element. Fusion elements may have functional properties derived from each of the original elements.
  • a translation enhancer is connected at its 5’ end to a spacer element.
  • a translation enhancer is connected at its 3’ end to a spacer element.
  • a stability element is connected at its 5’ end to a spacer element. In some embodiments, a stability element is connected at its 3’ end to a spacer element. In some embodiments, a translation enhancer is connected at its 5’ end to a stability element. In some embodiments, a translation enhancer is connected at its 3’ end to a translation stability.
  • GC content refers to the percentage of guanine (G) and cytosine (C) in a nucleic acid sequence.
  • the formula for calculation of the GC content is (G+C) I (A+G+C+U) x 100% (for RNA) or (G+C) I (A+G+C+T) x 100% (for DNA).
  • uridine content refers to the percentage of uridine (U) in a nucleic acid sequence.
  • the formula for calculation of the uridine content is U / (A+G+C+U) x 100%.
  • thymidine content refers to the percentage of thymidine (T) in a nucleic acid sequence.
  • heterologous is meant to occur in a context other than in the naturally occurring (native) context.
  • a “heterologous” polynucleotide sequence indicates that the polynucleotide sequence is being used in a way other than what is found in that sequence’s native genome.
  • a “heterologous promoter” is used to drive transcription of a sequence that is not one that is natively transcribed by that promoter; thus, a “heterologous promoter” sequence is often included in an expression construct by means of recombinant nucleic acid techniques.
  • heterologous is also used to refer to a given sequence that is placed in a non-naturally occurring relationship to another sequence; for example, a heterologous coding or non-coding nucleotide sequence is commonly inserted into a genome by genomic transformation techniques, resulting in a genetically modified or recombinant genome.
  • an impurity is an undesired substance present in a composition, e.g., a pharmaceutical composition as described herein.
  • an impurity is a process- related impurity.
  • an impurity is a product-related substance other than the desired product in the final composition, e.g., other than the active drug ingredient, e.g., circular polyribonucleotide, as described herein.
  • process-related impurity is a substance used, present, or generated in the manufacturing of a composition, preparation, or product that is undesired in the final composition, preparation, or product other than the linear polyribonucleotides described herein.
  • the process-related impurity is an enzyme used in the synthesis or circularization of polyribonucleotides.
  • product-related substance is a substance or byproduct produced during the synthesis of a composition, preparation, or product, or any intermediate thereof.
  • the product- related substance is deoxyribonucleotide fragments.
  • the product-related substance is deoxyribonucleotide monomers.
  • the product-related substance is one or more of: derivatives or fragments of polyribonucleotides described herein, e.g., fragments of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acids, monoribonucleic acids, diribonucleic acids, or triribonucleic acids.
  • innate immune system stimulator refers to a substance that induces an innate immunological response, in part, by inducing expression of one or more genes involved in innate immunity, including, but not limited to, a type I interferon (e.g., IFNa, INFp, and/or IFNy), a pro-inflammatory cytokine (e.g., IL-1 , IL-12, IL-18, TNF-a, and/or GM-CSF), retinoic-acid inducible gene-l (RIG-I, also known as DDX58), melanoma-differentiation-associated gene 5 (MDA5, also known as IFIH1 ), 2’-5’ oligoadenylate synthase 1 (OAS 1 ), OAS-like protein (OASL), and/or protein kinase R (PKR).
  • a type I interferon e.g., IFNa, INFp, and/or IFNy
  • An innate immune system stimulator function as an adjuvant, (e.g., when administered in combination with or formulated with a ribonucleotide that encodes an immunogen).
  • An innate immune system stimulator may be a separate molecule entity (e.g., not encoded by or incorporated as a sequence in a polyribonucleotide), for example, STING (e.g., caSTING), TLR3, TLR4, TLR9, TLR7, TLR8, TLR7, RIG-I/DDX58, and MDA-5/IFIH1 or a constitutively active mutant thereof.
  • An innate immune system stimulator may be encoded by (e.g., expressed from) a polyribonucleotide.
  • a polyribonucleotide may alternately or further include a ribonucleotide sequence that acts as an innate immune system stimulator (e.g., GU-rich motif, an AU-rich motif, a structured region including dsRNA, or an aptamer).
  • an innate immune system stimulator e.g., GU-rich motif, an AU-rich motif, a structured region including dsRNA, or an aptamer.
  • an immunogen refers to any molecule or molecular structure that includes one or more epitopes recognized, targeted, or bound by an antibody or a T cell receptor.
  • an immunogen induces an immune response in a subject (e.g., is immunogenic as defined herein).
  • An immunogen is capable of inducing an immune response in a subject, wherein the immune response refers to a series of molecular, cellular, and organismal events that are induced when an immunogen is encountered by the immune system.
  • the immune response may be humoral and/or cellular immune response. These may include the production of antibodies and the expansion of B- and T-cells.
  • the immunized subject can be monitored for the appearance of immune reactants directed at the specific immunogen. Immune responses to most immunogens induce the production of both specific antibodies and specific effector T cells.
  • the immunogen is foreign to a host. In some embodiments, the immunogen is not foreign to a host.
  • An immunogen may include all or a portion of a polypeptide, a polysaccharide, a polynucleotide, or a lipid.
  • An immunogen may also be a mixed polypeptide, polysaccharide, polynucleotide, and/or lipid.
  • an immunogen may be a polypeptide that has been translationally modified.
  • polypeptide immunogen refers to an immunogen that includes a polypeptide.
  • a polypeptide immunogen may also include one or more post-translational modifications, and/or may form a complex with one or more additional molecules, and/or may adopt a tertiary or quaternary structure, each of which may determine or affect the immunogenicity of the polypeptide.
  • the term “immunogenic” refers to a potential to induce a response to a substance in a particular immune response assay above a pre-determined threshold.
  • the assay can be, e.g., expression of certain inflammatory markers, production of antibodies, or an assay for immunogenicity as described herein.
  • an immune response may be induced when an immune system of an organism or a certain type of immune cells are exposed to an immunogen.
  • An immunogenic response may be assessed may evaluating the antibodies in the plasma or serum of a subject using a total antibody assay, a confirmatory test, titration and isotyping of the antibodies, and neutralizing antibody assessment.
  • a total antibody assay measures all the antibodies generated as part of the immune response in the serum or plasma of a subject that has been administered the immunogen.
  • the most commonly used test to detect antibodies is an ELISA (enzyme-linked immunosorbent assay), which detects antibodies in the tested serum that bind to the antibody of interest, including IgM, IgD, IgG, IgA, and IgE.
  • An immunogenic response can be further assessed by a confirmatory assay.
  • a confirmatory assay may be used to confirm the results of the total antibody assay.
  • a competition assay may be used to confirm that antibody is specifically binding to target and that the positive finding in the screening assay is not a result of non-specific interactions of the test serum or detection reagent with other materials in the assay.
  • An immunogenic response can be assessed by isotyping and titration.
  • An isotyping assay may be used to assess only the relevant antibody isotypes.
  • the expected isotypes may be IgM and IgG which may be specifically detected and quantified by isotyping and titration, and then compared to the total antibodies present.
  • An immunogenic response can be assessed by a neutralizing antibody assay (nAb).
  • a neutralizing antibody assay (nAb) may be used to determine if the antibodies produced in response to the immunogen neutralized the immunogen thereby inhibiting the immunogen from having an effect on the target and leading to abnormal pharmacokinetic behaviors.
  • An nAb assay is often a cell-based assay where the target cells are incubated with the antibody.
  • a variety of cell based nAb assays may be used including but not limited to Cell Proliferation, Viability, Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Cytopathic Effect Inhibition (CPE), Apoptosis, Ligand Stimulated Cell Signaling, Enzyme Activity, Reporter Gene Assays, Protein Secretion, Metabolic Activity, Stress and Mitochondrial Function.
  • Detection readouts include Absorbance, Fluorescence, Luminescence, Chemiluminescence, or Flow Cytometry.
  • a ligandbinding assay may also be used to measure the binding affinity of an immunogen and an antibody in vitro to evaluate neutralization efficacy.
  • induction of a cellular immune response may be assessed by measuring T cell activation in a subject using cellular markers on T cells obtained from the subject.
  • a blood sample, lymph node biopsy, or tissue sample can be collected from a subject and then T cells from this sample can be evaluated for one or more (e.g., 2, 3, 4 or more) activation markers: CD25, CD71 , CD26, CD27, CD28, CD30, CD154, CD40L, CD134, CD69, CD62L or CD44.
  • T cell activation can also be assessed using the same methods in an in vivo animal model.
  • This assay can also be performed by adding an immunogen to T cells in vitro (e.g., T cells obtained from a subject, animal model, repository, or commercial source) and measuring the aforementioned markers to evaluate T cell activation. Similar approaches can be used to assess the effect of and on activation of other immune cells, such as eosinophils (markers: CD35, CD11 b, CD66, CD69 and CD81 ), dendritic cells (makers: IL-8, MHC class II, CD40, CD80, CD83, and CD86), basophils (CD63, CD13, CD4, and CD203c), and neutrophils (CD11 b, CD35, CD66b and CD63). These markers can be assessed using flow cytometry, immunohistochemistry, in situ hybridization, and other assays that allow for measurement of cellular markers. Comparing results from before and after administration of an immunogen can be used to determine its effect.
  • T cells in vitro e.g., T cells obtained from a subject, animal model, repository, or
  • inducing an immune response refers to initiating, amplifying, or sustaining an immune response by a subject. Inducing an immune response may refer to an adaptive immune response or an innate immune response. The induction of an immune response may be measured as discussed above.
  • linear RNA As used herein, the terms “linear RNA,” “linear polyribonucleotide,” and “linear polyribonucleotide molecule” are used interchangeably and mean a monoribonucleotide molecule or polyribonucleotide molecule having a 5’ and 3’ end. One or both of the 5’ and 3’ ends may be free ends or joined to another moiety.
  • the linear RNA has a 5’ end or 3’ end that is modified or protected from degradation (e.g., by a 5’ end protectant or a 3’ end protectant).
  • the linear RNA has non-covalently linked 5’ or 3’ ends.
  • Linear RNA includes RNA that has not undergone circularization (e.g., is pre-circularized) and can be used as a starting material for circularization.
  • linear counterpart is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence similarity) as a circular polyribonucleotide and having two free ends (i.e., the uncircularized version (and its fragments) of the circularized polyribonucleotide).
  • the linear counterpart e.g., a pre-circularized version
  • the linear counterpart is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence similarity) and same or similar nucleic acid modifications as a circular polyribonucleotide and having two free ends (i.e., the uncircularized version (and its fragments) of the circularized polyribonucleotide).
  • the linear counterpart is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence similarity) and different or no nucleic acid modifications as a circular polyribonucleotide and having two free ends (i.e., the uncircularized version (and its fragments) of the circularized polyribonucleotide).
  • a fragment of the polyribonucleotide molecule that is the linear counterpart is any portion of linear counterpart polyribonucleotide molecule that is shorter than the linear counterpart polyribonucleotide molecule.
  • the linear counterpart further comprises a 5’ cap. In some embodiments, the linear counterpart further comprises a poly adenosine tail. In some embodiments, the linear counterpart further comprises a 3’ UTR. In some embodiments, the linear counterpart further comprises a 5’ UTR.
  • modified ribonucleotide is a nucleotide with at least one modification to the sugar, the nucleobase, or the internucleoside linkage.
  • composition is intended to also disclose that the circular polyribonucleotide included within a pharmaceutical composition can be used for the treatment of the human or animal body (e.g., veterinary use) by therapy. It is thus meant to be equivalent to “a circular polyribonucleotide for use in therapy”.
  • polyA based spacer element refers to a spacer element comprising an untranslated, contiguous region of nucleic acid molecules of at least 4 nucleotides in length and consisting of one or more individual adenine (A) residues in combination with one or more (A), thymine (T), cytosine (C), guanine (G), or uracil (U) residues.
  • the polyA based spacer element is a polyA region, which may be sequence of adenine residues.
  • the polyA based spacer element is a polyAT region, which is a combination of adenine and thymine residues.
  • the polyA based spacer element is a poly AU region, which may be a combination of adenine and uracil residues. In some embodiments, the polyA based spacer element is a polyAG region, which is a combination of adenine and guanine residues.
  • a polyA based spacer element comprises between 50% to 100% (e.g., between 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 70% to 90%, or 60% to 80%) adenine residues.
  • polynucleotide as used herein means a molecule including one or more nucleic acid subunits, or nucleotides, and can be used interchangeably with “nucleic acid” or “oligonucleotide.”
  • a polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof.
  • a nucleotide can include a nucleoside and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups.
  • a nucleotide can include a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups.
  • Ribonucleotides are nucleotides in which the sugar is ribose.
  • Polyribonucleotides or ribonucleic acids, or RNA can refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds.
  • Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.
  • a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof.
  • a polynucleotide is a short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc.
  • a polynucleotide molecule is circular.
  • a polynucleotide can have various lengths.
  • a nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 50 kb, or more.
  • a polynucleotide can be isolated from a cell or a tissue. As embodied herein, the polynucleotide sequences may include isolated and purified DNA/RNA molecules, synthetic DNA/RNA molecules, and synthetic DNA/RNA analogs.
  • Polydeoxyribonucleotides mean macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds.
  • a nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate.
  • a nucleotide means a deoxyribonucleoside polyphosphate, such as, e.g., a deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, which include detectable tags, such as luminescent tags or markers (e.g., fluorophores).
  • dNTP deoxyribonucleoside polyphosphate
  • dNTP deoxyribonucleoside triphosphate
  • dNTP deoxyribonucleoside triphosphate
  • dNTP deoxyribonucleoside triphosphate
  • dNTP deoxyribonucleoside triphosphate
  • dNTP deoxyribonucleoside triphosphat
  • Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e. , A or G, or variant thereof) or a pyrimidine (i.e., C, T or U, or variant thereof).
  • Polynucleotides e.g., polyribonucleotides or polydeoxyribonucleotides, may include one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s) and/or modified nucleotides.
  • modified nucleotides include, but are not limited to diaminopurine, 5-fl uorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
  • Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.
  • Nucleic acid molecules may also contain amine -modified groups, such as amino ally 1-dUTP (aa-dUTP) and aminohexhylacrylamide- dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N-hydroxy succinimide esters (NHS).
  • Alternatives to standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure can provide higher density in bits per cubic mm, higher safety (resistant to accidental or purposeful synthesis of natural toxins), easier discrimination in photoprogrammed polymerases, or lower secondary structure.
  • Such alternative base pairs compatible with natural and mutant polymerases for de novo and/or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. NAT. CHEM. BIOL. 2012 Jul ;8(7) :612-4, which is herein incorporated by reference for all purposes.
  • polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
  • Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide can be a single molecule or a multi-molecular complex such as a dimer, trimer, or tetramer. They can also comprise single chain or multichain polypeptides such as antibodies or insulin and can be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides.
  • polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • polyribonucleotide cargo herein includes any sequence including at least one polyribonucleotide.
  • the polyribonucleotide cargo includes one or multiple expression sequences, wherein each expression sequence encodes an immunogen.
  • the polyribonucleotide cargo includes one or multiple noncoding sequences, such as a polyribonucleotide having regulatory or catalytic functions.
  • the polyribonucleotide cargo includes a combination of expression and noncoding sequences.
  • the polyribonucleotide cargo includes one or more polyribonucleotide sequence described herein, such as one or multiple regulatory elements, internal ribosomal entry site (IRES) elements, or spacer elements.
  • IRS internal ribosomal entry site
  • the term “prevent” means to reduce the likelihood of developing a disease, disorder, or condition, or alternatively, to reduce the severity or frequency of symptoms in a subsequently developed disease or disorder.
  • the polyribonucleotides described herein can be administered to a subject who is at increased risk of developing a disease or disorder relative to a member of the general population in order to prevent the development of, or lessen the severity of, the disease or condition.
  • the polyribonucleotides described herein can be administered as a prophylactic (e.g., before development of any symptom or manifestation of a disease or disorder).
  • the terms “purify,” “purifying,” and “purification” refer to one or more steps or processes of removing impurities (e.g., a process-related impurity (e.g., an enzyme), a process- related substance (e.g., a deoxyribonucleotide fragment, a deoxyribonucleotide monomer)) or byproducts (e.g., linear RNA) from a sample containing a mixture circular RNA and linear RNA, among other substances, to produce a composition containing an enriched population of circular RNA with a reduced level of an impurity (e.g., a process-related impurity (e.g., an enzyme), a process-related substance (e.g., deoxyribonucleotide fragment, deoxyribonucleotide monomer)) or by-product (e.g., linear RNA) as compared to the original mixture or in which the linear RNA or substances have been reduced by 40% or more by mass
  • pure and “purity” refer to the extent to which an analyte (e.g., circular RNA) has been isolated and is free of other components.
  • purity of an isolated nucleic acid e.g., circular RNA
  • purity of a population of circular RNA indicates how much of the population is circular RNA by total mass of the isolated material, which may be determined using, e.g., pure circular RNA as a reference.
  • a level of purity found in the disclosure can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, greater than 95%, or greater than 99% (w/w).
  • the level of contaminants or impurities or by-products is no more than about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (w/w).
  • the phrase “substantially free of one or more impurities or by-products” refers to a property of a sample, such as a sample containing an enriched population of circular RNA, that is free of one or more impurities or by-products (e.g., one or more impurities or by-products disclosed herein) or contains a minimal amount of the one or more impurities or by-products.
  • a minimal amount of the one or more impurities or by-products may be no more than 20% (w/w) (e.g., no more than 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w/w), or less).
  • the sample or the enriched population of circular RNA is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount that is less than 15% (w/w) (e.g., no more than 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w/w), or less).
  • the sample or the enriched population of circular RNA is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount that is less than 10% (w/w) (e.g., no more than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w/w), or less).
  • the sample or the enriched population of circular RNA is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount that is less than 5% (w/w) (e.g., no more than 4%, 3%, 2%, 1% (w/w) or less).
  • the sample or the enriched population of circular RNA is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount that is less than 1% (no more than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% (w/w), or less).
  • regulatory element is a moiety, such as a nucleic acid sequence, that modifies expression of an expression sequence within the circular polyribonucleotide.
  • replication element is a sequence and/or motif useful for replication or that initiates transcription of the circular polyribonucleotide.
  • RNA equivalent refers to an RNA sequence that is the RNA equivalent of a DNA sequence.
  • An RNA equivalent of a DNA sequence therefore refers to a DNA sequence in which each of the thymidine (T) residues is replaced by a uridine (U) residue.
  • T thymidine
  • U uridine
  • the disclosure specifically contemplates that any of these DNA sequences may be converted to the corresponding RNA sequence and included in an RNA molecule described herein.
  • sequence identity is determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm. Sequences may then be referred to as “substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity.
  • the default scoring matrix used is a nwsgapdna.cmp scoring matrix and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).
  • Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121 -3752 USA, or EmbossWin version 2.10.0 (using the program “needle”).
  • percent identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc. Sequence identity refers to the sequence identity over the entire length of the sequence.
  • a “signal sequence” refers to a polypeptide sequence, e.g., between 10 and 45 amino acids in length, that is present at the N-terminus of a polypeptide sequence of a nascent protein which targets the polypeptide sequence to the secretory pathway.
  • spacer element refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacer elements may be present in between any of the nucleic acid elements described herein. Spacer element may also be present within a nucleic acid element described herein.
  • the term “stability element” refers to a polyribonucleotide element which increases the stability of the polyribonucleotide in comparison to a polyribonucleotide lacking the stability element.
  • the stability element may increase stability of the polyribonucleotide such that the polyribonucleotide degrades more slowly.
  • the term "subject" refers to an organism, such as an animal, plant, or microbe.
  • the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian).
  • the subject is a human.
  • the subject is a non-human mammal.
  • the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, bison, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit).
  • a non-human primate e.g., monkeys, apes
  • ungulate e.g., cattle, buffalo, bison, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys
  • carnivore e.g., dog, cat
  • rodent e.g., rat, mouse
  • lagomorph e.g., rabbit
  • the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).
  • avian taxa Galliformes e.g., chickens, turkeys, pheasants, quail
  • Anseriformes e.g., ducks, geese
  • Paleaognathae e.g., ostriches, emus
  • Columbiformes e.g., pigeons, doves
  • Psittaciformes e.g., par
  • the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc.
  • the subject is an invertebrate agricultural pest or an invertebrate that is parasitic on an invertebrate or vertebrate host.
  • the subject is a plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte.
  • the subject is a eukaryotic alga (unicellular or multicellular).
  • the subject is a plant of agricultural or horticultural importance, such as row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses.
  • translation enhancer refers to a polyribonucleotide element, which allows for translation of mRNA via recruitment of a ribosome, translation initiation factor directly or through interacting with RNA-binding protein.
  • the polyribonucleotides including a translation enhancer may, for example, demonstrate increased stability and/or increased expression of a polyribonucleotide cargo encoding an immunogen in comparison to a polyribonucleotide lacking the translation enhancer.
  • terminal element is a moiety, such as a nucleic acid sequence, that terminates translation of the expression sequence in the circular polyribonucleotide.
  • total ribonucleotide molecules means the total amount of any ribonucleotide molecules, including linear polyribonucleotide molecules, circular polyribonucleotide molecules, monomeric ribonucleotides, other polyribonucleotide molecules, fragments thereof, and modified variations thereof, as measured by total mass of the ribonucleotide molecules.
  • translation efficiency is a rate or amount of protein or peptide production from a ribonucleotide transcript.
  • translation efficiency can be expressed as amount of protein or peptide produced per given amount of transcript that codes for the protein or peptide, e.g., in a given period of time, e.g., in a given translation system, e.g., an in vitro translation system like rabbit reticulocyte lysate, or an in vivo translation system like a eukaryotic cell or a prokaryotic cell.
  • translation initiation sequence is a nucleic acid sequence that initiates translation of an expression sequence in the circular polyribonucleotide.
  • the terms “treat,” or “treating,” refers to a treatment of a disease or disorder (e.g., an infectious disease, a cancer, a toxicity, or an allergic reaction) in a subject.
  • the effect of treatment can include reversing, alleviating, reducing severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of the disease or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e. , not worsening) the state of the disease or disorder, and/or preventing the spread of the disease or disorder as compared to the state and/or the condition of the disease or disorder in the absence of the treatment.
  • a “variant” refers to a polypeptide which includes at least one alteration, e.g., a substitution, insertion, deletion, and/or fusion, at one or more residue positions, as compared to the parent or wild-type polypeptide.
  • a variant may include between 1 and 10, 10 and 20, 20 and 50, 50 and 100, or more alterations.
  • FIGS. 1A-1F are schematic diagrams of exemplary linear and circular polyribonucleotide constructs having spacer element(s) and/or expression augmenting element(s).
  • FIG. 2 is a table showing the circularization efficiency (%circRNA) of single spacer designs or dual spacer design constructs having a spacer element(s) between 50 and 300 nucleotides in length, wherein the construct encodes a Glue polypeptide or human erythropoietin.
  • FIG. 3A is a table of translation enhancers, including motifs and sequences.
  • FIG. 3B is a table showing the circularization efficiency (%circRNA) of single design and dual design constructs having a spacer element and/or a translation enhancer and encoding a Glue polypeptide.
  • FIG. 4A is a bar graph showing expression of Glue in HeLa cells transfected with circular RNAs having a spacer element and encoding a Glue polypeptide, 24 and 48 hours after transfection.
  • FIG. 4B is a graph showing expression of EPO in HeLa cells transfected with circular RNAs having a spacer element and encoding a human erythropoietin, 24 and 48 hours after transfection.
  • FIG. 5 is a bar graph showing expression of Glue in HeLa cells transfected with circular RNAs having a single spacer design or a dual spacer design and encoding a Glue polypeptide, 24 hours after transfection.
  • FIG. 6A is a bar graph showing expression of Glue in HeLa cells transfected with circular RNAs having a dual spacer design and encoding a Glue polypeptide, 4, 24, and 48 hours after transfection.
  • FIG. 6B is a bar graph showing expression of EPO in HeLa cells transfected with circular RNAs having a dual spacer design and encoding a human erythropoietin, 4, 24, and 48 hours after transfection.
  • FIG. 7A is a bar graph showing expression of Glue in A549 cells transfected with circular RNAs having a spacer element and a translation enhancer and encoding a Glue polypeptide.
  • FIG. 7B is a bar graph showing expression of EPO in A549 cells transfected with circular RNAs having a spacer element and a translation enhancer and encoding human erythropoietin, 24 hours after transfection.
  • FIG. 8 is a bar graph showing the expression of EPO in HEK cells transfected with a circular polyribonucleotide having a translation enhancer and encoding a human erythropoietin, at Day 1 and Day 2 after transfection.
  • FIG. 9 is a bar graph showing the expression of EPO in HeLa cells transfected with a circular polyribonucleotide having a translation enhancer and encoding a human erythropoietin, at Day 1 and Day 2 after transfection.
  • FIG. 10 is a bar graph showing in vivo expression of EPO encoded by a circular polyribonucleotide including a translation enhancer.
  • FIG. 11 is a graph showing the expression of EPO in A549 cells transfected with circular polyribonucleotides having spacer elements and/or translation enhancers and encoding a human erythropoietin with a CVB3 IRES, 24 hours after transfection.
  • FIG. 12 is a graph showing the expression of EPO in A549 cells transfected with circular polyribonucleotides having spacer elements and/or translation enhancers and encoding a human erythropoietin with an EMCV IRES, 24 hours after transfection.
  • FIG. 13 is a graph showing the expression of EPO in A549 cells transfected with circular polyribonucleotides having multiple spacer elements and multiple translation enhancers and encoding a human erythropoietin and a SARS-CoV-2 RBD polypeptide, 24 hours after transfection of the cells.
  • FIGS. 14A and 14B are graphs showing the time course of expression of EGFPd2 in A549 cells transfected with circular RNAs having a spacer element and a translation enhancer and encoding a EGFPd2 polypeptide.
  • FIG. 14A shows the expression of EGFPd2 over time.
  • FIG. 14B shows the area under the curve from 0 to 24 hours.
  • FIGS. 15A-15C are bar graphs showing expression of EPO in A549 cells transfected with circular RNAs having a spacer element and a translation enhancer and encoding a human erythropoietin, 24 hours after transfection of the cells.
  • FIGS. 16A and 16B are bar graphs showing expression of EPO in A549 cells transfected with circular RNAs having a spacer element and a translation enhancer and encoding a human erythropoietin, 24 hours after transfection.
  • FIGS. 17A-17C are bar graphs showing HiBiT expression in HEK cells transfected with circular RNAs having a spacer element and a translation enhancer and encoding a Factor 9-Albumin- HiBiT polypeptide, 24 hours after transfection.
  • FIGS. 18A and 18B are bar graphs showing the expression of Glue in HeLa cells transfected with different concentrations of circular RNA having a spacer element and a minimized or full length elF4g aptamer translation enhancer and encoding a Glue polypeptide, 48 hours after transfection.
  • FIG. 19 is a bar graph showing CFTR expression in HEK293T cells which were transfected with one of twelve (12) different circular RNAs encoding CFTR.
  • FIG. 20 is a bar graph showing SARS-CoV-2 Spike expression detected on the cell surface of HEK293T cells which were transfected with one of thirteen (13) different circular RNAs encoding SARS-CoV-2 Spike protein.
  • FIG. 21 is a bar graph showing VZV gE expression detected on the cell surface of HEK293T cells which were transfected with one of four different circular RNAs encoding VZV gE.
  • FIG. 22 is a graph showing the relative amount of circular polyribonucleotide present over time for circular polyribonucleotides having a 5’ spacer element and a 3’ RNA stability element and encoding a EGFPd2 polypeptide with a modified CVB3 IRES.
  • FIG. 23A-FIG. 23C are a series of bar graphs showing the concentration of polypeptide Factor 9-HiBiT encoded by circular polyribonucleotides having a 5’ spacer element and a 3’ translation enhancer and encoding a Factor 9-HiBiT, 24 hours post-transfection.
  • FIG. 24A and FIG. 24B are graphs showing the time course of expression data for EGFPd2 encoded by circular polyribonucleotides having spacer elements and/or translation enhancers and encoding EGFPd2 with a modified CVB3 IRES, where FIG. 24A shows EGFPd2 expression over time and FIG. 24B shows the area under the curve from 0 to 24 hours.
  • FIG. 25A and FIG. 25B are graphs showing the time course of expression data for EGFPd2 encoded by a circular polyribonucleotide having a 5’ translation enhancer and a 3’ spacer element and encoding EGFPd2 with a modified CVB3 IRES, where FIG. 25A shows EGFPd2 expression over time and FIG. 25B shows the area under the curve from 0 to 24 hours.
  • FIG. 26A and FIG. 26B are bar graphs showing the concentration of polypeptide B-HiBiT encoded by a circular polyribonucleotide having a 5’ spacer element and a 3’ translation enhancer and encoding polypeptide B-HiBiT with a EV69 IRES (FIG. 26A) or a modified CVB3 IRES (FIG. 26B), 24 hours post-transfection
  • FIG. 27 is a bar graph showing the concentration of polypeptide B-HiBiT encoded by circular polyribonucleotides having multiple translation enhancers or multiple spacer elements.
  • FIG. 28 shows the expression of polypeptide G encoded by circular polyribonucleotides having spacer elements and/or translation enhancers and encoding polypeptide G with a modified CVB3 IRES, 24 hours post-transfection.
  • FIG. 29A and FIG. 29B are bar graphs showing the area under the curve for GFPd2 (FIG. 29A) and concentration of polypeptide B-HiBiT (FIG. 29B) wherein the polypeptide is encoded by a circular polyribonucleotide having a single spacer design or a dual spacer design.
  • FIG. 30A and FIG. 30B are bar graphs showing the area under the curve for GFPd2 encoded by circular polyribonucleotides having a single spacer design or a dual spacer design.
  • FIG. 31 A and FIG. 31 B are bar graphs showing the area under the curve for polypeptide B- HiBiT encoded by circular polyribonucleotides having a single spacer design or a dual spacer design.
  • FIG. 32A-FIG. 32D are bar graphs showing the area under the curve for GFPd2 encoded by circular polyribonucleotides having a spacer element(s) and/or a translation enhancer(s), wherein the translation enhancer is FcIgG (FIG. 32A), TP53I3 (FIG. 32B), LSP1 (FIG. 32C), or Histone4E (FIG. 32D).
  • the translation enhancer is FcIgG (FIG. 32A), TP53I3 (FIG. 32B), LSP1 (FIG. 32C), or Histone4E (FIG. 32D).
  • FIG. 33A-FIG. 33D are bar graphs showing the concentration of polypeptide B-HiBiT encoded by circular polyribonucleotides having a spacer element(s) and/or a translation enhancer(s), wherein the translation enhancer is FcIgG (FIG. 33A), TP53I3 (FIG. 33B), LSP1 (FIG. 33C), or Histone4E (FIG. 33D).
  • FIG. 34A-FIG. 34D are bar graphs showing the area under the curve for GFPd2 encoded by circular polyribonucleotides having a translation enhancer, wherein the translation enhancer is TBR16 (FIG. 34A), 12S (FIG. 34B), TRAM1 - (FIG. 34C), and GPX4 (FIG. 34D).
  • TBR16 FIG. 34A
  • 12S FIG. 34B
  • TRAM1 - FIG. 34C
  • GPX4 FIG. 34D
  • FIG. 35A-FIG. 35D are bar graphs showing the concentration of polypeptide B-HiBiT encoded by circular polyribonucleotides having a translation enhancer, wherein the translation enhancer is TBR16 (FIG. 35A), 12S (FIG. 35B), TRAM1 - (FIG. 35C), and GPX4 (FIG. 35D).
  • compositions including polyribonucleotides having one or more expression augmenting elements or spacer elements.
  • the polyribonucleotides described herein are particularly useful in increasing the stability and/or increasing the expression of a polynucleotide cargo (e.g., encoding an immunogen) encoded by the polyribonucleotide.
  • the disclosure provides polyribonucleotides including a first post-circularization element; a first expression augmenting element having a length of at least 100 ribonucleotides; a polyribonucleotide cargo encoding an immunogen; a second expression augmenting element; and a second post-circularization element; and wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the disclosure also provides polyribonucleotides having a first post-circularization element; a first spacer element having a length of at least 100 ribonucleotides; a polyribonucleotide cargo encoding an immunogen; a second spacer element; and a second post-circularization element; and wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the disclosure also provides polyribonucleotides having a first post-circularization element; a first spacer element; a polyribonucleotide cargo encoding an immunogen; a second spacer element having a length of at least 100 ribonucleotides; and a second post-circularization element; and wherein the first postcircularization element and the second post-circularization element together form a circularization junction.
  • the polyribonucleotide constructs described herein may show increased stability, potentially increasing the length of time the polyribonucleotide may persist before degradation. Additionally, the polyribonucleotide constructs described herein may show increased expression of a polynucleotide cargo encoded by the polyribonucleotide.
  • Each of the DNA sequences described herein include the RNA equivalent sequence, as would be understood by one skilled in the art. Likewise, all RNA sequences described herein include the DNA equivalent sequence, as would be understood by one skilled in the art. The molecules, methods of producing, and uses thereof are described in more detail below.
  • the circular polyribonucleotide may include from 5’ to 3’ a first post-circularization element; one or more expression augmenting element; a polyribonucleotide cargo encoding an immunogen; and a second post-circularization element.
  • the circular polyribonucleotide includes from 5’ to 3’ a first post-circularization element; a polyribonucleotide cargo; one or more expression augmenting element; and a second post-circularization element.
  • the circular polyribonucleotide includes from 5’ to 3’ a first postcircularization element; a first expression augmenting element; a polyribonucleotide cargo; a second expression augmenting element; and a second post-circularization element.
  • the circular polyribonucleotide includes from 5’ to 3’ a first postcircularization element; a spacer element; a polyribonucleotide cargo; an expression augmenting element and a second post-circularization element. In some embodiments, the circular polyribonucleotide includes from 5’ to 3’ a first post-circularization element; a polyribonucleotide cargo encoding an immunogen; one or more expression augmenting element; and a second postcircularization element.
  • the circular polyribonucleotide includes from 5’ to 3’ a first post-circularization element; a first expression augmenting element; a polyribonucleotide cargo encoding an immunogen; a second expression augmenting element; and a second postcircularization element.
  • the circular polyribonucleotide includes from 5’ to 3’ a first post-circularization element; a first spacer element having a length of at least 100 ribonucleotides; a polyribonucleotide cargo encoding an immunogen; a second spacer element; and a second postcircularization element.
  • the circular polyribonucleotides as disclosed herein comprise a first post-circularization element; (b) a first spacer element having at least 100 ribonucleotides (e.g., at least 100, 120, 140, 160, 180, 200, 250, 300, 400, or 500 ribonucleotides).
  • the circular polyribonucleotide disclosed herein also includes a polyribonucleotide cargo encoding an immunogen and a second spacer element.
  • the second spacer element has a length of at least 100 ribonucleotides (e.g., at least 100, 120, 140, 160, 180, 200, 250, 300, 400, or 500 ribonucleotides) ribonucleotides.
  • the second spacer element may have a length of between 120 and 500 (e.g., between 120 and 400, 120 and 300, 120 and 200, 120 and 150, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 400 and 500, or 350 and 500) ribonucleotides.
  • the second spacer element has a length of between 100 and 300 (e.g., between 100 and 280, 100 and 260, 100 and 240, 100 and 220, 100 and 200, 100 and 180, 100 and 160, 100 and 140, 100 and 120, 120 and 300, 140 and 300, 160 and 300, 180 and 300, 200 and 300, 220 and 300, 240 and 300, 260 and 300, or 280 and 300) ribonucleotides.
  • 100 and 300 e.g., between 100 and 280, 100 and 260, 100 and 240, 100 and 220, 100 and 200, 100 and 180, 100 and 160, 100 and 140, 100 and 120, 120 and 300, 140 and 300, 160 and 300, 180 and 300, 200 and 300, 220 and 300, 240 and 300, 260 and 300, or 280 and 300
  • the second spacer element has a length of between 200 and 500 (e.g., between 200 and 450, 200 and 400, 200 and 350, 200 and 300, 200 and 250, 250 and 500, 300 and 500, 350 and 500, 400 and 500, 450 and 500, 300 and 500, or 300 and 400) ribonucleotides.
  • 200 and 500 e.g., between 200 and 450, 200 and 400, 200 and 350, 200 and 300, 200 and 250, 250 and 500, 300 and 500, 350 and 500, 400 and 500, 450 and 500, 300 and 500, or 300 and 400
  • the polyribonucleotides described herein may include one or more expression augmenting elements.
  • the circular or linear polyribonucleotides include between 1 and 5 expression augmenting elements (e.g., 1 , 2, 3, 4, or 5 expression augmenting elements).
  • the circular or linear polyribonucleotides may include a first expression augmenting element and a second expression augmenting element.
  • the one or more expression augmenting elements may include a translation enhancer, a stability element, a translation enhancer fused (e.g., joined, connected) to a spacer element, or a stability element fused (e.g., joined, connected) to a spacer element.
  • circular or linear polyribonucleotides includes a translation enhancer and a stability element.
  • polyribonucleotides described herein may include the combinations of elements as described in Table 1 in 5’ to 3’ order.
  • One or more spacer elements may be included between any one the elements described in Table 1 .
  • the spacer element may be fused to the 5’ end or the 3’ end of the Translation Enhancer or Stability Element
  • the polyribonucleotides described herein may include a first expression augmenting element or a second expression augmenting element.
  • the first expression augmenting element may include a translation enhancer.
  • the second augmenting element may include translation enhancer.
  • a circular or linear polyribonucleotide described herein includes one or more translation enhancers.
  • a translation enhancer may be fused (e.g., joined, connected) to a spacer element. Translation enhancers may be present in between any of the nucleic acid elements described herein. Translation enhancers may also be present within a nucleic acid element described herein.
  • a translation enhancer is a spacer element.
  • a translation enhancer is a stability element.
  • the translation enhancer is from a gene encoding an RNA binding protein. In some embodiments, the translation enhancer comprises nucleic acid sequence comprising a fragment from a gene encoding an RNA binding protein. In some embodiments, the translation enhancer has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% ⁇ 99%, or 100%) sequence identity to a nucleic acid sequence comprising a fragment from a gene encoding an RNA binding protein.
  • the translation enhancer is from a gene encoding a BYDV like-element (BTE), a translation enhancer element (TED), a PMV/PEMV-like translation enhancer (PTE), an l-shaped structure (ISS), a Y-shaped structure (YSS), a t-shaped structure (TSS), dumbbell shaped structure, viral RNA UTRs (including Dengue, West Nile, Zika, Rotavirus), EMCV, CVB3, hepatitis B virus posttranscriptional regulatory element, human genomic fragments, a histone mRNA sequence, a cyclin D mRNA sequence, or an elF4g aptamer sequence.
  • the translation enhancer element is from a plant virus.
  • the translation enhancer comprises nucleic acid sequence comprising a fragment from a plant virus. In some embodiments, the translation enhancer has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% ⁇ 99%, or 100%) sequence identity to a nucleic acid sequence comprising a fragment from a plant virus.
  • the plant virus is a Barley yellow dwarf virus (BYDV) like-element (BTE) translation enhancer.
  • Non-limiting examples of plant viruses with BTEs include BYDV, TNVD, OLV1 , LWSV, SCNMV, CRSV, TBTV, GRV, OMMV, BBSV, RSDaV, and OPMV.
  • BTEs bind to elF4g with high affinity.
  • a circular or linear polyribonucleotide comprises a BTE sequence described in TABLE 2.
  • the BTE comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 2, SEQ ID NOs: 1 -22.
  • the translation enhancer element is from a plant virus containing a TED.
  • plant viruses with TEDs include STNV, PLPV, PCRPV, ELV, RrLDV, PelRSV, and CbMV.
  • TEDs bind to elF4F with high affinity.
  • a circular or linear polyribonucleotide comprises a TED sequence described in TABLE 3.
  • the TED comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 3, SEQ ID NOs: 23-29.
  • the translation enhancer element is from a plant virus containing a Panicum mosaic virus/Pea enation mosaic virus (PMV/PEMV)-like translation enhancer (PTE) translation enhancer.
  • PMV/PEMV Panicum mosaic virus/Pea enation mosaic virus
  • PTE Panicum mosaic virus/Pea enation mosaic virus
  • PTEs bind to elF4E with high affinity.
  • a circular or linear polyribonucleotide comprises a PTE sequence described in TABLE 4.
  • the PTE comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 4, SEQ ID NOs:
  • the translation enhancer element is from a plant virus containing an ISS translation enhancer.
  • plant viruses with ISSs include MNeSV, MNSV264, CBV, MWLMV, JCSMV, and GoMVA.
  • ISSs bind to elF4E bound to elF4G.
  • a circular or linear polyribonucleotide comprises a TSS sequence described in TABLE
  • the TSS comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 5, SEQ ID NOs: 47-53
  • the translation enhancer element is from a plant virus containing a YSS translation enhancer.
  • plant viruses with YSSs include TBSV, CIRV, CymRSV, CNV, AMCV, PNSV, GALV, PLCV, PeLV, and LNV.
  • YSSs bind to elF4F.
  • a circular or linear polyribonucleotide comprises a YSS sequence described in TABLE
  • the YSS comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 6, SEQ ID NOs: 54-63.
  • the translation enhancer element is from a plant virus containing a TSS translation enhancer.
  • plant viruses with TSSs include TYMV, RCNM, TCV, and CCFV.
  • TSSs bind to the 60s ribosomal subunit.
  • a circular or linear polyribonucleotide comprises a TSS sequence described in TABLE 7.
  • the TSS comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 7, SEQ ID NOs: 64-67.
  • the translation enhancer element is from a plant virus containing a dumbbell-shaped translation enhancer.
  • a non-limiting example of a plant virus with a dumbbellshaped translation enhancer includes CABYV-X.
  • a circular or linear polyribonucleotide comprises a dumbbell-shaped translation enhancer sequence described in TABLE 8 SEQ ID NO: 68.
  • the dumbbell-shaped enhancer comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 8, SEQ ID NO: 68.
  • the translation enhancer element is from a gene from a mammal.
  • the translation enhancer comprises nucleic acid sequence comprising a fragment from a mammalian gene.
  • the translation enhancer has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% ⁇ 99%, or 100%) sequence identity to a nucleic acid sequence comprising a fragment from a mammalian gene.
  • the gene from a mammal may be, but is not limited to, histone or cyclin D mRNA sequences that binds to elF4E.
  • the translation enhancer element is from a synthetic sequence, including but not limited to, an elF4G aptamer that binds to elF4G.
  • the translation enhancer element is from a viral sequence.
  • Non-limiting examples of translation enhancers from viral sequences are HCV and DENV.
  • a circular or linear polyribonucleotide comprises a mammalian, synthetic, or viral translation enhancer sequence described in TABLE 8.
  • the mammalian, synthetic, or viral translation enhancer comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 8, SEQ ID NOs: 68-159, and 321 -335.
  • a circular or linear polyribonucleotide comprises a translation enhancer element described in TABLE 2-8.
  • the translation enhancer comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from TABLE 2-8.
  • the translation enhancer includes a nucleic acid sequence having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) sequence identity with the nucleic acid sequence of any one of SEQ ID NOs: 1 -159, 264-267, or 278-335.
  • the translation enhancer includes a nucleic acid sequence having at least 90% (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) identity with the nucleic acid sequence of any one of SEQ ID NOs: 1 -159, 264-267, or 278-335. In some embodiments, the translation enhancer includes a nucleic acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity) identity with the nucleic acid sequence of any one of SEQ ID NOs: 1 -159, 264-267, or 278-335. In some embodiments, the translation enhancer includes a nucleic acid sequence having a nucleic acid sequence of any one of SEQ ID NOs: 1 -159, 264-267, 278-335.
  • the translation enhancer element may be TBR16, 12S, TRAM1 , or GPX4.
  • the translation enhancer element may be a 5’ UTR or a 3’ UTR.
  • the 5’ UTR or 3’ UTR is a human translation enhancer.
  • the 5’ UTR or 3’ UTR comprises any one of the 5’ UTRs or 3’ UTRs described in Table 9, or a portion thereof.
  • the translation enhancer element may have between 20 and 750 ribonucleotides (e.g., between 20 and 700, 20 and 650, 20 and 600, 20 and 550, 20 and 500, 20 and 450, 20 and 400, 20 and 350, 20 and 300, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 50, 50 and 100, 50 and 750, 100 and 750, 150 and 750, 200 and 750, 250 and 750, 300 and 750, 350 and 750, 400 and 750, 450 and 750, 500 and 750, 550 and 750, 600 and 750, 650 and 750, and 700 and 750 ribonucleotides).
  • ribonucleotides e.g., between 20 and 700, 20 and 650, 20 and 600, 20 and 550, 20 and 500, 20 and 450, 20 and 400, 20 and 350, 20 and 300, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 50, 50 and 100, 50 and
  • translation enhancer may have a length of between 100 and 500 ribonucleotides (e.g., between 100 and 450, 100 and 400, 100 and 350, 100 and 300, 100 and 250, 100 and 200, 100 and 150, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, or 450 and 500 ribonucleotides).
  • 100 and 500 ribonucleotides e.g., between 100 and 450, 100 and 400, 100 and 350, 100 and 300, 100 and 250, 100 and 200, 100 and 150, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, or 450 and 500 ribonucleotides.
  • the expression augmenting element may include a translation enhancer fused to a spacer element.
  • the spacer element may be conjugated to the 5’ end of the translation enhancer. In some embodiments, the spacer element may be conjugated to the 3’ end of the translation enhancer.
  • the polyribonucleotides described herein may include a first expression augmenting element wherein the first expression augmenting element may include a stability element.
  • the second expression augmenting element may include a stability element.
  • the second spacer element may include an expression augmenting element.
  • One or more of the expression augmenting elements may be a stability element.
  • the stability element is a translation enhancer.
  • a stability element is a spacer element.
  • the stability element may be a 5’ UTR or a 3’ UTR. In some embodiments, the 5’ UTR or 3’
  • the 5’ UTR or 3’ UTR comprises any one of the 5’ UTRs or 3’ UTRs described in Table 9 below, or a portion thereof.
  • the 5’ UTR or 3’ UTR may be from a gene encoding a TRAM1 , a TMED2, a VAMP3, CRIP, an AP2A2, a PSMD5, a GPX4, or a PRKAB1 .
  • the 5’ UTR or 3’ UTR may be a 5’ UTR or 3’ UTR from a human beta actin, DDB2, TP53I3, FcIgG, LSP1 , AES, DRB4, or a mitochondrially encoded 12S rRNA.
  • the stability element may include between 50 and 2000 ribonucleotides (e.g., between 50 and 1500, 50 and 1000, 50 and 500, 50 and 100, 100 and 2000, 500 and 2000, 1000 and 2000, or 1500 and 2000).
  • the stability element may include between 20 and 750 ribonucleotides (e.g., between 20 and 700, 20 and 650, 20 and 600, 20 and 550, 20 and 500, 20 and 450, 20 and 400, 20 and 350, 20 and 300, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 50, 50 and 100, 50 and 750, 100 and 750, 150 and 750, 200 and 750, 250 and 750, 300 and 750, 350 and 750, 400 and 750, 450 and 750, 500 and 750, 550 and 750, 600 and 750, 650 and 750, and 700 and 750 nucleotides).
  • the stability element may have a length of between 100 and 500 ribonucleotides (e.g., between 100 and 450, 100 and 400, 100 and 350, 100 and 300, 100 and 250, 100 and 200, 100 and 150, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, or 450 and 500 ribonucleotides).
  • 100 and 500 ribonucleotides e.g., between 100 and 450, 100 and 400, 100 and 350, 100 and 300, 100 and 250, 100 and 200, 100 and 150, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, or 450 and 500 ribonucleotides.
  • the expression augmenting element may include a stability element fused to a spacer element.
  • the spacer element may be conjugated to the 5’ end of the stability element.
  • the spacer element not encoding an expression augmenting element may be conjugated to the 3’ end of the stability element.
  • the stability element may be any known stability element.
  • the stability element has a nucleic acid sequence having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences described in TABLE 9.
  • the stability element has a nucleic acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences described in TABLE 9.
  • the stability element has a nucleic acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences described in TABLE 9. In some embodiments, the stability element has a nucleic acid sequence of any one of the sequences described in TABLE 9.
  • a circular or linear polyribonucleotide includes untranslated regions (UTRs).
  • the stability element may include a UTR.
  • the translation enhancer may include a UTR.
  • the UTR is a 3’ UTR.
  • the UTR is a 5’ UTR.
  • UTRs of a genomic region including a gene may be transcribed but not translated.
  • a UTR may be included upstream of the translation initiation sequence of an expression sequence described herein.
  • a UTR may be included downstream of an expression sequence described herein.
  • one UTR for the first expression sequence is the same as or continuous with or overlapping with another UTR for a second expression sequence.
  • the intron is a human intron. In some embodiments, the intron is a full-length human intron, e.g., ZKSCAN1 .
  • a circular polyribonucleotide includes a poly-A sequence. Exemplary poly-A sequences are described in paragraphs [0202] - [0205] of International Patent Publication No WO2019/118919, which is hereby incorporated by reference in its entirety.
  • a circular polyribonucleotide lacks a poly-A sequence.
  • a circular or linear polyribonucleotide includes a UTR with one or more stretches of Adenosines and Uridines embedded within. These AU rich signatures may increase turnover rates of the expression product.
  • UTR AU rich elements may be useful to modulate the stability, or immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response) of the circular or linear polyribonucleotide.
  • immunogenicity e.g., the level of one or more markers of an immune or inflammatory response
  • one or more copies of an ARE may be introduced to the circular polyribonucleotide and the copies of an ARE may modulate translation and/or production of an expression product.
  • AREs may be identified and removed or engineered into the circular polyribonucleotide to modulate the intracellular stability and thus affect translation and production of the resultant protein.
  • any UTR from any gene may be incorporated into the respective flanking regions of the circular polyribonucleotide.
  • a circular polyribonucleotide lacks a 5’-UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular or linear polyribonucleotide lacks a 3’-UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular or linear polyribonucleotide lacks a poly-A sequence and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular or linear polyribonucleotide lacks a termination element and is competent for protein expression from its one or more expression sequences.
  • the circular or linear polyribonucleotide lacks an internal ribosomal entry site and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular or linear polyribonucleotide lacks a cap and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular or linear polyribonucleotide lacks a 5’- UTR, a 3’-UTR, and an IRES, and is competent for protein expression from its one or more expression sequences.
  • the circular or linear polyribonucleotide includes one or more of the following sequences: a sequence that encodes one or more miRNAs, a sequence that encodes one or more replication proteins, a sequence that encodes an exogenous gene, a sequence that encodes an immunogen, a regulatory element (e.g., translation modulator, e.g., translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids that targets endogenous genes (e.g., siRNA, IncRNAs, shRNA), and a sequence that encodes an immunogen.
  • a regulatory element e.g., translation modulator, e.g., translation enhancer or suppressor
  • a translation initiation sequence e.g., one or more regulatory nucleic acids that targets endogenous genes (e.g., siRNA, IncRNAs, shRNA), and a sequence that encodes an immunogen.
  • a circular or linear polyribonucleotide lacks a 5’-UTR. In some embodiments, the circular polyribonucleotide lacks a 3’-UTR. In some embodiments, the circular polyribonucleotide lacks a poly-A sequence. In some embodiments, the circular or linear polyribonucleotide lacks a termination element. In some embodiments, the circular or linear polyribonucleotide lacks an internal ribosomal entry site. In some embodiments, the circular or linear polyribonucleotide lacks degradation susceptibility by exonucleases.
  • the fact that the circular polyribonucleotide lacks degradation susceptibility can mean that the circular polyribonucleotide is not degraded by an exonuclease, or only degraded in the presence of an exonuclease to a limited extent, e.g., that is comparable to or similar to in the absence of exonuclease.
  • the circular polyribonucleotide is not degraded by exonucleases.
  • the circular polyribonucleotide has reduced degradation when exposed to exonuclease.
  • the circular polyribonucleotide lacks binding to a cap-binding protein. In some embodiments, the circular polyribonucleotide lacks a 5’ cap.
  • the polyribonucleotide includes one or more spacer elements.
  • either the first expression augmenting element or the second expression augmenting element may include a spacer element fused to a translation enhancer or a spacer element fused to a stability element.
  • the spacer may be fused to the 5’ end of the translation enhancer or stability element.
  • the spacer may be fused to the 3’ end of the translation enhancer or stability element.
  • a spacer element is a translation enhancer. Not all spacers are translation enhancers.
  • a spacer element is a stability element. Not all spacers are stability elements.
  • a spacer element refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacer elements may be present in between any of the nucleic acid elements described herein. Spacer element may also be present within a nucleic acid element described herein.
  • the circular polyribonucleotide may include a first post-circularization element; a first spacer element having a length of at least 100 ribonucleotides; a polyribonucleotide cargo encoding an immunogen; a second spacer element; and a second post-circularization element; and wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the circular polyribonucleotide may include a first post-circularization element; a first spacer element; a polyribonucleotide cargo encoding an immunogen; a second spacer element having a length of at least 100 ribonucleotides; and a second post-circularization element; and wherein the first post-circularization element and the second post-circularization element together form a circularization junction.
  • the spacer element has a length of at least 100 ribonucleotides.
  • the spacer element may have a length of between 100 to 500 (e.g., between 100 and 400, 100 and 300, 100 and 200, 200 and 500, 300 and 500, 400 and 500, 200 and 400, or 200 and 300) ribonucleotides.
  • the spacer element not including an expression augmenting element may have a length of between 120 and 500 (e.g., between 120 and 400, 120 and 300, 120 and 200, 120 and 150, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 400 and 500, or 350 and 500) ribonucleotides.
  • the spacer element includes between 110 and 500 (e.g., between 110 and 400, 110 and 300, 110 and 200, 110 and 150, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 400 and 500, or 350 and 500) ribonucleotides.
  • the spacer element includes between 100 and 300 (e.g., between 100 and 280, 100 and 260, 100 and 240, 100 and 220, 100 and 200, 100 and 180, 100 and 160, 100 and 140, 100 and 120, 110 and 300, 120 and 300, 140 and 300, 160 and 300, 180 and 300, 200 and 300, 220 and 300, 240 and 300, 260 and 300, or 280 and 300) ribonucleotides.
  • the spacer element has a length of between 200 and 500 (e.g., between 200 and 450, 200 and 400, 200 and 350, 200 and 300, 200 and 250, 250 and 500, 300 and 500, 350 and 500, 400 and 500, 450 and 500, 300 and 500, or 300 and 400) ribonucleotides. In some embodiments, the spacer element has at least 50 ribonucleotides.
  • the spacer element may have between 50 and 500 ribonucleotides (e.g., 50 and 100, 50 and 150, 50 and 200, 50 and 250, 50 and 300, 50 and 350, 50 and 400, 50 and 450, 100 and 500, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, and 450 and 500).
  • 50 and 500 ribonucleotides e.g., 50 and 100, 50 and 150, 50 and 200, 50 and 250, 50 and 300, 50 and 350, 50 and 400, 50 and 450, 100 and 500, 150 and 500, 200 and 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, and 450 and 500).
  • the spacer element has a length of about 50 (e.g., 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55) ribonucleotides. In some embodiments, the spacer element has a length of about 80 (e.g., 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, or 88) ribonucleotides.
  • 50 e.g., 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55
  • the spacer element has a length of about 80 (e.g., 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, or 88) ribonucleotides.
  • the spacer element has a length of about 100 (e.g., 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, or 110) ribonucleotides. In some embodiments, the spacer element has a length of about 120 (e.g., 108, 109, 110, 111 , 112, 113, 114,
  • the spacer element has a length of about 150 (e.g., 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156,
  • the spacer element has a length of about 200 (e.g., 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, or 220) ribonucleotides.
  • 200 e.g., 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 21
  • the polyribonucleotide includes a first spacer element and a second spacer element, wherein the first and second spacer are the same length. In some embodiments, the first spacer element and the second spacer element are about the same length.
  • the polyribonucleotide includes a first spacer element and a second spacer element, wherein the first spacer element and the second spacer elements are different lengths. In some embodiments, the difference between the length of the first spacer element and the length of the second region is 0 to 100 (e.g.
  • the difference between the length of the first spacer element and the length of the second spacer element is 0 to 50 (e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18 ,19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides.
  • the length of the first spacer is about 50 (e.g., 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55) ribonucleotides
  • the length of the second spacer is about 120 (e.g., 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , or 132) ribonucleotides.
  • the length of the first spacer is about 120 (e.g., 108, 109, 110, 111 , 112, 113, 114, 115,
  • a spacer element may be a polyA based spacer, or a polyA based spacer element.
  • a first spacer is a polyA based spacer, or a polyA based spacer element.
  • a second spacer is a polyA based spacer, or a polyA based spacer element.
  • the first spacer and the second spacer may be a polyA based spacer, or a polyA based spacer element.
  • a polyA based spacer, or a polyA based spacer element is equivalent to a polyA region (e.g., a polyAC region, polyAU region, polyAG region, or polyAT region), as described herein.
  • the first spacer element may consist of a polyA region comprising 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine residues.
  • the first spacer element may consist of a polyAC region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or cytosine residues.
  • the adenosine and cytosine residues may be present in any ratio to one another.
  • the first spacer element may consist of a polyAU region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or uridine residues.
  • the first spacer element may consist of a polyAG region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or guanosine residues.
  • the adenosine and guanosine residues may be present in any ratio to one another.
  • the first spacer element may consist of a polyAT region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or thymidine residues.
  • the adenosine and thymidine residues may be present in any ration to one another.
  • the first spacer element includes a polyAT region, wherein the polyAT region includes between 100 ribonucleotides and 150 ribonucleotides (e.g., 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 ribonucleotides).
  • the polyAT region includes between 100 ribonucleotides and 150 ribonucleotides (e.g., 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 ribonucleotides).
  • the second spacer element may consist of a polyA region comprising 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine residues.
  • the second spacer element may consist of a polyAC region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or cytosine residues.
  • the second spacer element may consist of a polyAU region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or uridine residues.
  • the adenosine and uridine residues may be present in any ratio to one another.
  • the second spacer element may consist of a polyAG region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or guanosine residues.
  • the adenosine and guanosine residues may be present in any ratio to one another.
  • the first spacer element may consist of a polyAT region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or thymidine residues.
  • the second spacer element includes a polyAT region, wherein the polyAT region includes between 100 ribonucleotides and 150 ribonucleotides (e.g., 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 ribonucleotides).
  • the adenosine and thymidine residues may be present in any ratio to one another.
  • the spacer element sequences can be a poly A region (e.g., polyA, polyAT, polyAU, polyAC, ro polyAG) or a random sequence.
  • the spacer element may be referred to based on a combination of nucleotide content and length.
  • an AU120 spacer element may consist of a polyAU region comprising between 80% to 100% adenosine or uridine residues and 120 nucleotides in length.
  • the spacer element has a nucleic acid sequence having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences described in TABLE 10.
  • the spacer element has a nucleic acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences described in TABLE 10.
  • the spacer element has a nucleic acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences described in TABLE 10. In some embodiments, the spacer element has a nucleic acid sequence of any one of the sequences described in TABLE 10.
  • a circular or linear polyribonucleotide described herein includes one or more internal ribosome entry site (IRES) elements.
  • the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences, where each expression sequence optionally encodes an immunogen.
  • the IRES is located between a heterologous promoter and the 5’ end of a coding sequence.
  • a suitable IRES element to include in a polyribonucleotide includes an RNA sequence capable of engaging a eukaryotic ribosome.
  • the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.
  • the IRES element is from the DNA of an organism including, but not limited to, a virus, a mammal, and a Drosophila.
  • viral DNA may be from, but is not limited to, picomavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA.
  • cDNA picomavirus complementary DNA
  • EMCV encephalomyocarditis virus
  • an IRES element is from an Antennapedia gene from Drosophila melanogaster.
  • the IRES sequence is an IRES sequence of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis Invicta virus 1 , Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1 , Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1 , Human Immunodeficiency Virus type 1 , Homalodisca coagulata virus-1 , Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 , Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus,
  • the IRES is an IRES sequence of Coxsackievirus B3 (CVB3).
  • the IRES is an IRES sequence of Encephalomyocarditis virus.
  • the IRES is an IRES sequence of Theiler's encephalomyelitis virus.
  • the IRES sequence has a modified sequence in comparison to the wild-type IRES sequence.
  • the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue
  • the last nucleotide of the wild-type IRES sequence is modified such that it is a cytosine residue.
  • the IRES sequence may be a CVB3 IRES sequence wherein the terminal adenosine residue is modified to cytosine residue.
  • the modified CVB3 IRES may have the nucleic acid sequence of: UUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGG UAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAACUGUAACUUAGAA GUAACACACACCGAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACU UCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAAAGCGU UCGUUAUCCGGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUG UUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACG GGCGACCGUGGCGGUGGCUGCGUUGCCUGCCCAUGGGGAAACCCAUGGGACG CUCUAAUACAGACAUGGUGCGAAGUCUAUUGAGCUAUUGAGCUAGUUGGUAGCUAUUGAG
  • the IRES sequence is an Enterovirus 71 (EV17) IRES.
  • the terminal guanosine residue of the EV17 IRES sequence is modified to a cytosine residue.
  • the modified EV71 IRES may have the nucleic acid sequence of:
  • the IRES sequence is a synthetic IRES.
  • a “synthetic IRES” is an IRES that is modified relative to a wildtype IRES in order to modulate its structure and/or activity.
  • an IRES that is modified to incorporate an aptamer sequence is a synthetic IRES.
  • the polyribonucleotide includes at least one IRES flanking at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the IRES flanks both sides of at least one (e.g., 2, 3, 4, 5 or more) expression sequence.
  • the polyribonucleotide includes one or more IRES sequences on one or both sides of each expression sequence, leading to separation of the resulting peptide(s) and or polypeptide(s).
  • a polyribonucleotide described herein may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.
  • a polyribonucleotide described herein includes an IRES (e.g., an IRES operably linked to a coding region).
  • the polyribonucleotide may include any IRES as described in Chen et al. Nature Biotechnology 41 :262-272, 2023, Chen et al. Mol. Cell 81 (20):4300- 4318, 2021 ; Jopling et al. Oncogene 20:2664-2670, 2001 ; Baranick et al. PNAS 105(12):4733-4738, 2008; Lang et al. Molecular Biology of the Cell 13(5) :1792-1801 , 2002; Dorokhov et al.
  • immunogens expressed from a circular or linear polyribonucleotide disclosed herein include a secreted immunogen, for example, an immunogen that naturally includes a signal sequence, or one that does not usually encode a signal sequence but is modified to contain one.
  • the immunogen(s) includes a secretion signal.
  • the secretion signal may be the naturally encoded secretion signal for a secreted immunogen.
  • the secretion signal may be a modified secretion signal for a secreted immunogen.
  • the immunogen(s) do not include a secretion signal.
  • a polyribonucleotide encodes multiple copies of the same immunogen (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more). In some embodiments, at least one copy of the polypeptide (e.g., immunogen) includes a signal sequence and at least one copy of the polypeptide does not include a signal sequence. In some embodiments, a circular polyribonucleotide encodes plurality of polypeptides (e.g., immunogens),, where at least one of the plurality of polypeptides includes a signal sequence and at least one copy of the plurality of polypeptides does not include a signal sequence.
  • the signal sequence is a wild-type signal sequence that is present on the N-terminus of the corresponding wild-type polypeptide (e.g., immunogen),, e.g., when expressed endogenously.
  • the signal sequence is heterologous to the polypeptide (e.g., immunogen),, e.g., is not present when the wild-type polypeptide is expressed endogenously.
  • a polyribonucleotide sequence encoding a polypeptide may be modified to remove the nucleotide sequence encoding a wild-type signal sequence and/or add a sequence encoding a heterologous signal sequence.
  • the circular polyribonucleotide may further include one or more adjuvants, each with or without a signal sequence.
  • the circular polyribonucleotide includes an adjuvant with or without a signal sequence.
  • the circular polyribonucleotide encodes at least one adjuvant and at least one polypeptide.
  • the at least one encoded adjuvant includes a signal sequence and the at least one encoded polypeptide does not include a signal sequence.
  • the at least one encoded adjuvant includes a signal sequence and the at least one encoded polypeptide includes a signal sequence.
  • the at least one encoded adjuvant does not include a signal sequence and the at least one encoded polypeptide includes a signal sequence.
  • neither the encoded adjuvant nor the encoded polypeptide includes a signal sequence.
  • the signal sequence is a wild-type signal sequence that is present on the N-terminus of the corresponding wild-type adjuvant, e.g., when expressed endogenously.
  • the signal sequence is heterologous to the adjuvant, e.g., is not present when the wild-type adjuvant is expressed endogenously.
  • a polyribonucleotide sequence encoding an adjuvant may be modified to remove the nucleotide sequence encoding a wild-type signal sequence and/or add a sequence encoding a heterologous signal sequence.
  • a polypeptide (e.g., immunogen or an adjuvant encoded by a polyribonucleotide) encoded by an expression sequence may include a signal sequence that directs the polypeptide to the secretory pathway.
  • the signal sequence may direct the polypeptide or adjuvant to reside in certain organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes).
  • the signal sequence directs the polypeptide or adjuvant to be secreted from the cell.
  • the signal sequence may be cleaved after secretion, resulting in a mature protein.
  • the signal sequence may become embedded in the membrane of the cell or certain organelles, creating a transmembrane segment that anchors the protein to the membrane of the cell, endoplasmic reticulum, or Golgi apparatus.
  • the signal sequence of a transmembrane protein is a short sequence at the N-terminal of the polypeptide.
  • the first transmembrane domain acts as the first signal sequence, which targets the protein to the membrane.
  • the secretion signal is human interleukin-2 (IL-2) secretion signal.
  • the IL-2 secretion signal has an amino acid sequence of at least 90% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 186).
  • the IL2 secretion signal has an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 186.
  • the IL-2 secretion signal has an amino acid sequence of at least 99% sequence identity to SEQ ID NO: 186.
  • the IL-2 secretion signal has an amino acid sequence of 100% sequence identity to SEQ ID NO: 186.
  • the secretion signal is Gaussia luciferase secretion signal.
  • the Gaussia luciferase secretion signal has an amino acid sequence of at least 90% sequence identity of MGVKVLFALICIAVAEAK (SEQ ID NO: 187).
  • the Gaussia luciferase secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 187.
  • the Gaussia luciferase secretion signal has an amino acid sequence of at least 99% sequence identity of SEQ ID NO: 187.
  • the Gaussia luciferase secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 187.
  • the secretion signal is an EPO (e.g., a human EPO) secretion signal.
  • the EPO secretion signal has an amino acid sequence of at least 90% sequence identity of MGVHECPAWLWLLLSLLSLPLGLPVLGA (SEQ ID NO: 188).
  • the EPO secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 188.
  • the 88. In some embodiments, the EPO secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 188.
  • the secretion signal is a wildtype SARS-CoV-2 secretion signal.
  • the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of at least 90% sequence identity of MFVFLVLLPLVSS (SEQ ID NO: 189).
  • the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 189.
  • the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of at least 99% sequence identity of SEQ ID NO: 189.
  • the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 189.
  • an adjuvant encoded by a polyribonucleotide includes a secretion signal.
  • a polypeptide encoded by a polyribonucleotide includes either a secretion signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence.
  • the polyribonucleotide described herein (e.g., the polyribonucleotide cargo encoding an immunogen of the polyribonucleotide) includes one or more regulatory elements.
  • the polyribonucleotide includes a regulatory element, e.g., a sequence that modifies expression of an expression sequence within the polyribonucleotide.
  • a regulatory element may include a sequence that is located adjacent to an expression sequence that encodes an expression product.
  • a regulatory element may be operably linked to the adjacent sequence.
  • a regulatory element may increase an amount of product expressed as compared to an amount of the expressed product when no regulatory element is present.
  • a regulatory element may be used to increase the expression of one or more polypeptide(s) and/or adjuvant(s) encoded by a polyribonucleotide.
  • a regulatory element may be used to decrease the expression of one or more polypeptide(s) encoded by a polyribonucleotide.
  • a regulatory element is used to increase expression of polypeptide and another regulatory element is used to decrease expression of another polypeptide on the same polyribonucleotide.
  • one regulatory element can increase an amount of product expressed for multiple expression sequences attached in tandem.
  • one regulatory element can enhance the expression of one or more expression sequences.
  • Multiple regulatory elements can also be used, for example, to differentially regulate expression of different expression sequences.
  • the regulatory element is a translation modulator.
  • a translation modulator can modulate translation of the expression sequence in the polyribonucleotide.
  • a translation modulator can be a translation enhancer or suppressor.
  • the polyribonucleotide includes at least one translation modulator adjacent to at least one expression sequence.
  • the polyribonucleotide includes a translation modulator adjacent to each expression sequence.
  • the translation modulator is present on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s).
  • the regulatory element is a microRNA (miRNA) or a miRNA binding site.
  • a regulatory element as provided herein includes a selective translation sequence.
  • selective translation sequence refers to a nucleic acid sequence that selectively initiates or activates translation of an expression sequence in the polyribonucleotide, for instance, certain riboswitch aptazymes.
  • a regulatory element can also include a selective degradation sequence.
  • selective degradation sequence refers to a nucleic acid sequence that initiates degradation of the polyribonucleotide, or an expression product of the polyribonucleotide.
  • the regulatory element is a translation modulator.
  • a translation modulator can modulate translation of the expression sequence in the polyribonucleotide.
  • a translation modulator can be a translation enhancer or suppressor.
  • a translation initiation sequence can function as a regulatory element.
  • a polyribonucleotide produces stoichiometric ratios of expression products. Rolling circle translation continuously produces expression products at substantially equivalent ratios. In some embodiments, the polyribonucleotide has a stoichiometric translation efficiency, such that expression products are produced at substantially equivalent ratios. In some embodiments, the polyribonucleotide has a stoichiometric translation efficiency of multiple expression products, e.g., products from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or more expression sequences. In some embodiments, the polyribonucleotide produces substantially different ratios of expression products.
  • the translation efficiency of multiple expression products may have a ratio of 1 :10,000; 1 :7000, 1 :5000, 1 :1000, 1 :700, 1 :500, 1 :100, 1 :50, 1 :10, 1 :5, 1 :4, 1 :3 or 1 :2.
  • the ratio of multiple expression products may be modified using a regulatory element.
  • a circular or linear polyribonucleotide of the disclosure can include a cleavage domain (e.g., a stagger element or a cleavage sequence).
  • a cleavage domain e.g., a stagger element or a cleavage sequence
  • the term “stagger element” is a moiety, such as a nucleotide sequence, that induces ribosomal pausing during translation.
  • the stagger element may include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof.
  • a circular or linear polyribonucleotide includes at least one stagger element adjacent to an expression sequence. In some embodiments, the circular or linear polyribonucleotide includes a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, leading to separation of the expression products. In some embodiments, the stagger element is a portion of the one or more expression sequences. In some embodiments, the circular or linear polyribonucleotide includes one or more expression sequences, and each of the one or more expression sequences is separated from a succeeding expression sequence by a stagger element on the circular or linear polyribonucleotide.
  • the stagger element prevents generation of a single polypeptide (a) from two rounds of translation of a single expression sequence or (b) from one or more rounds of translation of two or more expression sequences.
  • the stagger element is a sequence separate from the one or more expression sequences.
  • the stagger element includes a portion of an expression sequence of the one or more expression sequences.
  • a stagger element may be included to induce ribosomal pausing during translation.
  • the stagger element is at 3’ end of at least one of the one or more expression sequences.
  • the stagger element can be configured to stall a ribosome during rolling circle translation of the circular or linear polyribonucleotide.
  • the stagger element may include, but is not limited to a 2A-like, or CHYSEL (SEQ ID NO: 191 ) (cis-acting hydrolase element) sequence.
  • the stagger element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP, where Xi is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid (SEQ ID NO: 192).
  • stagger elements includes GDVESNPGP (SEQ ID NO: 193), GDIEENPGP (SEQ ID NO: 194), VEPNPGP (SEQ ID NO: 195), IETNPGP (SEQ ID NO: 196), GDIESNPGP (SEQ ID NO: 197), GDVELNPGP (SEQ ID NO: 198), GDIETNPGP (SEQ ID NO: 199), GDVENPGP (SEQ ID NO: 200), GDVEENPGP (SEQ ID NO: 201 ), GDVEQNPGP (SEQ ID NO: 202), IESNPGP (SEQ ID NO: 203), GDIELNPGP (SEQ ID NO: 204), HDIETNPGP (SEQ ID NO: 205), HDVETNPGP (SEQ ID NO: 206), HDVEMNPGP (SEQ ID NO: 207), GDMESNPGP (SEQ ID NO: 208), GDVETNPGP (SEQ ID NO: 209), GDIEQNPGP (SEQ ID NO:
  • a stagger element described herein cleaves an expression product, such as between G and P of the consensus sequence described herein.
  • the circular or linear polyribonucleotide includes at least one stagger element to cleave the expression product.
  • the circular or linear polyribonucleotide includes a stagger element adjacent to at least one expression sequence.
  • the circular or linear polyribonucleotide includes a stagger element after each expression sequence.
  • the circular or linear polyribonucleotide includes a stagger element that is present on one or both sides of each expression sequence, leading to translation of individual peptide(s) and or polypeptide(s) from each expression sequence.
  • a stagger element includes one or more modified nucleotides or unnatural nucleotides that induce ribosomal pausing during translation.
  • Unnatural nucleotides may include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Examples such as these are distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.
  • Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage I to the phosphodiester backbone), and any combination thereof that can induce ribosomal pausing during translation.
  • Some of the exemplary modifications provided herein are described elsewhere herein.
  • a stagger element is present in a circular or linear polyribonucleotide in other forms.
  • a stagger element includes a termination element of a first expression sequence in the circular or linear polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a first translation initiation sequence of an expression succeeding the first expression sequence.
  • the first stagger element of the first expression sequence is upstream of (5’ to) a first translation initiation sequence of the expression succeeding the first expression sequence in the circular or linear polyribonucleotide.
  • the first expression sequence and the expression sequence succeeding the first expression sequence are two separate expression sequences in the circular or linear polyribonucleotide.
  • the distance between the first stagger element and the first translation initiation sequence can enable continuous translation of the first expression sequence and its succeeding expression sequence.
  • the first stagger element includes a termination element and separates an expression product of the first expression sequence from an expression product of its succeeding expression sequences, thereby creating discrete expression products.
  • the circular or linear polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the succeeding sequence in the circular or linear polyribonucleotide is continuously translated, while a corresponding circular or linear polyribonucleotide including a stagger element of a second expression sequence that is upstream of a second translation initiation sequence of an expression sequence succeeding the second expression sequence is not continuously translated.
  • a stagger element includes a first termination element of a first expression sequence in the circular or linear polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a downstream translation initiation sequence.
  • the first stagger element is upstream of (5’ to) a first translation initiation sequence of the first expression sequence in the circular or linear polyribonucleotide.
  • the distance between the first stagger element and the first translation initiation sequence enables continuous translation of the first expression sequence and any succeeding expression sequences.
  • the first stagger element separates one round expression product of the first expression sequence from the next round expression product of the first expression sequences, thereby creating discrete expression products.
  • the circular or linear polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the first expression sequence in the circular or linear polyribonucleotide is continuously translated, while a corresponding circular or linear polyribonucleotide including a stagger element upstream of a second translation initiation sequence of a second expression sequence in the corresponding circular or linear polyribonucleotide is not continuously translated.
  • the distance between the second stagger element and the second translation initiation sequence is at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x greater in the corresponding circular or linear polyribonucleotide than a distance between the first stagger element and the first translation initiation in the circular or linear polyribonucleotide.
  • the distance between the first stagger element and the first translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater.
  • the distance between the second stagger element and the second translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater than the distance between the first stagger element and the first translation initiation.
  • the circular or linear polyribonucleotide includes more than one expression sequence.
  • the plurality of expression sequences encoded by a circular ribonucleotide may be separated by an IRES between each expression sequence.
  • a circular polyribonucleotide may include a first IRES operable linked to a first expression sequence and a second IRES operably linked to a second expression sequence.
  • the IRES may be the same IRES between all expression sequences.
  • the IRES may be different between expression sequences.
  • the plurality of expression sequences may be separated by a 2A selfcleaving peptide.
  • a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first expression sequence, a 2A, and a second expression sequences.
  • the 2A may have a sequence of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 212).
  • the plurality of expression sequences may be separated by a protease cleavage site (e.g., a furin cleavage site).
  • a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first expression sequence, a protease cleavage site (e.g., a furin cleavage site), and a second expression sequence.
  • the furin cleavage site may have a sequence of GRLRR (SEQ ID NO: 213).
  • the plurality of expression sequence may be separated by a 2A selfcleaving peptide and a protease cleavage site (e.g., a furin cleavage site).
  • a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first expression sequence, a 2A, a protease cleavage site (e.g., a furin cleavage site), and a second expression sequence.
  • a circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first expression sequence, a protease cleavage site (e.g., a furin cleavage site), a 2A, and a second expression sequence.
  • a tandem 2A and furin cleavage site may be referred to as a furin-2A (which includes furin-2A or 2A-furin, arranged in either orientation).
  • the plurality of expression sequences and/or adjuvants encoded by the circular ribonucleotide may be separated by both IRES and 2A sequences.
  • an IRES may be between one expression sequence and a second expression sequence while a 2A peptide may be between the second expression sequence and the third expression sequence.
  • the selection of a particular IRES or 2A self-cleaving peptide may be used to control the expression level of expression sequence under control of the IRES or 2A sequence. For example, depending on the IRES and or 2A peptide selected, expression on the polypeptide may be higher or lower.
  • a circular or linear polyribonucleotide includes at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to an expression sequence. In some embodiments, the cleavage sequence is between two expression sequences. In some embodiments, cleavage sequence is included in an expression sequence. In some embodiments, the circular or linear polyribonucleotide includes between 2 and 10 cleavage sequences. In some embodiments, the circular or linear polyribonucleotide includes between 2 and 5 cleavage sequences.
  • the multiple cleavage sequences are between multiple expression sequences; for example, a circular or linear polyribonucleotide may include three expression sequences two cleavage sequences such that there is a cleavage sequence in between each expression sequence.
  • the circular or linear polyribonucleotide includes a cleavage sequence, such as in an immolating circRNA or cleavable circRNA or self-cleaving circRNA.
  • the circular or linear polyribonucleotide includes two or more cleavage sequences, leading to separation of the circular or linear polyribonucleotide into multiple products, e.g., miRNAs, linear RNAs, smaller circular or linear polyribonucleotide, etc.
  • a cleavage sequence includes a ribozyme RNA sequence.
  • a ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNA, but they have also been found to catalyze the aminotransferase activity of the ribosome. Catalytic RNA can be “evolved” by in vitro methods. Similar to riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression.
  • a catalytic RNA or ribozyme can be placed within a larger non-coding RNA such that the ribozyme is present at many copies within the cell for the purposes of chemical transformation of a molecule from a bulk volume.
  • aptamers and ribozymes can both be encoded in the same non-coding RNA.
  • the cleavage sequence encodes a cleavable polypeptide linker.
  • a polyribonucleotide may encode two or more expression sequences are encoded by a single open-reading frame (ORF).
  • ORF open-reading frame
  • two or more expression sequences may be encoded by a single open-reading frame, the expression of which is controlled by an IRES.
  • the ORF further encodes a polypeptide linker, e.g., such that the expression product of the ORF encodes two or more expression sequences each separated by a sequence encoding a polypeptide linker (e.g., a linker of 5-200, 5 to 100, 5 to 50, 5 to 20, 50 to 100, or 50 to 200 amino acids).
  • the polypeptide linker may include a cleavage site, for example, a cleavage site recognized and cleaved by a protease (e.g., an endogenous protease in a subject following administration of the polyribonucleotide to that subject).
  • a protease e.g., an endogenous protease in a subject following administration of the polyribonucleotide to that subject.
  • a single expression product including the amino acid sequence of two or more expression sequences is cleaved upon expression, such that the two or more expression sequences are separated following expression.
  • protease cleavage sites are known to those of skill in the art, for example, amino acid sequences that act as protease cleavage sites recognized by a metalloproteinase (e.g., a matrix metalloproteinase (MMP), such as any one or more of MMPs 1 -28), a disintegrin and metalloproteinase (ADAM, such as any one or more of ADAMs 2, 7-12, 15, 17-23, 28-30 and 33), a serine protease, urokinase-type plasminogen activator, matriptase, a cysteine protease, an aspartic protease, or a cathepsin protease.
  • the protease is matriptase.
  • a circular or linear polyribonucleotide described herein is an immolating circular or linear polyribonucleotide, a cleavable circular or linear polyribonucleotide, or a self-cleaving circular or linear polyribonucleotide.
  • a circular or linear polyribonucleotide can deliver cellular components including, for example, RNA, IncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA.
  • a circular or linear polyribonucleotide includes miRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites; (iii) degradable linkers; (iv) chemical linkers; and/or (v) spacer element sequences.
  • circRNA includes siRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites (e.g., ADAR); (iii) degradable linkers (e.g., glycerol); (iv) chemical linkers; and/or (v) spacer element sequences.
  • self-cleavable elements include hammerhead, splicing element, hairpin, hepatitis delta virus (HDV), Varkud Satellite (VS), and glmS ribozymes.
  • the circular polyribonucleotide includes at least one stagger element adjacent to an expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and/or polypeptide(s). In some embodiments, the stagger element is a portion of the one or more expression sequences. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, and each of the one or more expression sequences is separated from a succeeding expression sequence by a stagger element on the circular polyribonucleotide.
  • the stagger element prevents generation of a single polypeptide (a) from two rounds of translation of a single expression sequence or (b) from one or more rounds of translation of two or more expression sequences.
  • the stagger element is a sequence separate from the one or more expression sequences.
  • the stagger element comprises a portion of an expression sequence of the one or more expression sequences.
  • a circular or linear polyribonucleotide encodes an expression sequence and includes a translation initiation sequence, e.g., a start codon.
  • the polyribonucleotide includes a translation initiation sequence operably linked to an expression sequence.
  • the translation initiation sequence includes a Kozak or Shine- Dalgarno sequence.
  • the translation initiation sequence includes a Kozak sequence.
  • the circular or linear polyribonucleotide includes the translation initiation sequence, e.g., Kozak sequence, adjacent to an expression sequence.
  • the translation initiation sequence is a non-coding start codon.
  • the translation initiation sequence e.g., Kozak sequence
  • the circular or linear polyribonucleotide includes at least one translation initiation sequence adjacent to an expression sequence.
  • the translation initiation sequence provides conformational flexibility to the circular or linear polyribonucleotide.
  • the translation initiation sequence is within a single stranded region of the circular or linear polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs [0163] - [0165] of International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
  • the circular or linear polyribonucleotide may include more than one start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons. Translation may initiate on the first start codon or may initiate downstream of the first start codon.
  • a circular or linear polyribonucleotide may initiate at a codon which is not the first start codon, e.g., AUG.
  • Translation of the circular or linear polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG.
  • translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions.
  • the translation of the polyribonucleotide may begin at alternative translation initiation sequence, such as ACG.
  • the polyribonucleotide translation may begin at alternative translation initiation sequence, CTG/CUG.
  • the polyribonucleotide translation may begin at alternative translation initiation sequence, GTG/GUG.
  • the polyribonucleotide may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g., CGG, GGGGCC, CAG, CTG.
  • RAN repeat-associated non-AUG
  • translation is initiated by eukaryotic initiation factor 4A (elF4A) treatment with Rocaglates (translation is repressed by blocking 43S scanning, leading to premature, upstream translation initiation and reduced protein expression from transcripts bearing the RocA- elF4A target sequence, see for example, nature.com/articles/nature17978).
  • elF4A eukaryotic initiation factor 4A
  • the polyribonucleotide described herein (e.g., the polyribonucleotide cargo encoding an immunogen of the polyribonucleotide) includes least one termination element.
  • the polyribonucleotide includes a termination element operably linked to an expression sequence.
  • the polynucleotide lacks a termination element.
  • the polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element.
  • the polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product.
  • the circular polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element.
  • the circular polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the circular polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product, e.g., peptides or polypeptides, due to lack of ribosome stalling or fall-off. In such an embodiment, rolling circle translation expresses a continuous expression product through each expression sequence.
  • a termination element of an expression sequence can be part of a stagger element.
  • one or more expression sequences in the circular polyribonucleotide includes a termination element.
  • rolling circle translation or expression of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide is performed.
  • the expression product may fall off the ribosome when the ribosome encounters the termination element, e.g., a stop codon, and terminates translation.
  • translation is terminated while the ribosome, e.g., at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.
  • the circular polyribonucleotide includes a termination element at the end of one or more expression sequences.
  • one or more expression sequences includes two or more termination elements in succession.
  • translation is terminated and rolling circle translation is terminated.
  • the ribosome completely disengages with the circular polyribonucleotide.
  • production of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide may require the ribosome to reengage with the circular polyribonucleotide prior to initiation of translation.
  • termination elements include an in-frame nucleotide triplet that signals termination of translation, e.g., UAA, UGA, UAG.
  • one or more termination elements in the circular polyribonucleotide are frame-shifted termination elements, such as but not limited to, off-frame or -1 and + 1 shifted reading frames (e.g., hidden stop) that may terminate translation.
  • Frame-shifted termination elements include nucleotide triples, TAA, TAG, and TGA that appear in the second and third reading frames of an expression sequence. Frame-shifted termination elements may be important in preventing misreads of mRNA, which is often detrimental to the cell.
  • the termination element is a stop codon.
  • the circular polyribonucleotide encodes a polyribonucleotide cargo.
  • a polyribonucleotide cargo described herein includes any sequence including at least one polyribonucleotide.
  • the polyribonucleotide cargo includes an expression sequence, a non-coding sequence, or an expression sequence and a non-coding sequence.
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide.
  • the polyribonucleotide cargo includes an IRES operably linked to an expression sequence encoding a polypeptide.
  • the polyribonucleotide cargo includes an expression sequence that encodes a polypeptide that has a biological effect on a subject.
  • a polyribonucleotide cargo may, for example, include at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1 ,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about
  • the polyribonucleotides cargo includes from 1 -20,000 nucleotides, 1 -10,000 nucleotides, 1 -5,000 nucleotides, 100-20,000 nucleotide, 100-10,000 nucleotides, 100-5,000 nucleotides, 500-20,000 nucleotides, 500-10,000 nucleotides, 500-5,000 nucleotides, 1 ,000-20,000 nucleotides, 1 ,000-10,000 nucleotides, or 1 ,000-5,000 nucleotides.
  • the polyribonucleotide cargo includes one or multiple expression (or coding) sequences, wherein each expression (or coding) sequence encodes a polypeptide.
  • the polyribonucleotide cargo includes one or multiple noncoding sequences.
  • the polyribonucleotide cargo consists entirely of non-coding sequence(s).
  • the polyribonucleotide cargo includes a combination of expression (or coding) and noncoding sequences.
  • the GC content of a nucleic acid sequence encoding a polypeptide is at least 51% (e.g., at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%). In some embodiments, the GC content of a nucleic acid sequence encoding a polypeptide is at most 52%, 53%, 54%, 55%, 56%, 57%, 58% or 59%, or 60%. In some embodiments, the GC content of a nucleic acid sequence encoding a polypeptide is 51% to 60%, 52% to 60%, 53% to 60%, 54% to 60%, 55% to 60%, 52% to 58%, 53% to 58%.
  • the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding polypeptide is more than 10% (e.g., more than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding a polypeptide is at most 30% (e.g., at most 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%).
  • the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding a polypeptide is 20% to 28%, 21 % to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21 % to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21 % to 23%, or 22% to 23%.
  • the GC content of an expression sequence encoding the polypeptide refers to the GO content of the expression sequence that exclusively encodes the polypeptide with no other coding regions that encode peptides other than the polypeptide.
  • the uridine content or thymidine of an expression sequence encoding the polypeptide refers to the uridine content of the expression sequence that exclusively encodes the polypeptide with no other coding regions that encode peptides other than the polypeptide.
  • the calculation of the GC content or the uridine (or thymidine) content of the expression sequence encoding the polypeptide only takes into account the continuous nucleic acid sequence that starts in a 5’ to 3’ direction from the first nucleoside of the start codon of the open reading frame that encodes the polypeptide to the last nucleoside of the stop codon of the same open reading frame.
  • the calculation of the GC content or the uridine (or thymidine) content of the expression sequence encoding the polypeptide only takes into account the continuous nucleic acid sequence that starts in a 5’ to 3’ direction from the first nucleoside of the codon that encodes the N-terminal end amino acid residue of the polypeptide to the last nucleoside of the codon that encodes the C-terminal end amino acid residue of the polypeptide.
  • the nucleic acid sequence encoding the polypeptide has a uridine content of more than 20%.
  • the uridine content of a nucleic acid sequence encoding polypeptide is more than 10% (e.g., more than 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, or 25%).
  • the uridine content of a nucleic acid sequence encoding polypeptide is at most 30% (e.g., at most 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 %, or 20%).
  • the uridine content of a nucleic acid sequence encoding polypeptide is 20% to 28%, 21 % to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21 % to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21 % to 23%, or 22% to 23%.
  • the nucleic acid sequence encoding the polypeptide has a uridine content of 20% to 28%.
  • polyribonucleotides made as described herein are used as effectors in therapy or agriculture.
  • a circular polyribonucleotide made by the methods described herein e.g., the cell-free methods described herein
  • a subject e.g., in a pharmaceutical, or agricultural composition
  • a circular polyribonucleotide made by the methods described herein e.g., the cell-free methods described herein
  • the polyribonucleotide includes any feature, or any combination of features as disclosed in International Patent Publication No. WO2019/1 18919, which is hereby incorporated by reference in its entirety.
  • the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the circular polyribonucleotide) includes one or more expression (or coding) sequences, wherein each expression sequence encodes a polypeptide.
  • the circular polyribonucleotide includes two, three, four, five, six, seven, eight, nine, ten or more expression (or coding) sequences.
  • Each encoded polypeptide may be linear or branched.
  • the polypeptide has a length from about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1 ,000 to about 2,500 amino acids, or any range therebetween.
  • the polypeptide has a length of less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1 ,500 amino acids, less than about 1 ,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less may be useful.
  • Polypeptides included herein may include naturally occurring polypeptides or non-naturally occurring polypeptides.
  • the polypeptide is or includes a functional fragment or variant of a reference polypeptide (e.g., an enzymatically active fragment or variant of an enzyme).
  • the polypeptide may be a functionally active variant of any of the polypeptides described herein with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a polypeptide described herein or a naturally occurring polypeptide.
  • the polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to a protein of interest.
  • polypeptides include, but are not limited to, a fluorescent tag or marker, an antigen, a therapeutic polypeptide, or a polypeptide for agricultural applications.
  • a therapeutic polypeptide may be a hormone, a neurotransmitter, a growth factor, an enzyme (e.g., oxidoreductase, metabolic enzyme, mitochondrial enzyme, oxygenase, dehydrogenase, ATP - independent enzyme, lysosomal enzyme, desaturase), a cytokine, an antigen binding polypeptide (e.g., antigen binding antibody or antibody-like fragments, such as single chain antibodies, nanobodies or other Ig heavy chain or light chain containing polypeptides), an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an interferon, an interleukin, and a thrombolytic.
  • an enzyme e.g., oxidoreductase, metabolic enzyme, mitochondrial enzyme, oxygenase, dehydrogenase, ATP - independent enzyme, lysosomal enzyme, desaturase
  • a cytokine e.g., an antigen
  • a polypeptide for agricultural applications may be a bacteriocin, a lysin, an antimicrobial polypeptide, an antifungal polypeptide, a nodule C-rich peptide, a bacteriocyte regulatory peptide, a peptide toxin, a pesticidal polypeptide (e.g., insecticidal polypeptide or nematocidal polypeptide), an antigen binding polypeptide (e.g., antigen binding antibody or antibody-like fragments, such as single chain antibodies, nanobodies or other Ig heavy chain or light chain containing polypeptides), an enzyme (e.g., nuclease, amylase, cellulase, peptidase, lipase, chitinase), a peptide pheromone, and a transcription factor.
  • an enzyme e.g., nuclease, amylase, cellulase, peptidase, lipase, chit
  • the circular polyribonucleotide expresses a non-human protein.
  • the circular polyribonucleotide expresses an antibody, e.g., an antibody fragment, or a portion thereof.
  • the antibody expressed by the circular polyribonucleotide can be of any isotype, such as IgA, IgD, Ig E, IgG, IgM.
  • the circular polyribonucleotide expresses a portion of an antibody, such as a light chain, a heavy chain, a Fc fragment, a CDR (complementary determining region), a Fv fragment, or a Fab fragment, a further portion thereof.
  • the circular polyribonucleotide expresses one or more portions of an antibody.
  • the circular polyribonucleotide can include more than one expression (or coding) sequence, each of which expresses a portion of an antibody, and the sum of which can constitute the antibody.
  • the circular polyribonucleotide includes one expression sequence coding for the heavy chain of an antibody, and another expression sequence coding for the light chain of the antibody.
  • the light chain and heavy chain can be subject to appropriate modification, folding, or other post-translation modification to form a functional antibody.
  • polypeptides include multiple polypeptides, e.g., multiple copies of one polypeptide sequence, or multiple different polypeptide sequences. In embodiments, multiple polypeptides are connected by linker amino acids or spacer amino acids.
  • the polynucleotide cargo includes a sequence encoding a signal peptide.
  • a signal peptide Many signal peptide sequences have been described, for example, the Tat (Twin-arginine translocation) signal sequence is typically an N-terminal peptide sequence containing a consensus SRRxFLK “twin-arginine” motif, which serves to translocate a folded protein containing such a Tat signal peptide across a lipid bilayer. See also, e.g., the Signal Peptide Database publicly available at www[dot]signalpeptide[dot]de.
  • Signal peptides are also useful for directing a protein to specific organelles; see, e.g., the experimentally determined and computationally predicted signal peptides disclosed in the Spdb signal peptide database, publicly available at proline, bic.nus.edu.sg/spdb.
  • the polynucleotide cargo includes sequence encoding a cell-penetrating peptide (CPP).
  • CPP cell-penetrating peptide
  • An example of a commonly used CPP sequence is a poly-arginine sequence, e.g., octoarginine or nonoarginine, which can be fused to the C-terminus of the CGI peptide.
  • the polynucleotide cargo includes sequence encoding a self-assembling peptide; see, e.g., Miki et al. (2021 ) Nature Communications, 21 :3412, DOI: 10.1038/s41467-021 - 23794-6.
  • the expression (or coding) sequence includes a poly-A sequence (e.g., at the 3’ end of an expression sequence).
  • the length of a poly-A sequence is greater than 10 nucleotides in length.
  • the poly-A sequence is greater than 15 nucleotides in length (e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400, 1 ,500, 1 ,600, 1 ,700, 1 ,800, 1 ,900, 2,000, 2,500, and 3,000 nucleotides).
  • the poly-A sequence is designed according to the descriptions of the poly-A sequence in [0202]-[0204] of International Patent Publication No. WO2019/118919A1 , which is incorporated herein by reference in its entirety.
  • the expression sequence lacks a poly-A sequence (e.g., at the 3’ end of an expression sequence).
  • a circular polyribonucleotide includes a polyA, lacks a polyA, or has a modified polyA to modulate one or more characteristics of the circular polyribonucleotide.
  • the circular polyribonucleotide lacking a polyA or having modified polyA improves one or more functional characteristics, e.g., immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response), half-life, and/or expression efficiency.
  • the circular polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the circular polyribonucleotide) includes at least one expression sequence encoding a therapeutic polypeptide.
  • a therapeutic polypeptide is a polypeptide that when administered to or expressed in a subject provides some therapeutic benefit. Administration to a subject or expression in a subject of a therapeutic polypeptide may be used to treat or prevent a disease, disorder, or condition or a symptom thereof.
  • the circular polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten or more therapeutic polypeptides.
  • the circular polyribonucleotide includes an expression sequence encoding a therapeutic protein.
  • the protein may treat the disease in the subject in need thereof.
  • the therapeutic protein can compensate for a mutated, under-expressed, or absent protein in the subject in need thereof.
  • the therapeutic protein can target, interact with, or bind to a cell, tissue, or virus in the subject in need thereof.
  • a therapeutic polypeptide can be a polypeptide that can be secreted from a cell, or localized to the cytoplasm, nucleus, or membrane compartment of a cell.
  • a therapeutic polypeptide may be a hormone, a neurotransmitter, a growth factor, an enzyme (e.g., oxidoreductase, metabolic enzyme, mitochondrial enzyme, oxygenase, dehydrogenase, ATP - independent enzyme, lysosomal enzyme, desaturase), a cytokine, a transcription factor, an antigen binding polypeptide (e.g., antigen binding antibody or antibody-like fragments, such as single chain antibodies, nanobodies or other Ig heavy chain or light chain containing polypeptides), an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an interferon, an interleukin, a thrombolytic, an antigen (e.g., a tumor, viral, or bacterial antigen), a nuclease (e.g., an endonuclease such as a Cas protein, e.g., Cas9), a membrane protein (e.g.,
  • the therapeutic polypeptide is an antibody, e.g., a full-length antibody, an antibody fragment, or a portion thereof.
  • the antibody expressed by the circular polyribonucleotide can be of any isotype, such as IgA, IgD, IgE, IgG, IgM.
  • the circular polyribonucleotide expresses a portion of an antibody, such as a light chain, a heavy chain, a Fc fragment, a CDR (complementary determining region), a Fv fragment, or a Fab fragment, a further portion thereof.
  • the circular polyribonucleotide expresses one or more portions of an antibody.
  • the circular polyribonucleotide can include more than one expression sequence, each of which expresses a portion of an antibody, and the sum of which can constitute the antibody.
  • the circular polyribonucleotide includes one expression sequence coding for the heavy chain of an antibody, and another expression sequence coding for the light chain of the antibody.
  • the light chain and heavy chain can be subject to appropriate modification, folding, or other post-translation modification to form a functional antibody.
  • circular polyribonucleotides made as described herein are used as effectors in therapy or agriculture.
  • a circular polyribonucleotide made by the methods described herein may be administered to a subject (e.g., in a pharmaceutical or agricultural composition).
  • the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian).
  • the subject is a human.
  • the method subject is a non-human mammal.
  • the subject is a nonhuman mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit).
  • a non-human primate e.g., monkeys, apes
  • ungulate e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys
  • carnivore e.g., dog, cat
  • rodent e.g., rat, mouse
  • lagomorph e.g., rabbit
  • the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).
  • avian taxa Galliformes e.g., chickens, turkeys, pheasants, quail
  • Anseriformes e.g., ducks, geese
  • Paleaognathae e.g., ostriches, emus
  • Columbiformes e.g., pigeons, doves
  • Psittaciformes e.g., par
  • the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc.
  • the subject is an invertebrate agricultural pest or an invertebrate that is parasitic on an invertebrate or vertebrate host.
  • the subject is a plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte.
  • the subject is a eukaryotic alga (unicellular or multicellular).
  • the subject is a plant of agricultural or horticultural importance, such as row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses.
  • the circular polyribonucleotide described herein includes at least one coding sequence encoding a secreted polypeptide effector.
  • exemplary secreted polypeptide effectors or proteins that may be expressed include, e.g., cytokines and cytokine receptors, polypeptide hormones and receptors, growth factors, clotting factors, therapeutic replacement enzymes and therapeutic non- enzymatic effectors, regeneration, repair, and fibrosis factors, transformation factors, and proteins that stimulate cellular regeneration, non-limiting examples of which are described herein, e.g., in the tables below.
  • an effector described herein comprises a cytokine of Table 2, or a functional variant or fragment thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 11 by reference to its UniProt ID.
  • the functional variant binds to the corresponding cytokine receptor with a Kd of no more than 10%, 20%, 30%, 40%, or 50% higher or lower than the Kd of the corresponding wild-type cytokine for the same receptor under the same conditions.
  • the effector comprises a fusion protein comprising a first region (e.g., a cytokine polypeptide of Table 11 or a functional variant or fragment thereof) and a second, heterologous region.
  • the first region is a first cytokine polypeptide of Table 11 .
  • the second region is a second cytokine polypeptide of Table 11 , wherein the first and second cytokine polypeptides form a cytokine heterodimer with each other in a wild-type cell.
  • the polypeptide of Table 11 or functional variant thereof comprises a signal sequence, e.g., a signal sequence that is endogenous to the effector, or a heterologous signal sequence.
  • an effector described herein comprises an antibody or fragment thereof that binds a cytokine of Table 11 .
  • the antibody molecule comprises a signal sequence.
  • an effector described herein comprises a hormone of Table 12, or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 12 by reference to its UniProt ID.
  • the functional variant binds to the corresponding receptor with a Kd of no more than 10%, 20%, 30%, 40%, or 50% higher than the Kd of the corresponding wild-type hormone for the same receptor under the same conditions.
  • the polypeptide of Table 12 or functional variant thereof comprises a signal sequence, e.g., a signal sequence that is endogenous to the effector, or a heterologous signal sequence.
  • an effector described herein comprises an antibody molecule (e.g., an scFv) that binds a hormone of Table 12. In some embodiments, an effector described herein comprises an antibody molecule (e.g., an scFv) that binds a hormone receptor of Table 12. In some embodiments, the antibody molecule comprises a signal sequence.
  • an effector described herein comprises a growth factor of Table 13, or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 13 by reference to its UniProt ID.
  • the functional variant binds to the corresponding receptor with a Kd of no more than 10%, 20%, 30%, 40%, or 50% higher than the Kd of the corresponding wild-type growth factor for the same receptor under the same conditions.
  • the polypeptide of Table 13 or functional variant thereof comprises a signal sequence, e.g., a signal sequence that is endogenous to the effector, or a heterologous signal sequence.
  • an effector described herein comprises an antibody or fragment thereof that binds a growth factor of Table 13.
  • an effector described herein comprises an antibody molecule (e.g., an scFv) that binds a growth factor receptor of Table 13.
  • the antibody molecule comprises a signal sequence.
  • an effector described herein comprises a polypeptide of Table 14, or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 14 by reference to its UniProt ID.
  • the functional variant catalyzes the same reaction as the corresponding wild-type protein, e.g., at a rate no less than 10%, 20%, 30%, 40%, or 50% lower or higher than the wild-type protein.
  • the polypeptide of Table 14 or functional variant thereof comprises a signal sequence, e.g., a signal sequence that is endogenous to the effector, or a heterologous signal sequence.
  • an effector described herein comprises an enzyme of Table 15, or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 15 by reference to its UniProt ID.
  • the functional variant catalyzes the same reaction as the corresponding wild-type protein, e.g., at a rate no less or no more than 10%, 20%, 30%, 40%, or 50% lower than the wild-type protein.
  • Table 15 Exemplary enzymatic effectors for enzyme deficiency
  • a therapeutic polypeptide described herein comprises a polypeptide of Table 16, or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 16 by reference to its UniProt ID. Table 16.
  • a functional variant thereof e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 16 by reference to its UniProt ID. Table 16.
  • Therapeutic polypeptides described herein also include growth factors, e.g., as disclosed in Table 17, or functional variants thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 17 by reference to its NCBI protein accession number. Also included are antibodies or fragments thereof against such growth factors, or miRNAs that promote regeneration and repair.
  • Therapeutic polypeptides described herein also include transformation factors, e.g., protein factors that transform fibroblasts into differentiated cell e.g., factors disclosed in Table 18 or functional variants thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 18 by reference to its UniProt ID.
  • transformation factors e.g., protein factors that transform fibroblasts into differentiated cell e.g., factors disclosed in Table 18 or functional variants thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 18 by reference to its UniProt ID.
  • Table 18 Polypeptides indicated for organ repair by transforming fibroblasts 1 Sequence available on the world wide web internet site ncbi.nlm.nih.gov/gene.
  • Therapeutic polypeptides described herein also include proteins that stimulate cellular regeneration e.g., proteins disclosed in Table 19 or functional variants thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in Table 19 by reference to its UniProt ID.
  • an effector described herein comprises CFTR gene of Table 20, or a functional variant or fragment thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a protein sequence disclosed in Table 20.
  • the circular polyribonucleotide comprises one or more expression sequences (coding sequences) and is configured for persistent expression in a cell of a subject in vivo.
  • the circular polyribonucleotide is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than an earlier time point.
  • the expression of the one or more expression sequences may be either maintained at a relatively stable level or may increase over time. The expression of the expression sequences may be relatively stable for an extended period of time.
  • the expression of the one or more expression sequences in the cell over a time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.
  • the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days.
  • the polyribonucleotide described herein includes at least one expression sequence encoding a plantmodifying polypeptide.
  • a plant-modifying polypeptide refers to a polypeptide that can alter the genetic properties (e.g., increase gene expression, decrease gene expression, or otherwise alter the nucleotide sequence of DNA or RNA), epigenetic properties, or physiological or biochemical properties of a plant in a manner that results in a change in the plant’s physiology or phenotype, e.g. ,an increase or decrease in the plant’s fitness.
  • the polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten or more different plant-modifying polypeptides, or multiple copies of one or more plant-modifying polypeptides.
  • a plant-modifying polypeptide may change the physiology or phenotype of, or increase or decrease the fitness of, a variety of plants, or can be one that affects such change(s) in one or more specific plants (e.g., a specific species or genera of plants).
  • polypeptides that can be used herein can include an enzyme (e.g., a metabolic recombinase, a helicase, an integrase, a RNAse, a DNAse, or a ubiquitination protein), a poreforming protein, a signaling ligand, a cell penetrating peptide, a transcription factor, a receptor, an antibody, a nanobody, a gene editing protein (e.g., CRISPR-Cas endonuclease, TALEN, or zinc finger), riboprotein, a protein aptamer, or a chaperone.
  • an enzyme e.g., a metabolic recombinase, a helicase, an integrase, a RNAse, a DNAse, or a ubiquitination protein
  • a poreforming protein e.g., a signaling ligand, a cell penetrating peptide,
  • the polyribonucleotide described herein includes at least one expression sequence encoding an agricultural polypeptide.
  • An agricultural polypeptide is a polypeptide that is suitable for an agricultural use.
  • an agricultural polypeptide is applied to a plant or seed (e.g., by foliar spray, dusting, injection, or seed coating) or to the plant’s environment (e.g., by soil drench or granular soil application), resulting in an alteration of the plant’s physiology, phenotype, or fitness.
  • Embodiments of an agricultural polypeptide include polypeptides that alter a level, activity, or metabolism of one or more microorganisms resident in or on a plant or non-human animal host, the alteration resulting in an increase in the host’s fitness.
  • the agricultural polypeptide is a plant polypeptide.
  • the agricultural polypeptide is an insect polypeptide.
  • the agricultural polypeptide has a biological effect when contacted with a non-human vertebrate animal, invertebrate animal, microbial, or plant cell.
  • the polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten or more agricultural polypeptides, or multiple copies of one or more agricultural polypeptides.
  • Embodiments of polypeptides useful in agricultural applications include, for example, bacteriocins, lysins, antimicrobial peptides, nodule C-rich peptides, and bacteriocyte regulatory peptides. Such polypeptides can be used to alter the level, activity, or metabolism of target microorganisms for increasing the fitness of insects, such as honeybees and silkworms.
  • Embodiments of agriculturally useful polypeptides include peptide toxins, such as those naturally produced by entomopathogenic bacteria (e.g., Bacillus thuringiensis, Photorhabdus luminescens, Serratia entomophila, or Xenorhabdus nematophila), as is known in the art.
  • Embodiments of agriculturally useful polypeptides include polypeptides (including small peptides such as cyclodipeptides or diketopiperazines) for controlling agriculturally important pests or pathogens, e.g., antimicrobial polypeptides or antifungal polypeptides for controlling diseases in plants, or pesticidal polypeptides (e.g., insecticidal polypeptides or nematicidal polypeptides) for controlling invertebrate pests such as insects or nematodes.
  • polypeptides including small peptides such as cyclodipeptides or diketopiperazines
  • antimicrobial polypeptides or antifungal polypeptides for controlling diseases in plants
  • pesticidal polypeptides e.g., insecticidal polypeptides or nematicidal polypeptides
  • invertebrate pests such as insects or nematodes.
  • Embodiments of agriculturally useful polypeptides include antibodies, nanobodies, and fragments thereof, e.g., antibody or nanobody fragments that retain at least some (e.g., at least 10%) of the specific binding activity of the intact antibody or nanobody.
  • Embodiments of agriculturally useful polypeptides include transcription factors, e.g., plant transcription factors; see., e.g., the “AtTFDB” database listing the transcription factor families identified in the model plant Arabidopsis thaliana), publicly available at agris-knowledgebase.org/AtTFDB/.
  • Embodiments of agriculturally useful polypeptides include nucleases, for example, exonucleases or endonucleases (e.g., Cas nucleases such as Cas9 or Cas12a).
  • Embodiments of agriculturally useful polypeptides further include cell-penetrating peptides, enzymes (e.g., amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones (for example, yeast mating pheromones, invertebrate reproductive and larval signaling pheromones, see, e.g., Altstein (2004) Peptides, 25:1373-1376).
  • enzymes e.g., amylases, cellulases, peptidases, lipases, chitinases
  • peptide pheromones for example, yeast mating pheromones, invertebrate reproductive and larval signaling phe
  • the polyribonucleotide described herein includes at least one termination element. In some embodiments, the polyribonucleotide includes a termination element operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination element.
  • the polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, the polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product.
  • the circular polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element.
  • the circular polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the circular polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product, e.g., peptides or polypeptides, due to lack of ribosome stalling or fall-off. In such an embodiment, rolling circle translation expresses a continuous expression product through each expression sequence.
  • a termination element of an expression sequence can be part of a stagger element.
  • one or more expression sequences in the circular polyribonucleotide includes a termination element.
  • rolling circle translation or expression of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide is performed.
  • the expression product may fall off the ribosome when the ribosome encounters the termination element, e.g., a stop codon, and terminates translation.
  • translation is terminated while the ribosome, e.g., at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.
  • the circular polyribonucleotide includes a termination element at the end of one or more expression sequences.
  • one or more expression sequences includes two or more termination elements in succession.
  • translation is terminated and rolling circle translation is terminated.
  • the ribosome completely disengages with the circular polyribonucleotide.
  • production of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide may require the ribosome to reengage with the circular polyribonucleotide prior to initiation of translation.
  • termination elements include an in-frame nucleotide triplet that signals termination of translation (e.g., UAA, UGA, UAG).
  • one or more termination elements in the circular. polyribonucleotide are frame-shifted termination elements, such as but not limited to, off-frame or -1 and + 1 shifted reading frames (e.g., hidden stop) that may terminate translation.
  • Frame-shifted termination elements include nucleotide triples, TAA, TAG, and TGA that appear in the second and third reading frames of an expression sequence. Frame-shifted termination elements may be important in preventing misreads of mRNA, which is often detrimental to the cell.
  • the termination element is a stop codon.
  • an expression sequence includes a poly-A sequence (e.g., at the 3’ end of an expression sequence, for example 3’ to a termination element).
  • the length of a poly-A sequence is greater than 10 nucleotides in length.
  • the poly-A sequence is greater than 15 nucleotides in length (e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400, 1 ,500, 1 ,600, 1 ,700, 1 ,800, 1 ,900, 2,000, 2,500, and 3,000 nucleotides).
  • 15 nucleotides in length e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400,
  • the poly-A sequence is designed according to the descriptions of the poly-A sequence in [0202]-[0204] of International Patent Publication No. WO2019/118919A1 , which is incorporated herein by reference in its entirety.
  • the expression sequence lacks a poly-A sequence (e.g., at the 3’ end of an expression sequence).
  • a circular polyribonucleotide includes a polyA, lacks a polyA, or has a modified polyA to modulate one or more characteristics of the circular polyribonucleotide.
  • the circular polyribonucleotide lacking a polyA or having modified polyA improves one or more functional characteristics, e.g., immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response), half-life, and/or expression efficiency.
  • a polyribonucleotide may include one or more substitutions, insertions and/or additions, deletions, and covalent modifications with respect to reference sequences, in particular, the parent polyribonucleotide, are included within the scope of this disclosure.
  • a polyribonucleotide includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.).
  • the one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999).
  • the RNA Modification Database 1999 update.
  • the first isolated nucleic acid includes messenger RNA (mRNA).
  • the polyribonucleotide includes at least one nucleoside selected from the group such as those described in [0311 ] of International Patent Publication No. WO2019/118919A1 , which is incorporated herein by reference in its entirety.
  • a polyribonucleotide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage I to the phosphodiester backbone).
  • One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro).
  • modifications e.g., one or more modifications
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • a polyribonucleotide includes at least one N(6)methyladenosine (m6A) modification to increase translation efficiency.
  • the m6A modification can reduce immunogenicity (e.g., reduce the level of one or more marker of an immune or inflammatory response) of the polyribonucleotide.
  • a modification may include a chemical or cellular induced modification.
  • RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.
  • chemical modifications to the ribonucleotides of a polyribonucleotide may enhance immune evasion.
  • the polyribonucleotide may be synthesized and/or modified by methods well established in the art, such as those described in Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, end modifications, e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), base modifications (e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners), removal of bases (abasic nucleotides), or conjugated bases.
  • the modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine.
  • base modifications may modulate expression, immune response, stability, subcellular localization, to name a few functional effects, of the polyribonucleotide.
  • the modification includes a bi-orthogonal nucleotide, e.g., an unnatural base.
  • a bi-orthogonal nucleotide e.g., an unnatural base.
  • sugar modifications e.g., at the 2' position or 4' position
  • replacement of the sugar one or more ribonucleotides of the polyribonucleotide may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages.
  • Specific examples of polyribonucleotide include, but are not limited to, polyribonucleotide including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages.
  • Polyribonucleotides having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • the polyribonucleotide will include ribonucleotides with a phosphorus atom in its internucleoside backbone.
  • Modified polyribonucleotide backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • the polyribonucleot
  • the modified nucleotides which may be incorporated into the polyribonucleotide, can be modified at the internucleoside linkage (e.g., phosphate backbone).
  • the phrases "phosphate” and “phosphodiester” are used interchangeably.
  • Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylenephosphonates).
  • the a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages.
  • Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • Phosphorothioate linked to the polyribonucleotide is expected to reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
  • a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'-0- (l-thiophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine (a- thio-cytidine), 5'-0-(l-thiophosphate)- guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0-(1 -thiophosphate)-pseudouridine).
  • alpha-thio-nucleoside e.g., 5'-0- (l-thiophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine (a- thio-cytidine), 5'-0-(l-thiophosphate)- guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0-(1 -thiophosphate)-pseud
  • internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.
  • a polyribonucleotide may include one or more cytotoxic nucleosides.
  • cytotoxic nucleosides may be incorporated into polyribonucleotide, such as bifunctional modification.
  • Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5- azacytidine, 4'-thio- aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, l-(2-C-cyano-2-deoxy-beta-D-arabino- pentofuranosyl)-cytosine, decitabine, 5- fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5- fluoro-l-(tetrahydrofuran-2- yl)pyrimidine-2,
  • Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D- arabinofuranosylcytosine, N4-octadecyl- 1 -beta-D- arabinofuranosylcytosine, N4- palmitoyl-l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5'-elaidic acid ester).
  • a polyribonucleotide may or may not be uniformly modified along the entire length of the molecule.
  • one or more or all types of nucleotides e.g., naturally occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU
  • the polyribonucleotide includes a pseudouridine.
  • the polyribonucleotide includes an inosine, which may aid in the immune system characterizing the polyribonucleotide as endogenous versus viral RNAs.
  • inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.
  • nucleotides in a polyribonucleotide are modified.
  • the modification may include an m6A, which may augment expression; an inosine, which may attenuate an immune response; pseudouridine, which may increase RNA stability, or translational readthrough (stagger element), an m5C, which may increase stability; and a 2,2,7-trimethylguanosine, which aids subcellular translocation (e.g., nuclear localization).
  • nucleotide modifications may exist at various positions in a polyribonucleotide.
  • nucleotide analogs or other modification(s) may be located at any position(s) of the polyribonucleotide, such that the function of the polyribonucleotide is not substantially decreased.
  • a modification may also be a non-coding region modification.
  • the polyribonucleotide may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.
  • any one or more of A, G, U or C) or any intervening percentage e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1 % to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 90% to 100%, and from 95% to 100%
  • RNA circle can include a DNA sequence of a naturally occurring nucleic acid sequence, a modified version thereof, or a DNA sequence encoding a synthetic polypeptide not normally found in nature (e.g., chimeric molecules or fusion proteins).
  • DNA and RNA molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant techniques, such as site- directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • classic mutagenesis techniques and recombinant techniques such as site- directed mutagenesis
  • chemical treatment of a nucleic acid molecule to induce mutations
  • restriction enzyme cleavage of a nucleic acid fragment ligation of nucleic acid fragments
  • PCR polymerase chain reaction
  • the circular polyribonucleotides may be prepared according to any available technique, including, but not limited to chemical synthesis and enzymatic synthesis.
  • a linear primary construct or linear polyribonucleotide for circularization may be cyclized or concatenated to create a circRNA described herein.
  • the linear polyribonucleotide for circularization may be cyclized in vitro prior to formulation and/or delivery.
  • the circular polyribonucleotide may be in a mixture with linear polyribonucleotides.
  • the linear polyribonucleotides have the same nucleic acid sequence as the circular polyribonucleotides.
  • the mechanism of cyclization or concatenation may occur through methods such as, e.g., chemical, enzymatic, splint ligation, or ribozyme-catalyzed methods.
  • the newly formed 5’-3’ linkage may be an intramolecular linkage or an intermolecular linkage.
  • a splint ligase such as a SplintR® ligase, can be used for splint ligation.
  • a single stranded polynucleotide such as a single-stranded DNA or RNA
  • splint can be designed to hybridize with both termini of a linear polyribonucleotide, so that the two termini can be juxtaposed upon hybridization with the single-stranded splint.
  • Splint ligase can thus catalyze the ligation of the juxtaposed two termini of the linear polyribonucleotide, generating a circRNA.
  • a DNA or RNA ligase may be used in the synthesis of the circular polynucleotides.
  • the ligase may be a circ ligase or circular ligase.
  • either the 5' or 3' end of the linear polyribonucleotide can encode a ligase ribozyme sequence such that during in vitro transcription, the resultant linear polyribonucleotide for circularization includes an active ribozyme sequence capable of ligating the 5' end of the linear polyribonucleotide for circularization to the 3' end of the linear polyribonucleotide for circularization.
  • the ligase ribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
  • a linear polyribonucleotide may be cyclized or concatenated by using at least one non-nucleic acid moiety.
  • the at least one non-nucleic acid moiety may react with regions or features near the 5' terminus or near the 3' terminus of the linear polyribonucleotide for circularization in order to cyclize or concatenate the linear polyribonucleotide for circularization.
  • the at least one non-nucleic acid moiety may be located in or linked to or near the 5' terminus or the 3' terminus of the linear polyribonucleotide for circularization.
  • the non-nucleic acid moieties may be homologous or heterologous.
  • the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage, or a cleavable linkage.
  • the non-nucleic acid moiety is a ligation moiety.
  • the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.
  • a linear polyribonucleotide for circularization may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase).
  • RppH RNA 5' pyrophosphohydrolase
  • apyrase an ATP diphosphohydrolase
  • the 5’ end of at least a portion of the linear polyribonucleotides includes a monophosphate moiety.
  • a population of polyribonucleotides including circular and linear polyribonucleotides is contacted with RppH prior to digesting at least a portion of the linear polyribonucleotides with a 5’ exonuclease and/or a 3’ exonuclease.
  • converting the 5' triphosphate of the linear polyribonucleotide for circularization into a 5' monophosphate may occur by a two-step reaction including: (a) contacting the 5' nucleotide of the linear polyribonucleotide for circularization with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphate.
  • a phosphatase e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase
  • a kinase e.g., Polynucleotide
  • linear polyribonucleotides for circularization may be cyclized or concatenated by self-splicing.
  • the linear polyribonucleotide may include a sequence that mediates self-ligation.
  • the linear polyribonucleotides may include loop E sequence (e.g., in PSTVd) to self-ligate.
  • the linear polyribonucleotide may include a HDV sequence, e.g., HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGAC UGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (SEQ ID NO: 234) (Beeharry et al 2004) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUUUUCUGUAAAGAGGAGACUGCUGGACUC GCCGCCCGAGCC (SEQ ID NO: 235), to self-ligate.
  • a HDV sequence e.g., HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUUUUCUGUAAAGAGGAGACUGCUGGACUC GCCGCCCGAGCC (SEQ ID NO: 235), to self-ligate.
  • the linear polyribonucleotides may include a self-circularizing intron, e.g., a 5' and 3’ slice junction, or a selfcircularizing catalytic intron such as a Group I, Group II, or Group III Introns.
  • group I intron self- splicing sequences may include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena, cyanobacterium Anabaena pre-tRNA-Leu gene, or a Tetrahymena pre-rRNA.
  • the polyribonucleotide includes catalytic intron fragments, such as a 3' half of Group I catalytic intron fragment and a 5' half of Group I catalytic intron fragment.
  • the first and second annealing regions may be positioned within the catalytic intron fragments.
  • Group I catalytic introns are self-splicing ribozymes that catalyze their own excision from mRNA, tRNA, and rRNA precursors via two-metal ion phorphoryl transfer mechanism.
  • the RNA itself self-catalyzes the intron removal without the requirement of an exogenous enzyme, such as a ligase.
  • the 3' half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a cyanobacterium Anabaena pre-tRNA-Leu gene, or a Tetrahymena pre-rRNA.
  • the 3' half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a Cyanobacterium Anabaena pre-tRNA-Leu gene, and the 3’ exon fragment includes the first annealing region and the 5’ exon fragment includes the second annealing region.
  • the first annealing region may include, e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11 , 12,
  • ribonucleotides and the second annealing region may include, e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11 , 12, 13, 14, or 15) ribonucleotides.
  • the 3' half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a Tetrahymena pre-rRNA, and the 3' half of Group I catalytic intron fragment includes the first annealing region and the 5’ exon fragment includes the second annealing region. In some embodiments, the 3' exon includes the first annealing region and the 5’ half of Group I catalytic intron fragment includes the second annealing region.
  • the first annealing region may include, e.g., from 6 to 50, e.g., from 10 to 16 (e.g., 10, 11 , 12, 13, 14, 15, or 16) ribonucleotides
  • the second annealing region may include, e.g., from 6 to 50, e.g., from 10 to 16 (e.g., 10, 11 , 12, 13,
  • the 3' half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a cyanobacterium Anabaena pre-tRNA-Leu gene, a Tetrahymena pre-rRNA, or a T4 phage td gene.
  • the 3' half of Group I catalytic intron fragment and the 5’ Group I catalytic intron fragment are from a T4 phage td gene.
  • the 3' exon fragment may include the first annealing region and the 5’ half of Group I catalytic intron fragment may include the second annealing region.
  • the first annealing region may include, e.g., from 2 to 16, e.g., 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16) ribonucleotides
  • the second annealing region may include, e.g., from 2 to 16, e.g., 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16) ribonucleotides.
  • the 3' half of Group I catalytic intron fragment is the 5’ terminus of the linear polynucleotide.
  • the 5' half of Group I catalytic intron fragment is the 3’ terminus of the linear polyribonucleotide.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCA AGACGAGGGTAAAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAG AATG-3’ (SEQ ID NO: 236).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATC TAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT-3’ (SEQ ID NO: 237).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 236 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 237.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- CTTCTGTTGATATGGATGCAGTTCACAGACTAAATGTCGGTCGGGGAAGATGTATTCTTCTCATA AGATATAGTCGGACCTCCTTAATGGGAGCTAGCGGATGAAGTGATGCAACACTGGAGCCGCT GGGAACTAATTTGTATGCGAAAGTATATTGATTAGTTTTGGAGTACTCG-3’ (SEQ ID NO: 238).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AAATAGCAATATTTACCTTTGGAGGGAAAAGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAAC CAATAGATTGCATCGGTTTAAAAGGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTT CAGTACCAAGTCTCAGGGGAAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGG ACATGGTCCTAACCACGCAGCCAAGTCCTAAGTCAACAGAT-3’ (SEQ ID NO: 239).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 238 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 239.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- GGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAG ACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATT CCGGGGTAAGATTAACGACCTTATCTGAACATAATG-3’ (SEQ ID NO: 240).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- TAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTA GACAATCCCGTGCTAAATTGTAGGACT-3’ (SEQ ID NO: 241 ).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 240 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 241.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- TAAACAACTAACAGCTTTAGAAGGTGCAGAGACTAGACGGGAGCTACCCTAACGGATTCAGCCG AGGGTAAAGGGATAGTCCAATTCTCAACATCGCGATTGTTGATGGCAGCGAAAGTTGCAGAGAGAG AATGAAAATCCGCTGACTGTAAAGGTCGTGAGGGTTCGAGTCCCTCCGCCCCCA-3’ (SEQ ID NO: 242).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- ACGGTAGACGCAGCGGACTTAGAAAACTGGGCCTCGATCGCGAAAGGGATCGAGTGGCAGCTC TCAAACTCAGGGAAACCTAAAACTTTAAACATTMAAGTCATGGCAATCCTGAGCCAAGCTAAAGC- 3’ (SEQ ID NO: 243).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 242 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 243.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- TTAAACTCAAAATTTAAAATCCCAAATTCAAAATTCCGGGAAGGTGCAGAGACTCGACGGGAGCT ACCCTAACGTAAAGCCGAGGGTAAAGGGAGAGTCCAATTCTCAAAGCCTGAAGTTGCTGAAGCA ACAAGGCAGTAGTGAAAGCTGCGAGAGAATGAAAATCCGTTGACTGTAAAAAGTCGTGGGGGTT CAAGTCCCCCCACCCCC-3’ (SEQ ID NO: 244).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTTAAGTGGAAGCTCTC AAATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGCCAAGCCCAGAAAATTTAG ACTTGAGATTTGATTTTGGAG-3’ (SEQ ID NO: 245).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 244 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 245.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- GGCTTTCAATTTGAAATCAGAAATTCAAAATTCAGGGAAGGTGCAGAGACTCGACGGGAGCTACC CTAACGTAAAGGCGAGGGTAAAGGGAGAGTCCAATTCTTAAAGCCTGAAGTTGTGCAAGCAACA AGGCAACAGTGAAAGCTGTGGAAGAATGAAAATCCGTTGACCTTAAACGGTCGTGGGGGTTCAA GTCCCCCCACCCCC-3’ (SEQ ID NO: 246).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTCAAGTGGAAGCTCTC AAATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGCCAAGCCCGGAAATTTTAG AATCAAGATTTTATTTT-3’ (SEQ ID NO: 247).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 246 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 247.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGG GACAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCG AAGGTCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA-3’ (SEQ ID NO: 248).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGG GACAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCG AAGGTCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA-3’ (SEQ ID NO: 249).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 248 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 249.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- ACAACAGATAACTTACTAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCT AACGTCAAGACGAGGGTAAAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTG CGGGAGAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA-3’ (SEQ ID NO: 250).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCAAACTC AGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAG TTAGTAAGTT-3’ (SEQ ID NO: 251 ).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 250 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 251.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AACAACAGATAACTTACTAGTTACTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAAC GTCAAGACGAGGGTAAAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGG GAGAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA-3’ (SEQ ID NO: 252).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTC AGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAG TT-3’ (SEQ ID NO: 253).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 252 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 253.
  • the Group I catalytic intron fragment is from the T4 phage nrdB gene or nrdD gene.
  • the 3' half of Group I catalytic intron fragment of includes a sequence having at least 80% sequence identity to 5’- TTGCAAAACAAGGTTCAACGACTAGTCTTCGGACGTAGGGTCAAGCGACTCGAAATGGGGAGAA TCCCTCCGGGATTGTGATATAGTCTGGACTGCATGGTAACATGCAGCAGTTCATAAGAGAACGG GTTGAGAATTAGCGAGCTCAATCGAACATACG-3’ (SEQ ID NO: 254).
  • the 3' half of Group I catalytic intron fragment of (A) includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- TTGCAAAACAAGGTTCAACGACTAGTCTTCGGACGTAGGGTCAAGCGACTCGAAATGGGGAGAA TCCCTCCGGGATTGTGATATAGTCTGGACTGCATGGTAACATGCAGCAGTTCATAAGAGAACGG GTTGAGAATTAGCGAGCTCAATCGAACATACG-3’ (SEQ ID NO: 254).
  • the 5' half of Group I catalytic intron fragment from the T4 phage nrdB gene. In some embodiments, the 3' half of Group I catalytic intron fragment is from the T4 phage nrdB gene and the 5' half of Group I catalytic intron fragment is from the T4 phage nrdB gene.
  • the 5' half of Group I catalytic intron includes a sequence having at least 80% sequence identity to 5’- AAAATGCGCCTTTAAACGGTAACGTTTATCGAAAACTCCTTTAATTGCTGGAAAGTCCTTTATGGA AAACTAGCAGCCAAGGTTTTGCTT-3’ (SEQ ID NO: 255).
  • the 5' half of Group I catalytic intron includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- AAAATGCGCCTTTAAACGGTAACGTTTATCGAAAACTCCTTTAATTGCTGGAAAGTCCTTTATGGA AAACTAGCAGCCAAGGTTTTGCTT-3’ (SEQ ID NO: 255).
  • the 3' half of Group I catalytic intron fragment is from the T4 phage nrdD gene.
  • the 3' half of Group I catalytic intron fragment includes a sequence having at least 80% sequence identity to 5’- CAGTAGCTGTAAATGCCCAACGACTATCCCTGATGAATGTAAGGGAGTAGGGTCAAGCGACCCG AAACGGCAGACAACTCTAAGAGTTGAAGATATAGTCTGAACTGCATGGTGACATGCAGCTGTTTA TCCTCGTATAAATATGAATACGAGGTGAAACGATGAAATGAATTACATTGTTTCATATAAACGGGT AGAGAAGTAGCGAACTCTACTGAACACATTG-3’ (SEQ ID NO: 256).
  • the 3' half of Group I catalytic intron fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- CAGTAGCTGTAAATGCCCAACGACTATCCCTGATGAATGTAAGGGAGTAGGGTCAAGCGACCCG AAACGGCAGACAACTCTAAGAGTTGAAGATATAGTCTGAACTGCATGGTGACATGCAGCTGTTTA TCCTCGTATAAATATGAATACGAGGTGAAACGATGAAATGAATTACATTGTTTCATATAAACGGGT AGAGAAGTAGCGAACTCTACTGAACACATTG-3’ (SEQ ID NO: 256).
  • the 5' half of Group I catalytic intron fragment is from the T4 phage nrdD gene. In some embodiments, the 3' half of Group I catalytic intron fragment is from the T4 phage nrdD gene and the 5' half of Group I catalytic intron fragment is from the T4 phage nrdD gene.
  • the 5' half of Group I catalytic intron includes a sequence having at least 80% sequence identity to 5’- TAACGTAAGTCAAGCTCATGTAAAATCTGCCTAAAACGGGAAACTCTCACTGAGACAATCCGTTG CTAAATCAG-3’ (SEQ ID NO: 257).
  • the 5' half of Group I catalytic intron includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- TAACGTAAGTCAAGCTCATGTAAAATCTGCCTAAAACGGGAAACTCTCACTGAGACAATCCGTTG CTAAATCAG-3’ (SEQ ID NO: 257).
  • the 3’ exon fragment includes a sequence having at least 80% sequence identity to 5’-GTACCTTTAACTTCCATAAGAACATGGAAATCATGGAAGGTAATGCCAAG- 3’ (SEQ ID NO: 258).
  • the 3’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- GTACCTTTAACTTCCATAAGAACATGGAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 258).
  • the 3’ exon fragment includes a sequence having at least 80% sequence identity to 5’- GTACCTTTAACTTCCAAAAGATACATAAAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 259).
  • the 3’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- GTACCTTTAACTTCCAAAAGATACATAAAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 259).
  • the 5’ exon fragment includes a sequence having at least 80% sequence identity to 5’-TTTTTATGTATCTTTTGCGT-3’ (SEQ ID NO: 260).
  • the 5’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’-TTTTTATGTATCTTTTGCGT-3’ (SEQ ID NO: 260).
  • the 3’ exon fragment Includes a sequence having at least 80% sequence identity to 5’- ATGAAGTGAACACGTTATTCAGTTCAAACGGACAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 261 ).
  • the 3’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- ATGAAGTGAACACGTTATTCAGTTCAAACGGACAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 261 ).
  • the 3’ exon fragment includes a sequence having at least 80% sequence identity to 5’- ATGAAGTGAACACGTTACATAAGCTTGGAATGCAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 262). In some embodiments, the 3’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- ATGAAGTGAACACGTTACATAAGCTTGGAATGCAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 262).
  • the 5’ exon fragment includes a sequence having at least 80% sequence identity to 5’- TGCATTCCAAGCTTATGAGT -3’ (SEQ ID NO: 263).
  • the 5’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- TGCATTCCAAGCTTATGAGT -3’ (SEQ ID NO: 263).
  • a linear polyribonucleotide for circularization may be cyclized or concatenated by a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near, or linked to the 5' and 3' ends of the linear polyribonucleotide for circularization.
  • one or more linear polyribonucleotides is cyclized or concatenated by intermolecular forces or intramolecular forces.
  • intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, Van der Waals forces, and London dispersion forces.
  • Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation and antibonding.
  • a linear polyribonucleotide for circularization may include a ribozyme RNA sequence near the 5' terminus and near the 3' terminus.
  • the ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme.
  • the peptides covalently linked to the ribozyme RNA sequence near the 5’ terminus and the 3 ‘terminus may associate with each other causing a linear polyribonucleotide to cyclize or concatenate.
  • the peptides covalently linked to the ribozyme RNA near the 5' terminus and the 3' terminus may cause the linear primary construct or linear mRNA to cyclize or concatenate after being subjected to ligation using various methods known in the art such as, but not limited to, protein ligation.
  • ribozymes for use in the linear primary constructs or linear polyribonucleotides of the present invention or a non-exhaustive listing of methods to incorporate or covalently link peptides are described in US patent application No. US20030082768, the contents of which is here in incorporated by reference in its entirety.
  • chemical methods of circularization may be used to generate the circular polyribonucleotide.
  • Such methods may include but are not limited to click chemistry (e.g., alkyne and azide-based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal- imine crosslinking, base modification, and any combination thereof.
  • the 5'-end and the 3'-end of a linear polyribonucleotide for circularization includes chemically reactive groups that, when close together, may form a new covalent linkage between the 5'-end and the 3'-end of the molecule.
  • the 5'-end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a linear RNA molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'73'-amide bond.
  • the circular polyribonucleotide may be produced using a deoxyribonucleotide template transcribed in a cell-free system (e.g., by in vitro transcription) to a produce a linear RNA.
  • the linear polyribonucleotide produces a splicing-compatible polyribonucleotide, which may be self-spliced to produce a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide (e.g., in a cell-free system) by providing a linear polyribonucleotide; and selfsplicing linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide; thereby producing a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding the linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide, wherein the transcribing occurs in a solution under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.
  • the linear polyribonucleotide comprises a 5’ split-intron and a 3’ split-intron (e.g., a selfsplicing construct for producing a circular polyribonucleotide). In some embodiments, the linear polyribonucleotide comprises a 5’ annealing region and a 3’ annealing region.
  • Suitable conditions for in vitro transcriptions and or self-splicing may include any conditions (e.g., a solution or a buffer, such as an aqueous buffer or solution) that mimic physiological conditions in one or more respects.
  • suitable conditions include between 0.1 -100mM Mg2+ ions or a salt thereof (e.g., 1 -1 OOmM, 1 -50mM, 1 -20mM, 5- 50mM, 5-20 mM, or 5-15mM).
  • suitable conditions include between 1 -1 OOOmM K+ ions or a salt thereof such as KCI (e.g., 1 -1 OOOmM, 1 -500mM, 1 -200mM, 50- 500mM, 100-500mM, or 100-300mM).
  • suitable conditions include between 1 -1 OOOmM Cl- ions or a salt thereof such as KCI (e.g., 1 -1 OOOmM, 1 -500mM, 1 -200mM, 50- 500mM, 100-500mM, or 100-300mM).
  • suitable conditions include between 0.1 -1 OOmM Mn2+ ions or a salt thereof such as MnCI2 (e.g., 0.1 -100mM, 0.1 -50mM, 0.1 -20mM, 0.1 -10mM, 0.1 -5mM, 0.1 -2mM, 0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5-5mM, 0.5-2mM, or 0.1 -1 OmM).
  • MnCI2 e.g., 0.1 -100mM, 0.1 -50mM, 0.1 -20mM, 0.1 -10mM, 0.1 -5mM, 0.1 -2mM, 0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5-5mM, 0.5-2mM, or 0.1 -1 OmM.
  • suitable conditions include dithiothreitol (DTT) (e.g., 1 -1000 pM, 1 -500 pM, 1 -200pM, 50- 500pM, 100-500pM, 100-300pM, 0.1 - 100mM, 0.1 -50mM, 0.1 -20mM, 0.1 -10mM, 0.1 -5mM, 0.1 -2mM, 0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5-5mM, 0.5-2mM, or 0.1 -1 OmM).
  • DTT dithiothreitol
  • suitable conditions include between 0.1 mM and 10OmM ribonucleoside triphosphate (NTP) (e.g., 0.1 -100 mM, 0.1 -50mM, 0.1 -1 OmM, 1 - 10OmM, 1 -50mM, or 1 -1 OmM).
  • NTP ribonucleoside triphosphate
  • suitable conditions include a pH of 4 to 10 (e.g., pH of 5 to 9, pH of 6 to 9, or pH of 6.5 to 8.5).
  • suitable conditions include a temperature of 4°C to 50°C (e.g., 10°C to 40°C, 15 °C to 40°C, 20°C to 40°C, or 30°C to 40°C),
  • the linear polyribonucleotide is produced from a deoxyribonucleic acid, e.g., a deoxyribonucleic acid described herein, such as a DNA vector, a linearized DNA vector, or a cDNA.
  • the linear polyribonucleotide is transcribed from the deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).
  • the circular polyribonucleotide may be produced in a cell, e.g., a prokaryotic cell or a eukaryotic cell.
  • an exogenous polyribonucleotide is provided to a cell (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding for the transcription of a linear polyribonucleotide described here).
  • the linear polyribonucleotides may be transcribed in the cell from an exogenous DNA molecule provided to the cell.
  • the linear polyribonucleotide may be transcribed in the cell from an exogenous recombinant DNA molecule transiently provided to the cell.
  • the exogenous DNA molecule does not integrate into the cell’s genome.
  • the linear polyribonucleotide is transcribed in the cell from a recombinant DNA molecule that is incorporated into the cell’s genome.
  • the cell is a prokaryotic cell.
  • the prokaryotic cell including the polyribonucleotides described herein is a bacterial cell or an archaeal cell.
  • the prokaryotic cell including the polyribonucleotides described herein may be E coli, halophilic archaea (e.g., Haloferax volcaniii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira) spp., and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp.
  • the prokaryotic cells may be grown in a culture medium.
  • the prokaryotic cells may be contained in a bioreactor.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a unicellular eukaryotic cell.
  • the unicellular eukaryotic is a unicellular fungal cell such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp, and Pichia spp.).
  • the unicellular eukaryotic cell is a unicellular animal cell.
  • a unicellular animal cell may be a cell isolated from a multicellular animal and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular animal cell is dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. A unicellular plant cell may be a cell isolated from a multicellular plant and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular plant cell is dedifferentiated. In some embodiments, the unicellular plant cell is from a plant callus. In embodiments, the unicellular cell is a plant cell protoplast.
  • the unicellular eukaryotic cell is a unicellular eukaryotic algal cell, such as a unicellular green alga, a diatom, an euglenid, or a dinoflagellate.
  • Non-limiting examples of unicellular eukaryotic algae of interest include Du naliella salina, Chlorella vulgaris, Chlorel la zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris spp., Protosiphon botryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas spp.
  • the unicellular eukaryotic cell is a protist cell.
  • the unicellular eukaryotic cell is a protozoan cell.
  • the eukaryotic cell is a cell of a multicellular eukaryote.
  • the multicellular eukaryote may be selected from the group consisting of a vertebrate animal, an invertebrate animal, a multicellular fungus, a multicellular alga, and a multicellular plant.
  • the eukaryotic organism is a human.
  • the eukaryotic organism is a non-human vertebrate animal.
  • the eukaryotic organism is an invertebrate animal.
  • the eukaryotic organism is a multicellular fungus.
  • the eukaryotic organism is a multicellular plant.
  • the eukaryotic cell is a cell of a human or a cell of a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., bovids including cattle, buffalo, bison, sheep, goat, and musk ox; pig; camelids including camel, llama, and alpaca; deer, antelope; and equids including horse and donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare).
  • a non-human primate e.g., monkeys, apes
  • ungulate e.g., bovids including cattle, buffalo, bison, sheep, goat, and musk ox
  • pig camelids including camel, llama, and alpaca
  • the eukaryotic cell is a cell of a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).
  • avian taxa Galliformes e.g., chickens, turkeys, pheasants, quail
  • Anseriformes e.g., ducks, geese
  • Paleaognathae e.g., ostriches, emus
  • Columbiformes e.g., pigeons, doves
  • the eukaryotic cell is a cell of an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc.
  • the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte.
  • the eukaryotic cell is a cell of a eukaryotic multicellular alga.
  • the eukaryotic cells may be grown in a culture medium.
  • the eukaryotic cells may be contained in a bioreactor.
  • any method of producing a circular polyribonucleotide described herein may be performed in a bioreactor.
  • a bioreactor refers to any vessel in which a chemical or biological process is carried out which involves organisms or biochemically active substances derived from such organisms. Bioreactors may be compatible with the cell-free methods for production of circular RNA described herein.
  • a vessel for a bioreactor may include a culture flask, a dish, or a bag that may be single use (disposable), autoclavable, or sterilizable.
  • a bioreactor may be made of glass, or it may be polymer-based, or it may be made of other materials.
  • bioreactors include, without limitation, stirred tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibromixers, fluidized bed reactors, and membrane bioreactors.
  • the mode of operating the bioreactor may be a batch or continuous processes.
  • a bioreactor is continuous when the reagent and product streams are continuously being fed and withdrawn from the system.
  • a batch bioreactor may have a continuous recirculating flow, but no continuous feeding of reagents or product harvest.
  • the method may be performed in a volume of 1 liter (L) to 50 L, or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more).
  • the method may be performed in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L.
  • a bioreactor may produce at least 1g of circular RNA.
  • a bioreactor may produce 1 -200g of circular RNA (e.g., 1 -10g, 1 -20g, 1 -50g, 10-50g, 10-100g, 50-100g, or 50-200g of circular RNA).
  • the amount produced is measured per liter (e.g., 1 -200g per liter), per batch or reaction (e.g., 1 -200g per batch or reaction), or per unit time (e.g., 1 -200g per hour or per day).
  • more than one bioreactor may be utilized in series to increase the production capacity (e.g., one, two, three, four, five, six, seven, eight, or nine bioreactors may be used in series).
  • circularization efficiency is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, circularization efficiency is at least about 40%. In some embodiments, circularization efficiency is between about 10% and about 100%; for example, circularization efficiency is about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 99%. In some embodiments, circularization efficiency is between about 20% and about 80%. In some embodiments, circularization efficiency is between about 30% and about 60%. In some embodiments, circularization efficiency is about 40%.
  • the linear polyribonucleotide is substantively enriched or pure (e.g., purified) prior to self-splicing the linear polyribonucleotide.
  • the linear polyribonucleotide is not purified prior to self-splicing the linear polyribonucleotide.
  • the resulting circular RNA is purified.
  • Purification may include separating or enriching the desired reaction product from one or more undesired components, such as any unreacted stating material, byproducts, enzymes, or other reaction components.
  • purification of linear polyribonucleotide following transcription in a cell-free system may include separation or enrichment from the DNA template prior to self-splicing the linear polyribonucleotide.
  • Purification of the circular RNA product following splicing may be used to separate or enrich the circular polyribonucleotide from its corresponding linear polyribonucleotide.
  • Methods of purification of polyribonucleotides are known to those of skill in the art and include enzymatic purification or by chromatography.
  • the methods of purification result in a circular polyribonucleotide that has less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) linear polyribonucleotides.
  • the reference criterion for the amount of linear polyribonucleotide molecules present in a preparation is the presence of no more than 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 1 pg/ ml, 10 pg/ml, 50 pg/ml, 100 pg/ml, 200 g/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 600 pg/ml, 600 pg/ml, 1
  • the reference criterion for the amount of circular polyribonucleotide molecules present in a preparation is at least 30% (w/w), 40% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 85% (w/w), 90% (w/w), 91 % (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w), 96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.1% (w/w), 99.2% (w/w), 99.3% (w/w), 99.4% (w/w), 99.5% (w/w), 99.6% (w/w), 99.7% (w/w), 99.8% (w/w), 99.9% (w/w), or 100% (w/w) molecules of the total ribonucleotide molecules in the preparation.
  • the reference criterion for the amount of linear polyribonucleotide molecules present in a preparation is no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), 40% (w/w), 50% (w/w) linear polyribonucleotide molecules of the total ribonucleotide molecules in the preparation.
  • the reference criterion for the amount of nicked polyribonucleotide molecules present in a preparation is no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), or 15% (w/w) nicked polyribonucleotide molecules of the total ribonucleotide molecules in the preparation.
  • the reference criterion for the amount of combined nicked and linear polyribonucleotide molecules present in a preparation is no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), 40% (w/w), 50% (w/w) combined nicked and linear polyribonucleotide molecules of the total ribonucleotide molecules in the preparation.
  • a preparation e.g., pharmaceutical preparation
  • a preparation is an intermediate preparation of a final circular polyribonucleotide drug product.
  • a preparation e.g., pharmaceutical preparation
  • a preparation is a drug substance or active pharmaceutical ingredient (API).
  • a preparation is a drug product for administration to a subject.
  • a preparation e.g., pharmaceutical preparation
  • a preparation of circular polyribonucleotides is (before, during or after the reduction of linear polyribonucleotide) further processed to substantially remove DNA, protein contamination (e.g., cell protein such as a host cell protein or protein process impurities), endotoxin, mononucleotide molecules, and/or a process-related impurity.
  • protein contamination e.g., cell protein such as a host cell protein or protein process impurities
  • endotoxin e.g., mononucleotide molecules
  • a process-related impurity e.g., endotoxin, mononucleotide molecules, and/or a process-related impurity.
  • the linear polyribonucleotide comprises the elements as described below as described herein.
  • Linear polyribonucleotides described herein are a polyribonucleotide molecule having a 5’ and 3’ end.
  • the linear RNA has a free 5’ end or 3’ end.
  • the linear RNA has a 5’ end or 3’ end that is modified or protected from degradation.
  • the linear RNA has non-covalently linked 5’ or 3’ ends.
  • the linear RNA is an mRNA.
  • the linear polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1 ,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides
  • the linear polyribonucleotides of the disclosure may include any element or combination of elements described herein, e.g., any element or combination of elements described above with respect to circular polyribonucleotides.
  • a linear polyribonucleotide may include any one or more IRES, signal sequence, regulatory element, cleavage domain, translation initiation sequence, untranslated region, termination element, or modification as described herein (e.g., with respect to circular polyribonucleotide described above).
  • a linear polyribonucleotide may include such elements in any number or configuration described herein (e.g., with respect to circular polyribonucleotide described above).
  • a polyribonucleotide described herein may be administered to a subject (e.g., in a pharmaceutical composition).
  • a circular or linear polyribonucleotide described herein may be administered to a subject (e.g., in a pharmaceutical composition).
  • a circular or linear polyribonucleotide described herein is administered to a subject in an immunogenic composition.
  • Immunogenic compositions of the disclosure may include a diluent or a carrier, adjuvant, or any combination thereof. Immunogenic compositions of the disclosure may also include one or more immunoregulatory agents, e.g., one or more adjuvants. The adjuvants may include a TH1 adjuvant and/or a TH2 adjuvant, further discussed below.
  • the immunogenic composition includes a diluent free of any carrier and is used for naked delivery of the circular polyribonucleotide to a subject.
  • the immunogenic composition includes a diluent free of any carrier and is used for naked delivery of the linear polyribonucleotide to a subject.
  • Immunogenic compositions of the disclosure are used to raise an immune response in a subject.
  • the immune response is preferably protective and preferably involves an antibody response (usually including IgG) and/or a cell-mediated immune response.
  • a subject is immunized with an immunogenic composition including a circular polyribonucleotide of the disclosure to induce an immune response.
  • a subject is immunized with an immunogenic composition including a linear polyribonucleotide including an immunogen to stimulate production of antibodies that bind to the immunogen.
  • the immunogenic compositions are vaccine compositions.
  • Vaccines according to the disclosure may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection) but will typically be prophylactic.
  • the subject is a mammal.
  • the subject is an animal, preferably a mammal, e.g., a human.
  • the subject is a human.
  • the subject is a non-human mammal, e.g., selected from a cow (e.g., dairy and beef cattle), a sheep, a goat, a pig, a horse, a dog, or a cat.
  • the subject is a bird, e.g., a hen or rooster, turkey, parrot.
  • the animal is not a mouse or a rabbit or a cow.
  • the immunogenic composition is for prophylactic use, the human is a child (e.g. a toddler or infant) or a teenager.
  • the immunogenic composition is for therapeutic use, the human is a teenager or an adult.
  • An immunogenic composition intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
  • Immunogenic composition prepared according to the disclosure may be used to treat both children and adults.
  • a human subject may be less than 1 year old, less than 5 years old, 1 -5 years old, 5-15 years old, 15-55 years old, or at least 55 years old.
  • a human subject for receiving the immunogenic compositions are the elderly (e.g., >50 years old, >60 years old, and >65 years), the young (e.g., ⁇ 5 years old), hospitalized patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, or immunodeficient patients.
  • the immunogenic compositions are not suitable solely for these groups, however, and may be used more generally in a population.
  • the subject is further immunized with an adjuvant. In some embodiments the subject is further immunized with a vaccine.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide described herein (e.g., in a pharmaceutical or agricultural composition) may be administered in a single dose or in multiple doses to a cell, tissue, or subject.
  • a method of administering multiple doses of a composition of a polyribonucleotide described herein comprises providing two or more compositions, over a period of time, to a cell, tissue or subject (e.g., a mammal).
  • a composition of a polyribonucleotide described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • multiple doses of a composition of a polyribonucleotide described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • the methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of a composition of a polyribonucleotide described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition).
  • a composition of a polyribonucleotide described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • a pharmaceutical or agricultural composition e.g., in a pharmaceutical or agricultural composition
  • compositions of a polyribonucleotide described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • a predetermined interval e.g., hours, days, weeks or months
  • the present invention provides methods which comprise sequentially administering to the subject a single initial dose of a composition of a polyribonucleotide described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition), followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition.
  • a composition of a polyribonucleotide described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • secondary doses of the composition e.g., in a pharmaceutical or agricultural composition
  • the terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of a composition of a polyribonucleotide described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition).
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”);
  • the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of a composition of a polyribonucleotide described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition), and in certain embodiments, may differ from one another in terms of frequency of administration.
  • a polyribonucleotide described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • a pharmaceutical or agricultural composition e.g., in a pharmaceutical or agricultural composition
  • the amount of a composition of a polyribonucleotide described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition) contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • one or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
  • each secondary and/or tertiary dose is administered after the immediately preceding dose.
  • the phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of the composition of a polyribonucleotide described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition) which is administered to a subject prior to the administration of the very next dose in the sequence with no intervening doses.
  • each secondary and/or tertiary dose is administered every day, every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the immediately preceding dose.
  • each secondary and/or tertiary dose is administered every 0.5 weeks, 1 week, 2 weeks, 3 weeks, or 4 weeks after the immediately preceding dose.
  • the methods according to this aspect of the invention may comprise administering to a subject any number of secondary and/or tertiary doses of a composition of a polyribonucleotide described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition).
  • a composition of a polyribonucleotide described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • a pharmaceutical or agricultural composition e.g., in a pharmaceutical or agricultural composition.
  • only a single secondary dose is administered to the subject.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the subject.
  • only a single tertiary dose is administered to the subject.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are
  • the frequency at which the secondary and/or tertiary doses are administered to a subject can vary over the course of the treatment regimen.
  • the frequency of administration may also be adjusted during the course of treatment.
  • multiple doses are provided to produce a level of the composition or express a level of the encoded polypeptide in a cell, tissue or subject. In some embodiments, multiple doses are provided to produce or maintain a level of the composition, or to produce or maintain a level of the encoded polypeptide, in a cell, tissue or subject for a period of time, for instance, for at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 days, or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 21 , or 24 months, or at least 1 , 2, 3, 4, or 5 years.
  • the method comprises providing (e.g., administering) at least a first composition and a second composition to the cells, tissue, or subject (e.g., a mammal, e.g., a human).
  • the method further comprises providing (e.g., administering) a third composition, fourth composition, fifth composition, sixth composition, seventh composition, eighth composition, ninth composition, tenth composition, or more.
  • additional compositions are provided for the duration of the life of the cell.
  • additional compositions are provided (e.g., administered) while the cell, tissue or subject obtains a benefit from the composition.
  • a first composition in a multiple dosing regimen comprises a first amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) disclosed herein.
  • a second composition in a multiple dosing regimen comprises a second amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) disclosed herein.
  • a third composition, a fourth composition, a fifth composition, a sixth composition, a seventh composition, an eighth composition, a ninth composition, a tenth composition, or more in a multiple dosing regimen comprises a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) disclosed herein.
  • the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the second amount of the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the first amount of the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the third amount of the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the third amount of the polyribonucleotide is the same as the first amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide).
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the polyribonucleotide is the same as the first amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide).
  • the second amount of the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the third amount of the polyribonucleotide is less than the first amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide).
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the polyribonucleotide is less than the first amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide).
  • the second amount of the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the first amount of the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the third amount of the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the first amount of the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the polyribonucleotide is greater than the first amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide).
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of the second composition varies by no more than 1%, 5%, 10%, 15%, 20%, or 25% of an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of the first composition.
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of the second composition is no more than 1%, 5%, 10%, 15%, 20%, or 25% less than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of the first composition.
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a second composition is from 0.1 -fold to 1000-fold higher than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a first composition.
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a second composition is 0.1 -fold, 1 -fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a first composition.
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a subsequent composition is 0.1 -fold, 1 -fold, 5-fold, 10- fold, 100-fold, or 1000-fold higher than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a first composition.
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a second composition is from 0.1 -fold to 1000-fold lower than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a first composition.
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a second composition is 0.1 -fold, 1 -fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide, linear polyribonucleotide) of a subsequent composition is 0.1 -fold, 1 -fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a first composition.
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a subsequent composition is from 0.1 -fold to 1000-fold higher or lower than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a first composition.
  • an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a subsequent composition is 0.1 -fold, 1 -fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher or lower than an amount of the polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) of a first composition.
  • a first composition comprises 1 -fold polyribonucleotide molecule (e.g., circular polyribonucleotide), a second composition comprises 5-fold polyribonucleotide (e.g., circular polyribonucleotide) compared to the first composition, and a third composition comprises 0.2-fold polyribonucleotide (e.g., circular polyribonucleotide) compared to the first composition.
  • the second composition comprises at least 5-fold polyribonucleotide (e.g., circular polyribonucleotide) compared to an amount of polyribonucleotide (e.g., circular polyribonucleotide) of a first composition.
  • the first composition comprises a higher amount of the polyribonucleotide (e.g., circular polyribonucleotide) than the second composition. In some embodiments, the first composition comprises a higher amount of the polyribonucleotide (e.g., circular polyribonucleotides) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition.
  • the polyribonucleotide e.g., circular polyribonucleotide
  • the plurality (e.g., two or more) of compositions of a polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) that are administered in a multiple dosing regimen as described herein are the same compositions. In some embodiments, the plurality (e.g., two or more) of compositions of a polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) that are administered in a multiple dosing regimen as described herein, are different compositions.
  • the same compositions comprise the polyribonucleotides (e.g., circular polyribonucleotides, linear polyribonucleotides).
  • the different compositions comprise the polyribonucleotides (e.g., circular polyribonucleotides, linear polyribonucleotides), or a combination thereof.
  • a composition of the polyribonucleotide e.g., a circular polyribonucleotide, linear polyribonucleotide disclosed herein can induce a response in a subject.
  • the method of administering the polyribonucleotide includes administering to a subject in need thereof the polyribonucleotide for multiple times (multiple doses), e.g., at Ieast 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 100, 150, 200, or 500 times, with an interval of from 1 day to 56 days, such as about 49 days, 42 days, 35 days, 28 days, 21 days, 14 days, or 7 days.
  • the method provided herein includes administering to a subject in need thereof the polyribonucleotide for at least 3 times, with an interval of about 7 days.
  • a level of the encoded polypeptide is maintained at a level with variation of less than 50%, 40%, 30%, 20%, or 10% for a period of longer than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 18, or 20 weeks after the last dose.
  • a level of the encoded polypeptide is maintained at a first level for a period of longer than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first level is higher than a level of the encoded polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).
  • a level of the encoded polypeptide is maintained at a first level for a period of longer than 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first level is higher than a level of the encoded polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).
  • a circular polyribonucleotide as described herein may be included in a composition (e.g., pharmaceutical composition) with a carrier or without a carrier.
  • the linear polyribonucleotide as described herein may be included in a composition (e.g., pharmaceutical composition) with a carrier or without a carrier.
  • compositions described herein may be formulated for example including a carrier, such as a pharmaceutical carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to a subject in need thereof (e.g., a human or non-human agricultural or domestic animal, e.g., cattle, dog, cat, horse, poultry).
  • a carrier such as a pharmaceutical carrier and/or a polymeric carrier, e.g., a liposome
  • transfection e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers
  • electroporation or other methods of membrane disruption e.g., nucleofection
  • viral delivery e.g., lentivirus, retrovirus, adenovirus, AAV
  • microinjection e.g., lentivirus, retrovirus, adenovirus, AAV
  • microinjection e.g., lentivirus, retrovirus, adenovirus, AAV
  • microinjection e.g., lentivirus, retrovirus, adenovirus, AAV
  • microinjection e.g., lentivirus, retrovirus, adenovirus, AAV
  • microinjection e.g., lentivirus, retrovirus, adenovirus, AAV
  • microinjection e.g., lentivirus, retrovirus, adenovirus, AAV
  • microprojectile bombardment e gun
  • fugene
  • compositions can be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • suitable aqueous and nonaqueous compositions which may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polyribonucleotide described herein (e.g., circular polyribonucleotide or linear polyribonucleotide)) in the required amount in an appropriate solvent with one or a combination of ingredients e.g., as described herein, as required, followed by sterilization microfiltration.
  • the active compound e.g., a polyribonucleotide described herein (e.g., circular polyribonucleotide or linear polyribonucleotide)
  • an appropriate solvent e.g., a combination of ingredients e.g., as described herein, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g., from those described herein.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously steri le-filtered solution thereof.
  • Polyribonucleotides e.g., circular polyribonucleotides, linear polyribonucleotides
  • a composition that protects against rapid release such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • a composition of the disclosure can be, for example, an immediate release form or a controlled release formulation.
  • An immediate release formulation can be formulated to allow the compounds (e.g., agents, such as a circular polyribonucleotide or linear polyribonucleotide) to act rapidly.
  • Nonlimiting examples of immediate release formulations include readily dissolvable formulations.
  • a controlled release formulation can be a pharmaceutical formulation that has been adapted such that release rates and release profiles of the active agent can be matched to physiological and chronotherapeutic requirements or, alternatively, has been formulated to effect release of an active agent at a programmed rate.
  • Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin), other gelling agents (e.g., gel-forming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through), granules within a matrix, polymeric mixtures, and granular masses.
  • hydrogels e.g., of synthetic or natural origin
  • other gelling agents e.g., gel-forming dietary fibers
  • matrix-based formulations e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through
  • compositions can include aqueous solutions of the active compounds (e.g., polyribonucleotides (e.g., circular polyribonucleotide or linear polyribonucleotide) in water soluble form.
  • Suspensions of the active compounds can be prepared as oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension can also contain suitable stabilizers or agents which increase the solubility of the agents to allow for the preparation of highly concentrated solutions.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.
  • compositions comprising the polyribonucleotides described herein include formulating the polyribonucleotides with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition.
  • Solid compositions include, for example, powders, dispersible granules, and cachets.
  • Liquid compositions include, for example, solutions in which a polyribonucleotide is dissolved, emulsions comprising a polyribonucleotide, or a solution containing liposomes, micelles, or nanoparticles comprising a polyribonucleotide as disclosed herein.
  • Semi-solid compositions include, for example, gels, suspensions, and creams.
  • compositions can be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.
  • Non-limiting examples of dosage forms suitable for use in the disclosure include liquid, powder, gel, nanosuspension, nanoparticle, microgel, aqueous or oily suspensions, emulsion, and any combination thereof.
  • the formulation contains a thermal stabilizer, such as a sugar or sugar alcohol, for example, sucrose, sorbitol, glycerol, trehalose, or mannitol, or any combination thereof.
  • a thermal stabilizer such as a sugar or sugar alcohol, for example, sucrose, sorbitol, glycerol, trehalose, or mannitol, or any combination thereof.
  • the stabilizer is a sugar.
  • the sugar is sucrose, mannitol, or trehalose.
  • polyribonucleotides are delivered in a naked delivery formulation.
  • a naked delivery formulation delivers a polyribonucleotide (e.g., a circular polyribonucleotide or linear polyribonucleotide) to a cell without the aid of a carrier and without covalent modification of the polyribonucleotide (e.g., circular polyribonucleotide or linear polyribonucleotide) or partial or complete encapsulation of the polyribonucleotide (e.g., circular polyribonucleotide or linear polyribonucleotide).
  • the circular polyribonucleotide molecule as described herein is delivered to a cell, tissue, or subject, includes administering the composition (e.g., pharmaceutical composition) as described herein to the cell, tissue, or subject.
  • the method of delivering is an in vivo method.
  • a method of delivery of a circular polyribonucleotide as described herein includes parenterally administering to a subject in need thereof, the composition, drug substance, or drug product as described herein to the subject in need thereof.
  • a method of delivering a circular polyribonucleotide to a cell or tissue of a subject includes administering parenterally to the cell or tissue the composition, drug substance, or drug product as described herein.
  • the circular polyribonucleotide is in an amount effective to elicit a biological response in the subject.
  • the circular polyribonucleotide is an amount effective to have a biological effect on the cell or tissue in the subject.
  • the composition, drug substance, or drug product is administered parenterally.
  • the composition, drug substance, or drug product is administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, intralymphatically, subcutaneously, or intramuscularly.
  • parenteral administration is intravenously, intramuscularly, ophthalmically, subcutaneously, intradermally, or topically.
  • the composition, drug substance, or drug product as described herein is administered intravenously. In some embodiments, the composition, drug substance, or drug product as described herein is administered intramuscularly. In some embodiments, the composition, drug substance, or drug product as described herein is administered subcutaneously. In some embodiments, the composition, drug substance, or drug product as described herein is administered topically. In some embodiments, the composition, drug substance, or drug product is administered intratracheally.
  • the composition, drug substance, or drug product as described herein is administered orally. In some embodiments, the composition, drug substance, or drug product as described herein is administered nasally (e.g., intranasally). In some embodiments, the composition, drug substance, or drug product as described herein is administered by inhalation. In some embodiments, the composition, drug substance, or drug product as described herein is administered opthalmically. In some embodiments, the composition, drug substance, or drug product as described herein is administered rectally. In some embodiments, the composition, drug substance, drug product is administered via intraocular administration, intracochlear (inner ear) administration, or intratracheal administration.
  • the composition, drug substance, or drug product is administered by injection.
  • the administration can be systemic administration or local administration.
  • the composition, drug substance, or drug product is administered via intraocular administration, intracochlear (inner ear) administration, or intratracheal administration.
  • the composition, drug substance, or drug product is administered prenatally, neonatally or postnatally.
  • any of the methods of delivery as described herein are performed with a carrier. In some embodiments, any methods of delivery as described herein are performed without the aid of a carrier in a naked delivery formulation.
  • the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is an immune cell. In some embodiments, the tissue is a connective tissue, a muscle tissue, a nervous tissue, or an epithelial tissue. In some embodiments, the tissue is an organ (e.g., liver, lung, spleen, kidney, etc.). In some embodiments, the subject is a mammal.
  • a pharmaceutical formulation disclosed herein can comprise: (i) a compound (e.g., circular polyribonucleotide) disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and (v) a stabilizer.
  • a pharmaceutical formulation disclosed herein can comprise: (i) a compound (e.g., linear polyribonucleotide) disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and (v) a stabilizer.
  • the pharmaceutical formulation disclosed herein is a stable liquid pharmaceutical formulation.
  • an adjuvant enhances the immune responses (humoral and/or cellular) elicited in a subject who receives the adjuvant and/or an immunogenic composition including the adjuvant.
  • an adjuvant is administered to a subject as disclosed herein.
  • an adjuvant is used in the methods described herein to produce an immune response as described herein.
  • an adjuvant and polyribonucleotide are co-administered in separate compositions.
  • an adjuvant is mixed or formulated with a polyribonucleotide in a single composition and administered to a subject.
  • an adjuvant and circular or linear polyribonucleotide are co-administered in separate compositions.
  • an adjuvant is mixed or formulated with a linear or circular polyribonucleotide in a single composition to obtain an immunogenic composition that is administered to a subject.
  • An adjuvant may be formulated with a polyribonucleotide in the same pharmaceutical composition.
  • An adjuvant may be administered separately (e.g., as a separate pharmaceutical composition) in combination with a polyribonucleotide.
  • Adjuvants may be a TH1 adjuvant and/or a TH2 adjuvant. Further adjuvants contemplated by this disclosure include, but are not limited to, one or more of the following:
  • Mineral-containing compositions suitable for use as adjuvants in the disclosure include mineral salts, such as aluminum salts, and calcium salts.
  • the disclosure includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc., or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.).
  • Calcium salts include calcium phosphate (e.g., the “CAP”).
  • Aluminum salts include hydroxides, phosphates, sulfates, and the like.
  • Oil emulsion compositions suitable for use as adjuvants in the disclosure include squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80 and 0.5% Span, formulated into submicron particles using a microfluidizer), AS03 (a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion), Montanide formulations (e.g. Montanide ISA 51 , Montanide ISA 720), incomplete Freunds adjuvant (I FA), complete Freund’s adjuvant (CFA), and incomplete Freund’s adjuvant (I FA).
  • squalene-water emulsions such as MF59 (5% Squalene, 0.5% Tween 80 and 0.5% Span, formulated into submicron particles using a microfluidizer
  • AS03 a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion
  • Small molecules suitable for use as adjuvants in the disclosure include imiquimod or 847, resiquimod or R848, or gardiquimod.
  • Polymeric nanoparticles suitable for use as an adjuvant in the disclosure include poly(a-hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones or polyester-amides, and combinations thereof.
  • Saponin i.e., a glycoside, polycyclic aglycones attached to one or more sugar side chains.
  • Saponin formulations suitable for use as an adjuvant in the disclosure include purified formulations, such as QS21 , as well as lipid formulations, such as ISCOMs and ISCOMs matrix.
  • QS21 is marketed as STIMULON (TM).
  • Saponin formulations may also include a sterol, such as cholesterol. Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs).
  • ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs.
  • the ISCOM includes one or more of QuilA, QHA & QHC.
  • the ISCOMS may be devoid of additional detergent.
  • Adjuvants suitable for use in the disclosure include non-toxic derivatives of enterobacterial lipopolysaccharide (LPS). Such derivatives include monophosphoryl lipid A (MPLA), glucopyranosyl lipid A (GLA) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g., RC-529.
  • Liposomes suitable for use as an adjuvant in the disclosure include virosomes and CAF01 .
  • Lipid nanoparticles lipid nanoparticles
  • Adjuvants suitable for use in the disclosure include lipid nanoparticles (LNPs) and their components.
  • Lipopeptides i.e., compounds including one or more fatty acid residues and two or more amino acid residues. Lipopeptide suitable for use as an adjuvant in the disclosure include Pam2 (Pam2CSK4) and Pam3 (Pam3CSK4).
  • Glycolipids suitable for use as an adjuvant in the disclosure include cord factor (trehalose dimycolate).
  • Peptides and peptidoglycans derived from (synthetic or purified) gram-negative or grampositive bacteria, such as MDP (N-acetyl-muramyl-L-alanyl-D-isoglutamine) are suitable for use as an adjuvant in the disclosure.
  • Carbohydrates (carbohydrate containing) or polysaccharides suitable for use as an adjuvant include dextran (e.g., branched microbial polysaccharide), dextran-sulfate, lentinan, zymosan, betaglucan, deltin, mannan, and chitin.
  • RNA based adjuvants include dextran (e.g., branched microbial polysaccharide), dextran-sulfate, lentinan, zymosan, betaglucan, deltin, mannan, and chitin.
  • RNA based adjuvants suitable for use in the disclosure are poly IC, poly IC:LC, hairpin RNAs with or without a 5’triphosphate, viral sequences, polyU containing sequence, dsRNA natural or synthetic RNA sequences, and nucleic acid analogs (e.g., cyclic GMP- AMP or other cyclic dinucleotides e.g., cyclic di-GMP, immunostimulatory base analogs e.g., C8- substituted and N7,C8-disubstituted guanine ribonucleotides).
  • the adjuvant is the linear polyribonucleotide counterpart of the circular polyribonucleotide described herein.
  • DNA based adjuvants suitable for use in the disclosure include CpGs, dsDNA, and natural or synthetic immunostimulatory DNA sequences.
  • Proteins or peptides suitable for use as an adjuvant in the disclosure include flagellin-fusion proteins, MBL (mannose-binding lectin), cytokines, and chemokines.
  • Viral particles suitable for use as an adjuvant include virosomes (phospholipid cell membrane bilayer).
  • An adjuvant for use in the disclosure may be bacterial derived, such as a flagellin, LPS, or a bacterial toxin (e.g., enterotoxins (protein), e.g., heat-labile toxin or cholera toxin).
  • An adjuvant for use in the disclosure may be a hybrid molecule such as CpG conjugated to imiquimod.
  • An adjuvant for use in the disclosure may be a fungal or oomycete microbe-associated molecular patterns (MAMPs), such as chitin or beta-glucan.
  • MAMPs microbe-associated molecular patterns
  • an adjuvant is an inorganic nanoparticle, such as gold nanorods or silica-based nanoparticles (e.g., mesoporous silica nanoparticles (MSN)).
  • an adjuvant is a multi-component adjuvant or adjuvant system, such as AS01 , AS03, AS04 (MLP5 + alum), CFA (complete Freund’s adjuvant: IFA + peptiglycan + trehalose dimycolate), CAF01 (two component system of cationic liposome vehicle (dimethyl dioctadecyl-ammonium (DDA)) stabilized with a glycolipid immunomodulator (trehalose 6,6- dibehenate (TDB), which can be a synthetic variant of cord factor located in the mycobacterial cell wall).
  • DDA dimethyl dioctadecyl-ammonium
  • a subject is administered a circular or linear polyribonucleotide encoding one or more immunogens in combination with an adjuvant.
  • the term “in combination with” as used throughout the description includes any two compositions administered as part of a vaccination regimen. This may include, for example, a polyribonucleotide and an adjuvant formulated as a single pharmaceutical composition. This also includes, for example, a polyribonucleotide and an adjuvant administered to a subject as separate compositions according to a defined vaccination regimen. An adjuvant may be administered to a subject before, at the same time, or after the administration of a polyribonucleotide.
  • An adjuvant may be administered within 1 day, 2 days, 5 days, 10 days, 20 days, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months before or after administration of a polyribonucleotide.
  • An adjuvant may be administered by the same route of administration (e.g., intramuscularly, subcutaneously, intravenously, intraperitoneally, topically, or orally) or a different route than a polyribonucleotide.
  • a second agent is also administered to the subject, e.g., a second vaccine is also administered to a subject.
  • a composition that is administered to a subject includes a circular polyribonucleotide described herein and a second vaccine.
  • a vaccine and circular polyribonucleotide are coadministered in separate compositions. The vaccine is simultaneously administered with the circular polyribonucleotide immunization, administered before the circular polyribonucleotide immunization, or after the circular polyribonucleotide immunization.
  • a subject is immunized with a non-circular polyribonucleotide vaccine (e.g., protein subunit vaccine) and an immunogenic composition including a circular polyribonucleotide.
  • a subject is immunized with a nonpolyribonucleotide vaccine for a first microorganism (e.g., pneumococcus) and an immunogenic composition including a circular polyribonucleotide as disclosed herein.
  • a vaccine can be any bacterial infection vaccine or viral infection vaccine.
  • a vaccine is a pneumococcal polysaccharide vaccine, such as PCV13 or PPSV23.
  • the vaccine is an influenza vaccine.
  • the vaccine is an RSV vaccine (e.g., palivizumap).
  • a composition that is administered to a subject includes a linear polyribonucleotide and a vaccine.
  • a vaccine and linear polyribonucleotide are co-administered in separate compositions.
  • the vaccine is simultaneously administered with the linear polyribonucleotide immunization, administered before the linear polyribonucleotide immunization, or after the linear polyribonucleotide immunization.
  • a subject is immunized with a polyribonucleotide (e.g., non-linear polyribonucleotide) vaccine (e.g., protein subunit vaccine) and an immunogenic composition including a linear polyribonucleotide as disclosed herein including a sequence encoding an immunogen.
  • a subject is immunized with a non-polyribonucleotide vaccine for a first microorganism (e.g., pneumococcus) and an immunogenic composition including a linear polyribonucleotide as disclosed herein including a sequence encoding an immunogen.
  • a vaccine can be any bacterial infection vaccine or viral infection vaccine.
  • a vaccine is a pneumococcal polysaccharide vaccine, such as PCV13 or PPSV23.
  • the vaccine is an influenza vaccine.
  • the vaccine is an RSV vaccine (e.g., palivizumap).
  • methods of the disclosure include immunizing a subject with an immunogenic composition including a circular polyribonucleotide as disclosed herein.
  • an immunogen is expressed from the circular polyribonucleotide.
  • immunization induces an immune response in a subject against the immunogen expressed from the circular polyribonucleotide.
  • immunization induces an immune response in a subject (e.g., induces the production of antibodies that bind to the immunogen expressed from the circular polyribonucleotide).
  • an immunogenic composition includes the circular polyribonucleotide and a diluent, carrier, first adjuvant or a combination thereof in a single composition.
  • the subject is further immunized with a second adjuvant. In some embodiments, the subject is further immunized with a vaccine. In some embodiments, the first and/or second adjuvant may be encoded by a separate circular polyribonucleotide.
  • methods of the disclosure include immunizing a subject with an immunogenic composition including a linear polyribonucleotide as disclosed herein.
  • an immunogen is expressed from the linear polyribonucleotide.
  • immunization induces an immune response in a subject against the immunogen expressed from the linear polyribonucleotide.
  • immunization induces the production of antibodies that bind to the immunogen expressed from the linear polyribonucleotide.
  • immunization induces a cell-mediated immune response.
  • an immunogenic composition includes the linear polyribonucleotide and a diluent, carrier, first adjuvant or a combination thereof in a single composition.
  • the subject is further immunized with a second adjuvant.
  • the subject is further immunized with a vaccine.
  • the subject is immunized with one or more immunogenic composition(s) including any number of circular polyribonucleotides.
  • the subject is immunized with, for example, one or more immunogenic composition(s) including at least 1 circular polyribonucleotide.
  • a non-human animal having a non-humanized immune system is immunized with, for example, one or more immunogenic composition(s) including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 20 different circular polyribonucleotides, or more different circular polyribonucleotides.
  • a subject is immunized with one or more immunogenic composition(s) including at most 1 circular polyribonucleotide.
  • a non-human animal having a humanized immune system is immunized with one or more immunogenic composition(s) including at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 20 different circular polyribonucleotides, or less than 21 different circular polyribonucleotides.
  • a subject is immunized with one or more immunogenic composition(s) including about 1 circular polyribonucleotide.
  • a non-human animal having a humanized immune system is immunized with one or more immunogenic composition(s) including about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, or about 20 different circular polyribonucleotides.
  • a subject is immunized with one or more immunogenic composition(s) including about 1 -20, 1 -15, 1 -10, 1 -9, 1 -8, 1 -7, 1 -6, 1 -5, 1 -4, 1 -3, 1 -2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 5-10, 10-15, or 15-20 different circular polyribonucleotides.
  • Different circular polyribonucleotides have different sequences from each other. For example, they can include or encode different immunogens, overlapping immunogens, similar immunogens, or the same immunogens (for example, with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the disclosure).
  • the two or more different circular polyribonucleotides can be in the same or different immunogenic compositions and immunized at the same time or at separate times.
  • the immunogenic compositions including two or more different circular polyribonucleotides can be administered to the same anatomical location or different anatomical locations.
  • the subject can be immunized with one or more immunogenic composition(s) including any number of linear polyribonucleotides.
  • the subject is immunized with, for example, one or more immunogenic composition(s) including at least 1 linear polyribonucleotide.
  • a non-human animal having a non-humanized immune system is immunized with, for example, one or more immunogenic composition(s) including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 20 different linear polyribonucleotides, or more different linear polyribonucleotides.
  • a subject is immunized with one or more immunogenic composition(s) including at most 1 linear polyribonucleotide.
  • a non-human animal having a humanized immune system is immunized with one or more immunogenic composition(s) including at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 20 different linear polyribonucleotides, or less than 21 different linear polyribonucleotides.
  • a subject is immunized with one or more immunogenic composition(s) including about 1 linear polyribonucleotide.
  • a non-human animal having a humanized immune system is immunized with one or more immunogenic composition(s) including about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, or about 20 different linear polyribonucleotides.
  • a subject is immunized with one or more immunogenic composition(s) including about 1 -20, 1 -15, 1 -10, 1 -9, 1 -8, 1 -7, 1 -6, 1 -5, 1 -4, 1 -3, 1 -2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 5-10, 10-15, or 15-20 different linear polyribonucleotides.
  • Different linear polyribonucleotides can have different sequences from each other. For example, they can include or encode different immunogens, overlapping immunogens, similar immunogens, or the same immunogens (for example, with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the disclosure).
  • the two or more different linear polyribonucleotides can be in the same or different immunogenic compositions and immunized at the same time or at separate times.
  • the immunogenic compositions including two or more different linear polyribonucleotides can be administered to the same anatomical location or different anatomical locations.
  • the two or more different linear polyribonucleotides can include or encode immunogens from the same source, different source, or different combinations of sources disclosed herein.
  • the two or more different linear polyribonucleotides can include or encode immunogens from the same virus or from different viruses, for example, different isolates.
  • the subject is immunized with one or more immunogenic composition(s) including any number of circular polyribonucleotides and one or more immunogenic composition(s) including any number of linear polyribonucleotides as disclosed herein.
  • an immunogenic composition disclosed herein includes one or more circular polyribonucleotides and one or more linear polyribonucleotides as disclosed herein.
  • an immunogenic composition includes a circular polyribonucleotide and a diluent, a carrier, a first adjuvant, or a combination thereof.
  • an immunogenic composition includes a circular polyribonucleotide described herein and a carrier or a diluent free of any carrier.
  • an immunogenic composition including a circular polyribonucleotide with a diluent free of any carrier is used for naked delivery of the circular polyribonucleotide to a subject.
  • an immunogenic composition includes a circular polyribonucleotide described herein and a first adjuvant.
  • a subject is further administered a second adjuvant.
  • An adjuvant enhances the innate immune response, which in turn, enhances the adaptive immune response in a subject.
  • An adjuvant can be any adjuvant as discussed below.
  • an adjuvant is formulated with the circular polyribonucleotide as a part of an immunogenic composition.
  • an adjuvant is not part of an immunogenic composition including the circular polyribonucleotide.
  • an adjuvant is administered separately from an immunogenic composition including the circular polyribonucleotide.
  • the adjuvant is coadministered (e.g., administered simultaneously) or administered at a different time than an immunogenic composition including the circular polyribonucleotide to the subject.
  • the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an immunogenic composition including the circular polyribonucleotide.
  • the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an immunogenic composition including the circular polyribonucleotide.
  • the adjuvant is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an immunogenic composition including the circular polyribonucleotide.
  • the adjuvant is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, before an immunogenic composition including the circular polyribonucleotide.
  • the adjuvant is administered to the same anatomical location or different anatomical location as the immunogenic composition including the circular polyribonucleotide.
  • an immunogenic composition includes a linear polyribonucleotide and a diluent, a carrier, a first adjuvant, or a combination thereof.
  • an immunogenic composition includes a linear polyribonucleotide described herein and a carrier or a diluent free of any carrier.
  • an immunogenic composition including a linear polyribonucleotide with a diluent free of any carrier is used for naked delivery of the linear polyribonucleotide to a subject.
  • an immunogenic composition includes a linear polyribonucleotide described herein and a first adjuvant.
  • a subject is further administered a second adjuvant.
  • An adjuvant enhances the innate immune response, which in turn, enhances the adaptive immune response in a subject.
  • An adjuvant can be any adjuvant as discussed below.
  • an adjuvant is formulated with the linear polyribonucleotide as a part of an immunogenic composition.
  • an adjuvant is not part of an immunogenic composition including the linear polyribonucleotide.
  • an adjuvant is administered separately from an immunogenic composition including the linear polyribonucleotide.
  • the adjuvant is coadministered (e.g., administered simultaneously) or administered at a different time than an immunogenic composition including the linear polyribonucleotide to the subject.
  • the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an immunogenic composition including the linear polyribonucleotide.
  • the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an immunogenic composition including the linear polyribonucleotide.
  • the adjuvant is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an immunogenic composition including the linear polyribonucleotide.
  • the adjuvant is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, before an immunogenic composition including the linear polyribonucleotide.
  • the adjuvant is administered to the same anatomical location or different anatomical location as the immunogenic composition including the linear polyribonucleotide.
  • a subject is further immunized with a second agent, e.g., a vaccine (as described below) that is not a circular polyribonucleotide.
  • a second agent e.g., a vaccine (as described below) that is not a circular polyribonucleotide.
  • the vaccine is co-administered (e.g., administered simultaneously) or administered at a different time than an immunogenic composition including the circular polyribonucleotide to the subject.
  • the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an immunogenic composition including the circular polyribonucleotide.
  • the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an immunogenic composition including the circular polyribonucleotide.
  • the vaccine is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an immunogenic composition including the circular polyribonucleotide.
  • the vaccine is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, before an immunogenic composition including the circular polyribonucleotide.
  • a subject is further immunized with a second agent, e.g., a vaccine (as described below) that is not a linear polyribonucleotide.
  • a second agent e.g., a vaccine (as described below) that is not a linear polyribonucleotide.
  • the vaccine is co-administered (e.g., administered simultaneously) or administered at a different time than an immunogenic composition including the linear polyribonucleotide to the subject.
  • the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an immunogenic composition including the linear polyribonucleotide.
  • the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an immunogenic composition including the linear polyribonucleotide.
  • the vaccine is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an immunogenic composition including the linear polyribonucleotide.
  • the vaccine is administered 1 , 2, 3, 4, 5, 6, 7, 14, 21 , 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, before an immunogenic composition including the linear polyribonucleotide.
  • a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof any suitable number of times to achieve a desired response.
  • a prime-boost immunization strategy can be utilized to elicit systemic and/or mucosal immunity.
  • a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure, for example, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 15 times, or more.
  • a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, or at most 20 times, or less.
  • an immunogenic composition e.g., adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, or at most 20 times, or less.
  • vaccine e.g., protein subunit vaccine
  • a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times.
  • an immunogenic composition e.g., protein subunit vaccine
  • vaccine e.g., protein subunit vaccine
  • a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure once. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure twice. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure three times. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure four times.
  • an immunogenic composition, adjuvant, vaccine e.g., protein subunit vaccine
  • a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure five times. In some embodiments, a subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure seven times.
  • an immunogenic composition, adjuvant, vaccine e.g., protein subunit vaccine
  • Suitable time intervals can be selected for spacing two or more immunizations.
  • the time intervals can apply to multiple immunizations with the same immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combination thereof, for example, the same immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combination thereof, can be administered in the same amount or a different amount, via the same immunization route or a different immunization route.
  • the time intervals can apply to multiple immunizations with a different immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combination thereof, for example, a different immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combination thereof, can be administered in the same amount or a different amount, via the same immunization route or a different immunization route.
  • the time intervals can apply to immunizations with different agents, for example, a first immunogenic composition including a first circular polyribonucleotide and a second immunogenic composition including a second circular polyribonucleotide.
  • the time intervals can apply to immunizations with different agents, for example, a first immunogenic composition including a first circular polyribonucleotide and a second immunogenic composition including a protein immunogen (e.g., a protein subunit).
  • the time intervals can apply to a first immunogenic composition including a first linear polyribonucleotide and a second immunogenic composition including a second linear polyribonucleotide.
  • the time intervals between immunizations can be the same or different.
  • At least 1 at least 2, at least 3, at least 4, at least 5, at least 6, at least
  • At most 1 at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 24, at most 36, or at most 72 hours, or less elapse between two immunizations.
  • the method includes pre-administering to the subject an agent to improve immunogenic responses to a circular polyribonucleotide including a sequence encoding an immunogen.
  • the agent is the immunogen as disclosed herein (e.g., a protein immunogen).
  • the method includes administering the protein immunogen from 1 to 7 days prior to administration of the circular polyribonucleotide including the sequence encoding the protein immunogen.
  • the protein immunogen is administered 1 , 2, 3, 4, 5, 6, or 7 days prior to administration of the circular polyribonucleotide including the sequence encoding the protein immunogen.
  • the method includes administering the protein immunogen from 1 to 7 days prior to administration of the linear polyribonucleotide including the sequence encoding the protein immunogen.
  • the protein immunogen is administered 1 , 2, 3, 4, 5, 6, or 7 days prior to administration of the linear polyribonucleotide including the sequence encoding the protein immunogen.
  • the protein immunogen may be administered as a protein preparation, encoded in a plasmid (pDNA), presented in a virus-like particle (VLP), formulated in a lipid nanoparticle, or the like.
  • the method includes administering to the subject an agent to improve immunogenic responses to a circular polyribonucleotide including a sequence encoding an immunogen after the subject has been administered the circular polyribonucleotide including a sequence encoding an immunogen.
  • the agent is the immunogen as disclosed herein (e.g., a protein immunogen).
  • the circular polyribonucleotide includes a sequence encoding a protein immunogen.
  • the method includes administering the protein immunogen within 1 year (e.g., within 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, and 1 month) of administering the circular polyribonucleotide including a sequence encoding the immunogen to the subject.
  • the method includes administering any one of the circular polyribonucleotides described herein or any one of the immunogenic compositions described herein and a protein subunit to the subject.
  • the protein immunogen has the same amino acid sequence as the immunogen encoded by circular polyribonucleotide.
  • the polypeptide immunogen may correspond to (e.g., shares 90%, 95%, 96%, 97%, 98%, or 100%) amino acid sequence identity with a polypeptide immunogen encoded by a sequence of the circular polyribonucleotide.
  • the protein immunogen has a different amino acid sequence from the amino acid sequence of the immunogen encoded by the circular polyribonucleotide.
  • the polypeptide immunogen may share less than 90% (e.g., 80%, 70%, 30%, 20%, or 10%) amino acid sequence identity with the polypeptide immunogen encoded by a sequence of the circular polyribonucleotide.
  • a subject can be immunized with an immunogenic composition, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, at any suitable number anatomical sites.
  • the same immunogenic composition, an adjuvant, a vaccine (e.g., protein subunit vaccine), or a combination thereof can be administered to multiple anatomical sites, different immunogenic compositions including the same or different circular polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccine) or a combination thereof can be administered to different anatomical sites, different immunogenic compositions including the same or different circular polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccines) or a combination thereof can be administered to the same anatomical site, or any combination thereof.
  • an immunogenic composition including a circular polyribonucleotide can be administered in to two different anatomical sites, and/or an immunogenic composition including a circular polyribonucleotide can be administered to one anatomical site, and an adjuvant can be administered to a different anatomical site.
  • the same immunogenic composition, an adjuvant, a vaccine (e.g., protein subunit vaccine), or a combination thereof can be administered to multiple anatomical sites, different immunogenic compositions including the same or different linear polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccine) or a combination thereof can be administered to different anatomical sites, different immunogenic compositions including the same or different linear polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccines) or a combination thereof can be administered to the same anatomical site, or any combination thereof.
  • an immunogenic composition including a linear polyribonucleotide can be administered in to two different anatomical sites, and/or an immunogenic composition including a linear polyribonucleotide can be administered to one anatomical site, and an adjuvant can be administered to a different anatomical site.
  • Immunization at any two or more anatomical routes can be via the same route of immunization (e.g., intramuscular) or by two or more routes of immunization.
  • an immunogenic composition including a circular polyribonucleotide, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure is immunized to at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 anatomical sites of a subject.
  • an immunogenic composition including a circular polyribonucleotide, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure is immunized to at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 anatomical sites of the subject, or less.
  • an immunogenic composition including a circular polyribonucleotide or an adjuvant of the disclosure is immunized to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 anatomical sites of a subject.
  • an immunogenic composition including a linear polyribonucleotide, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure is immunized to at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 anatomical sites of a subject.
  • an immunogenic composition including a linear polyribonucleotide, an adjuvant, or a vaccine (e.g., protein subunit vaccine), or a combination thereof, of the disclosure is immunized to at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 anatomical sites of the subject, or less.
  • an immunogenic composition including a linear polyribonucleotide or an adjuvant of the disclosure is immunized to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 anatomical sites of a subject.
  • Immunization can be by any suitable route.
  • immunization routes include intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intracerebroventricular, intracisternal, or intraparenchymal, e.g., injection and infusion.
  • immunization can be via inhalation. Two or more immunizations can be done by the same route or by different routes.
  • a subject can be immunized with at least about 1 ng, at least about 10 ng, at least about 100 ng, at least about 1 pg, at least about 10 pg, at least about, at least about 100 pg, at least about 1 mg, at least about 10 mg, at least about 100 mg, or at least about 1 g of a circular polyribonucleotide.
  • a subject can be immunized with at most about 1 ng, at most about 10 ng, at most about 100 ng, at most about 1 pg, at most about 10 pg, at most about, at most about 100 pg, at most about 1 mg, at most about 10 mg, at most about 100 mg, or at most about 1 g of a circular polyribonucleotide.
  • a subject can be immunized with about 1 ng, about 10 ng, about 100 ng, about 1 pg, about 10 pg, about, about 100 pg, about 1 mg, about 10 mg, about 100 mg, or about 1 g of a circular polyribonucleotide.
  • the method further includes evaluating the subject for antibody response to the immunogen. In some embodiments, the evaluating is before and/or after administration of the circular polyribonucleotide including a sequence encoding an immunogen.
  • a polyribonucleotide e.g., a circular polyribonucleotide, linear polyribonucleotide described herein is formulated in composition, e.g., a composition for delivery to a human subject.
  • the polyribonucleotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • a composition includes a polyribonucleotide (e.g., a circular polyribonucleotide or a linear polyribonucleotide) and a diluent, a carrier, an adjuvant, or a combination thereof.
  • a composition includes a polyribonucleotide (e.g., a circular polyribonucleotide or a linear polyribonucleotide) described herein and a carrier or a diluent free of any carrier.
  • a composition including a polyribonucleotide e.g., a circular polyribonucleotide or a linear polyribonucleotide
  • a diluent free of any carrier is used for naked delivery of the polyribonucleotide to a subject.
  • Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product.
  • a pharmaceutical formulation disclosed herein can include: (i) a compound (e.g., circular polyribonucleotide) disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and/or (v) a stabilizer.
  • the pharmaceutical formulation disclosed herein is a stable liquid pharmaceutical formulation.
  • the pharmaceutical formulation disclosed herein includes protamine or a protamine salt (e.g., protamine sulfate).
  • compositions may optionally include one or more additional active substances, e.g., therapeutically and/or prophylactically active substances.
  • Pharmaceutical compositions may optionally include an inactive substance that serves as a vehicle or medium for the compositions described herein (e.g., compositions including circular polyribonucleotides, such as any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database).
  • Pharmaceutical compositions of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
  • Nonlimiting examples of an inactive substance include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, polymers, peptides, proteins, cells, hyaluronidases, dispersing agents, granulating agents, disintegrating agents, binding agents, buffering agents (e.g., phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures thereof.
  • solvents e.g., phosphate buffered saline (PBS)
  • PBS phosphate buffered saline
  • compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • compositions described herein may be formulated either in pure form or together with one or more additional agents (such as excipient, delivery vehicle, carrier, diluent, stabilizer, etc.) to facilitate application or delivery of the compositions.
  • additional agents such as excipient, delivery vehicle, carrier, diluent, stabilizer, etc.
  • excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, and mineral oil.
  • the composition includes a delivery vehicle or carrier.
  • the delivery vehicle includes an excipient.
  • excipients include, but are not limited to, solid or liquid carrier materials, solvents, stabilizers, slow-release excipients, colorings, and surface-active substances (surfactants).
  • the delivery vehicle is a stabilizing vehicle.
  • the stabilizing vehicle includes a stabilizing excipient.
  • Exemplary stabilizing excipients include, but are not limited to, epoxidized vegetable oils, antifoaming agents, e.g., silicone oil, preservatives, viscosity regulators, binding agents and tackifiers.
  • the composition is microencapsulated in a polymer bead delivery vehicle.
  • the delivery vehicle contains a pH buffer.
  • the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.
  • the composition may be formulated into emulsifiable concentrates, suspension concentrates, directly sprayable or dilutable solutions, coatable pastes, diluted emulsions, spray powders, soluble powders, dispersible powders, wettable powders, dusts, granules, encapsulations in polymeric substances, microcapsules, foams, aerosols, carbon dioxide gas preparations, tablets, resin preparations, paper preparations, nonwoven fabric preparations, or knitted or woven fabric preparations.
  • the composition is a liquid.
  • the composition is a solid.
  • the composition is an aerosol, such as in a pressurized aerosol can.
  • the composition is present in the waste (such as feces) of the pest.
  • the composition is present in or on a live pest.
  • the delivery vehicle is the food or water of the host. In other instances, the delivery vehicle is a food source for the host. In some instances, the delivery vehicle is a food bait for the host. In some instances, the composition is a comestible agent consumed by the host. In some instances, the composition is delivered by the host to a second host and consumed by the second host. In some instances, the composition is consumed by the host or a second host, and the composition is released to the surrounding of the host or the second host via the waste (such as feces) of the host or the second host.
  • the composition includes at least any of 0.1 %, 0.5%, 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more active ingredients (such as phage, lysin or bacteriocin).
  • active ingredients such as phage, lysin or bacteriocin.
  • the concentrated agents are preferred as commercial products, the final user normally uses diluted agents, which have a substantially lower concentration of active ingredient.
  • any of the formulations described herein may be used in the form of a bait, a coil, an electric mat, a smoking preparation, a fumigant, or a sheet.
  • a composition e.g., pharmaceutical composition
  • the polyribonucleotide can be present in either linear or circular form.
  • the composition e.g., pharmaceutical composition
  • Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, Onamer M, or other agents known to those skilled in the art. In ophthalmic products, e.g., such preservatives can be employed at a level of from 0.004% to 0.02%. In the compositions described herein the preservative, e.g., benzalkonium chloride, can be employed at a level of from 0.001% to less than 0.01%, e.g., from 0.001% to 0.008%, preferably about 0.005% by weight.
  • compositions can be susceptible to RNase that can be abundant in ambient environment.
  • Compositions e.g., pharmaceutical compositions
  • compositions can include reagents that inhibit RNase activity, thereby preserving the polyribonucleotide from degradation.
  • the composition e.g., pharmaceutical composition
  • the polyribonucleotide, and cell-penetrating agent and/or diluents or carriers, vehicles, excipients, or other reagents in the composition provided herein can be prepared in RNase-free environment.
  • the composition can be formulated in RNase-free environment.
  • a composition provided herein can be sterile.
  • the composition can be formulated as a sterile solution or suspension, in suitable vehicles, known in the art.
  • the composition can be sterilized by conventional, known sterilization techniques, e.g., the composition can be sterile filtered.
  • a composition e.g., pharmaceutical composition
  • a physiological salt such as sodium salt can be included in a composition provided herein.
  • Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, or the like.
  • a pharmaceutical composition is formulated with one or more pharmaceutically acceptable salts.
  • the one or more pharmaceutically acceptable salts can include those of the inorganic ions, such as, for example, sodium, potassium, calcium, magnesium ions, and the like.
  • Such salts can include salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid.
  • the polyribonucleotide can be present in either linear or circular form.
  • a composition e.g., pharmaceutical composition
  • a composition e.g., pharmaceutical composition
  • the composition e.g., pharmaceutical composition
  • the polyribonucleotide can be present in either linear or circular form. Detergents/surfactants
  • a composition e.g., pharmaceutical composition
  • the one or more detergents and/or surfactants can be present only at trace amounts.
  • the composition can include less than 1 mg/ml of each of octoxynol-10 and polysorbate 80.
  • Non-ionic surfactants can be used herein.
  • Surfactants can be classified by their “HLB” (hydrophile/lipophile balance). In some cases, surfactants have a HLB of at least 10, at least 15, and/or at least 16.
  • the polyribonucleotide can be present in either linear or circular form.
  • a composition e.g., pharmaceutical composition
  • a composition includes a circular polyribonucleotide and a diluent.
  • a composition e.g., pharmaceutical composition
  • a composition (e.g., pharmaceutical composition) comprises a circular polyribonucleotide as described herein in a vesicle or other membrane-based carrier.
  • a composition (e.g., pharmaceutical composition) comprises a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier.
  • a diluent can be a non-carrier excipient.
  • a non-carrier excipient serves as a vehicle or medium for a composition, such as a polyribonucleotide as described herein.
  • Non-limiting examples of a non-carrier excipient include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, polymers, peptides, proteins, cells, hyaluronidases, dispersing agents, granulating agents, disintegrating agents, binding agents, buffering agents (e.g., phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures thereof.
  • buffering agents e.g., phosphate buffered saline (PBS)
  • PBS phosphate buffered sa
  • a non-carrier excipient can be any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database that does not exhibit a cellpenetrating effect.
  • a non-carrier excipient can be any inactive ingredient suitable for administration to a non-human animal, for example, suitable for veterinary use. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • the polyribonucleotide (e.g., circular polyribonucleotide or linear polyribonucleotide) is delivered as a naked delivery formulation, such as including a diluent.
  • a naked delivery formulation delivers a polyribonucleotide (e.g., circular polyribonucleotide or linear polyribonucleotide), to a cell without the aid of a carrier and without covalent modification of the polyribonucleotide or partial or complete encapsulation of the polyribonucleotide.
  • a naked delivery formulation is a formulation that is free from a carrier and wherein the polyribonucleotide is without a covalent modification that binds a moiety that aids in delivery to a cell or without partial or complete encapsulation of the polyribonucleotide.
  • a polyribonucleotide without a covalent modification that binds a moiety that aids in delivery to a cell is a polyribonucleotide that is not covalently bound to a moiety, such as a protein, small molecule, a particle, a polymer, or a biopolymer that aids in delivery to a cell.
  • a polyribonucleotide without covalent modification that binds a moiety that aids in delivery to a cell does not contain a modified phosphate group.
  • a polyribonucleotide without a covalent modification that binds a moiety that aids in delivery to a cell does not contain phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.
  • a naked delivery formulation is free of any or all transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers.
  • a naked delivery formulation is free from phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2- dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), N-
  • a naked delivery formulation includes a non-carrier excipient.
  • a noncarrier excipient serves as a vehicle or medium for a polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) described herein.
  • a non-carrier excipient includes an inactive ingredient that does not exhibit a cell-penetrating effect.
  • a non-carrier excipient includes a buffer, for example PBS.
  • a non-carrier excipient is a solvent, a non-aqueous solvent, a dispersion media, a diluent, a suspension aid, a surface-active agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersing agent, a granulating agent, a disintegrating agent, a binding agent, a buffering agent, a lubricating agent, or an oil, or mixtures thereof.
  • a non-carrier excipient is a pharmaceutically acceptable excipient.
  • a non-carrier excipient can be any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database that does not exhibit a cell-penetrating effect.
  • a naked delivery formulation includes a diluent, such as a parenterally acceptable diluent.
  • a diluent e.g., a parenterally acceptable diluent
  • a diluent e.g., a parenterally acceptable diluent
  • an RNA solubilizing agent include water, ethanol, methanol, acetone, formamide, and 2-propanol.
  • Examples of a buffer include 2-(N- morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2- ethanesulfonic acid) (PIPES), 2-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate.
  • Examples of an isotonic agent include glycerin, mannitol, polyethylene glycol, prop
  • the formulation includes a cell-penetrating agent.
  • the formulation is a topical formulation and includes a cell-penetrating agent.
  • the cellpenetrating agent can include organic compounds such as alcohols having one or more hydroxyl function groups.
  • the cell-penetrating agent includes an alcohol such as, but not limited to, monohydric alcohols, polyhydric alcohols, unsaturated aliphatic alcohols, and alicyclic alcohols.
  • the cell-penetrating agent can include one or more of methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzyl alcohol, specially denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol, menthol, polyethylene glycols (PEG)-400, ethoxylated fatty acids, or hydroxyethylcellulose.
  • the cell-penetrating agent includes ethanol.
  • the cellpenetrating agents can include any cell-penetrating agent in any amount or in any formulation as described in WO 2020/180751 or WO 2020/180752, which are hereby incorporated by reference in their entirety.
  • composition e.g., a pharmaceutical composition
  • a disclosure includes any one of the polyribonucleotides described herein and a carrier.
  • a composition (e.g., pharmaceutical composition) comprises a circular polyribonucleotide as described herein in a vesicle or other membrane-based carrier.
  • a composition e.g., pharmaceutical composition comprises a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier.
  • a composition e.g., pharmaceutical composition
  • the composition (e.g., pharmaceutical composition) includes the polyribonucleotide in liposomes or other similar vesicles.
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and an impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, JOURNAL OF DRUG DELIVERY, vol. 2011 , Article ID 469679, 12 pages, 2011 . doi :10.1155/2011/469679 for review).
  • BBB blood brain barrier
  • Vesicles can be made from several diverse types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference).
  • vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, JOURNAL OF DRUG DELIVERY, vol. 2011 , Article ID 469679, 12 pages, 2011. doi :10.1155/2011/469679 for review).
  • Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., NATURE BIOTECH, 15:647-52, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.
  • a composition (e.g., a pharmaceutical composition) of the disclosure includes a polyribonucleotide and lipid nanoparticles, for example lipid nanoparticles described herein.
  • Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a polyribonucleotide molecule as described herein.
  • Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of the SLN, improve drug stability and loading capacity, and prevent drug leakage.
  • Polymer nanoparticles (PNPs) are a key component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release.
  • Lipid-polymer nanoparticles a new type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes.
  • a PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell offers good biocompatibility. As such, the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs.
  • Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122 see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122.
  • carriers include carbohydrate carriers (e.g., an anhydride- modified phytoglycogen or glycogen-type material), protein carriers (e.g., a protein covalently linked to the polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or transfection reagent).
  • carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, and anhydride-modified phytoglycogen beta-dextrin.
  • Non-limiting examples of cationic carriers include lipofectamine, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-Dioleoyl-3- Trimethylammonium-Propane(DOTAP), N-[ 1 -(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl- 3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-
  • Exosomes can also be used as drug delivery vehicles for polyribonucleotides described herein.
  • Ex vivo differentiated red blood cells can also be used as a carrier for polyribonucleotides described herein. See, e.g., International Patent Publication Nos. WO2015/073587; WO2017/123646; WO2017/123644; WO2018/102740; WO2016/183482; WO2015/153102; WO2018/151829;
  • Fusosome compositions e.g., as described in International Patent Publication No. WO2018/208728, can also be used as carriers to deliver a polyribonucleotide molecule described herein.
  • Virosomes and virus-like particles can also be used as carriers to deliver a polyribonucleotide molecule described herein to targeted cells.
  • Plant nanovesicles and plant messenger packs e.g., as described in International Patent Publication Nos. WO2011/097480, WO2013/070324, WO2017/004526, or W02020/041784 can also be used as carriers to deliver polyribonucleotides described herein.
  • Microbubbles can also be used as carriers to deliver a polyribonucleotide molecule described herein. See, e.g., US7115583; Beeri, R. et al., CIRCULATION. 2002 Oct 1 ;106(14):1756-59; Bez, M. et al., NAT PROTOC. 2019 Apr; 14(4): 1015-26; Hernot, S. et al., ADV DRUG DELIV REV. 2008 Jun 30; 60(10): 1153-66; Rychak, J.J. et al., ADV DRUG DELIV REV. 2014 Jun; 72: 82-93.
  • microbubbles are albumin-coated perfluorocarbon microbubbles.
  • the carrier including the polyribonucleotides described herein may include a plurality of particles.
  • the particles may have median article size of 30 to 700 nanometers (e.g., 30 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 100 to 500, 50 to 500, or 200 to 700 nanometers).
  • the size of the particle may be optimized to favor deposition of the payload, including the polyribonucleotide into a cell. Deposition of the polyribonucleotide into certain cell types may favor different particle sizes.
  • the particle size may be optimized for deposition of the polyribonucleotide into immunogen presenting cells.
  • the particle size may be optimized for deposition of the polyribonucleotide into dendritic cells.
  • the particle size may be optimized for depositions of the polyribonucleotide into draining lymph node cells.
  • compositions, methods, and delivery systems provided by the present disclosure may employ any suitable carrier or delivery modality described herein, including, in certain embodiments, lipid nanoparticles (LNPs).
  • Lipid nanoparticles include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of International Patent Publication No. WO2019/217941 ; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol).
  • ionic lipids such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids)
  • conjugated lipids such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of International Patent Publication No. WO2019/217941 ; incorporated
  • Lipids that can be used in nanoparticle formations include, for example those described in Table 4 of International Patent Publication No. WO2019/217941 , which is incorporated by reference — e.g., a lipid-containing nanoparticle can include one or more of the lipids in Table 4 of International Patent Publication No. WO2019/217941 .
  • Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of International Patent Publication No. WO2019/217941 , incorporated by reference.
  • conjugated lipids when present, can include one or more of PEG- diacylglycerol (DAG) (such as 1 -(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG- DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'- di(tetradecanoyloxy)propyl-1 -0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)-1 ,2-distearoyl-sn-
  • DAG P
  • WO2019/051289 (incorporated by reference), and combinations of the foregoing. Additional exemplary PEG-lipid conjugates are described, for example, in US Patent No. 5,885,613, US Patent No. 6,287,591 , US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US 2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, US2018/0028664, and International Patent Publication No. WO2017/099823, the contents of all of which are incorporated herein by reference in their entirety.
  • sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009/127060 or US2010/0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.
  • the lipid particle includes an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol.
  • the amounts of these components can be varied independently and to achieve desired properties.
  • the lipid nanoparticle includes an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids.
  • the ratio of total lipid to nucleic acid can be varied as desired.
  • the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1 .
  • the lipid to nucleic acid ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1 :1 to about 25:1 , from about 10:1 to about 14:1 , from about 3:1 to about 15:1 , from about 4:1 to about 10:1 , from about 5:1 to about 9:1 , or about 6:1 to about 9:1 .
  • the amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher.
  • the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg/ml to about 30 mg/mL.
  • lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein includes,
  • an LNP including Formula (i) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (ii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (iii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (v) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (vi) is used to deliver a polyribonucleotide
  • composition e.g., a circular polyribonucleotide, a linear polyribonucleotide composition described herein to cells.
  • an LNP including Formula (viii) is used to deliver a polyribonucleotide
  • composition e.g., a circular polyribonucleotide, a linear polyribonucleotide composition described herein to cells.
  • an LNP including Formula (ix) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • X 1 is O, NR 1 , or a direct bond
  • X 2 is C2-5 alkylene
  • R 1 is H or Me
  • R 3 is C1 -3 alkyl
  • R 2 is C1 -3 alkyl
  • X 1 is NR 1 , R 1 and R 2 taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R 2 taken together with R 3 and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring
  • Y 1 is C2- 12 alkylene
  • Y 2 is selected from
  • R 4 is C1 -15 alkyl
  • Z 1 is C1 -6 alkylene or a direct bond
  • R 5 is C5-9 alkyl or C6-10 alkoxy
  • R 6 is C5-9 alkyl or C6-10 alkoxy
  • W is methylene or a direct bond
  • R 4 is linear C5 alkyl
  • Z 1 is C2 alkylene
  • Z 2 is absent
  • W is methylene
  • R 7 is H
  • R 5 and R 6 are not Cx alkoxy.
  • an LNP including Formula (xii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (xi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP includes a compound of Formula (xiii) and a compound of Formula (xiv).
  • an LNP including Formula (xv) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including a formulation of Formula (xvi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a lipid compound used to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein is made by one of the following reactions: 5
  • an LNP including Formula (xxi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • the LNP of Formula (xxi) is an LNP described by International Patent Publication No. WO2021/113777 (e.g., a lipid of Formula (1 ) such as a lipid of Table 1 of International Patent Publication No. WO2021/113777). in which each n is independently an integer from 2-15; Li and L3 are each independently -OC(O)-* or - C(O)O-*, in which indicates the attachment point to R1 or R3.
  • R1 and R3 are each independently a linear or branched C9-C20 alkyl or C9-C20 alkenyl, optionally substituted by one or more substituents selected from a group consisting of oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxy
  • R2 is selected from a group consisting of:
  • an LNP including Formula (xxii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • the LNP of Formula (xxii) is an LNP described by International Patent Publication No. WO2021/113777 (e.g., a lipid of Formula (2) such as a lipid of Table 2 of International Patent Publication No. WO2021/113777). in which each n is independently an integer from 1 -15;
  • Ri and R2 are each independently selected from a group consisting of:
  • Rs is selected from a group consisting of:
  • an LNP including Formula (xxiii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • the LNP of Formula (xxiii) is an LNP described by International Patent Publication No. WO2021/113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of International Patent Publication No. WO2021/113777). in which X is selected from -O-, -S-, or -OC(O)-*, in which * indicates the attachment point to
  • Ri is selected from a group consisting of: and R2 is selected from a group consisting of:
  • a composition described herein e.g., a nucleic acid (e.g., a circular polyribonucleotide, a linear polyribonucleotide) or a protein
  • an LNP that includes an ionizable lipid.
  • the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo- 6-(undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of U.S. Patent No. 9,867,888 (incorporated by reference herein in its entirety).
  • the ionizable lipid is 9Z , 12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01 ), e.g., as synthesized in Example 13 of International Patent Publication No. WO2015/095340 (incorporated by reference herein in its entirety).
  • the ionizable lipid is Di((Z)-non-2-en-1 -yl) 9- ((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., as synthesized in Example 7, 8, or 9 of US2012/0027803 (incorporated by reference herein in its entirety).
  • the ionizable lipid is 1 ,1 ’-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl) amino)ethyl)piperazin-1 -yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of International Patent Publication No. WO2010/053572 (incorporated by reference herein in its entirety).
  • the ionizable lipid is Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17-tetradecahydro-1 H- cyclopenta[a]phenanthren-3-yl 3-(1 H-imidazol-4- yl)propanoate, e.g., Structure (I) from International Patent Publication No. W02020/106946 (incorporated by reference herein in its entirety).
  • ICE Imidazole cholesterol ester
  • an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated.
  • the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.
  • Exemplary cationic lipids include one or more amine group(s) which bear the positive charge.
  • the lipid particle includes a cationic lipid in formulation with one or more of neutral lipids, ionizable amine- containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids.
  • the cationic lipid may be an ionizable cationic lipid.
  • An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0.
  • a lipid nanoparticle may include a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid.
  • a lipid nanoparticle may include between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a nucleic acid (e.g., RNA (e.g., a circular polyribonucleotide, a linear polyribonucleotide)) described herein, encapsulated within or associated with the lipid nanoparticle.
  • the nucleic acid is co-formulated with the cationic lipid.
  • the nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP including a cationic lipid.
  • the nucleic acid may be encapsulated in an LNP, e.g., an LNP including a cationic lipid.
  • the lipid nanoparticle may include a targeting moiety, e.g., coated with a targeting agent.
  • the LNP formulation is biodegradable.
  • a lipid nanoparticle including one or more lipid described herein, e.g., Formula (i), (ii), (ii), (vii) and/or (ix) encapsulates at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule.
  • Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of International Patent Publication No. WO2019/051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016/031 1759; I of US201503761 15 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or HA of US20170210967; l-c of US20150140070; A of US2013/0178541 ; I of US2013/0303587 or US2013/0123338; I of US2015/0141678; II, III, IV, or V of US2015/0239926; I of US2017/01 19904; I or II of International Patent Publication No.
  • WO2013/016058 A of WO2012/162210; I of US2008/042973; I, II, III, or IV of US2012/01287670; I or II of US2014/0200257; I, II, or III of US2015/0203446; I or III of US2015/0005363; I, IA, IB, IC, ID, II, HA, IIB, HO, HD, or HI-XXIV of US2014/0308304; of US2013/0338210; I, II, HI, or IV of International Patent Publication No.
  • Exemplary lipids further include a lipid of any one of Tables 1 -16 of International Patent Publication No. WO2021/113777.
  • the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31 - tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of International Patent Publication No. WO2019/051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of International Patent Publication No. WO2019/051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13,16- dien-1 -amine (Compound 32), e.g., as described in Example 11 of International Patent Publication No. WO2019/051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of International Patent Publication No. WO2019/051289A9 (incorporated by reference herein in its entirety).
  • non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl.
  • Additional exemplary lipids include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.
  • Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).
  • Other examples of non-cationic lipids suitable for use in the lipid nanoparticles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • Other non-cationic lipids are described in International Patent Publication No. WO2017/099823 or US
  • the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018/0028664, incorporated herein by reference in its entirety.
  • the non-cationic lipid can include, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle.
  • the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , or 8:1 ).
  • the lipid nanoparticles do not include any phospholipids.
  • the lipid nanoparticle can further include a component, such as a sterol, to provide membrane integrity.
  • a component such as a sterol
  • a sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof.
  • cholesterol derivatives include polar analogues such as 5a-cholestanol, 5p-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl’(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a- cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4'-hydroxy)-butyl ether.
  • exemplary cholesterol derivatives are described in PCT publication W02009/127060 and US patent publication US2010/0130588, each of which is incorporated herein by reference in its entirety.
  • the component providing membrane integrity such as a sterol
  • such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
  • the lipid nanoparticle can include a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization.
  • PEG polyethylene glycol
  • exemplary conjugated lipids include, but are not limited to, PEG- lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof.
  • the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)- conjugated lipid.
  • PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1 -(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG- dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'- di(tetradecanoyloxy)propyl-1 -0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3
  • PEG-lipid conjugates are described, for example, in U.S. Patent No. 5,885,613, U.S. Patent No. 6,287,591 , US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, US2018/0028664, and International Patent Publication No. WO2017/099823, the contents of all of which are incorporated herein by reference in their entirety.
  • a PEG-lipid is a compound of Formula III, lll-a-l, lll-a-2, lll-b-1 , lll-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety.
  • a PEG-lipid is of Formula II of US20150376115 or US2016/0376224, the content of both of which is incorporated herein by reference in its entirety.
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG- dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG-dilaurylglycamide, PEG- dimyristylglycamide, PEG- dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1 ’[8'- (Cholest-5-en-3[beta]-oxy)carboxamido-3', 6'-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB(3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-
  • the PEG-lipid includes PEG-DMG, 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid includes a structure selected from:
  • lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid.
  • polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.
  • Exemplary conjugated lipids, i.e. , PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of International Patent Publication No. WO2019/051289A9, the contents of all of which are incorporated herein by reference in their entirety.
  • the PEG or the conjugated lipid can include 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG-conjugated lipid can be varied as needed.
  • the lipid particle can include 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non-cationic lipid by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition.
  • the composition includes 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10- 20% non- cationic-lipid by mole or by total weight of the composition.
  • the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic lipid, by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition.
  • the composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% noncationic lipid by mole or by total weight of the composition.
  • the composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition.
  • the formulation may also be a lipid nanoparticle formulation, for example including 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1 -20% cholesterol by mole or by total weight of the
  • the lipid particle formulation includes ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50: 10:38.5: 1 .5. In some other embodiments, the lipid particle formulation includes ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5: 1 .5.
  • the lipid particle includes ionizable lipid, non-cationic lipid (e.g., phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non- cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.
  • non-cationic lipid e.g., phospholipid
  • a sterol e.g., cholesterol
  • PEG-ylated lipid e.g., PEG-ylated lipid
  • the lipid particle includes ionizable lipid I non-cationic- lipid / sterol I conjugated lipid at a molar ratio of 50:10:38.5: 1 .5.
  • the disclosure provides a lipid nanoparticle formulation including phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.

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Abstract

La présente divulgation concerne des compositions comprenant des polyribonucléotides présentant un ou plusieurs éléments d'augmentation d'expression ou éléments d'espacement. Les polyribonucléotides de la présente divulgation peuvent augmenter la stabilité du polyribonucléotide et/ou peuvent augmenter l'expression d'une cargaison polynucléotidique codée par le polyribonucléotide.
PCT/US2024/020317 2023-03-15 2024-03-15 Compositions immunogènes et leurs utilisations WO2024192422A1 (fr)

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Citations (117)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001813A1 (fr) 1990-07-25 1992-02-06 Syngene, Inc. Extension circulaire pour generer plusieurs copies complementaires d'un acide nucleique
US5426180A (en) 1991-03-27 1995-06-20 Research Corporation Technologies, Inc. Methods of making single-stranded circular oligonucleotides
US5712128A (en) 1992-01-13 1998-01-27 Duke University Enzymatic RNA molecules
US5766903A (en) 1995-08-23 1998-06-16 University Technology Corporation Circular RNA and uses thereof
US5773244A (en) 1993-05-19 1998-06-30 Regents Of The University Of California Methods of making circular RNA
US5885613A (en) 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US6210931B1 (en) 1998-11-30 2001-04-03 The United States Of America As Represented By The Secretary Of Agriculture Ribozyme-mediated synthesis of circular RNA
US6287591B1 (en) 1997-05-14 2001-09-11 Inex Pharmaceuticals Corp. Charged therapeutic agents encapsulated in lipid particles containing four lipid components
US20030077829A1 (en) 2001-04-30 2003-04-24 Protiva Biotherapeutics Inc.. Lipid-based formulations
US20030082768A1 (en) 1998-04-17 2003-05-01 Whitehead Institute For Biomedical Research Use of a ribozyme to join nucleic acids and peptides
US6693086B1 (en) 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
US20050175682A1 (en) 2003-09-15 2005-08-11 Protiva Biotherapeutics, Inc. Polyethyleneglycol-modified lipid compounds and uses thereof
US20060008378A1 (en) 2004-04-30 2006-01-12 Kunihiro Imai Sterilization method
US7115583B2 (en) 1996-06-28 2006-10-03 Avi Biopharma, Inc. Microbubble compositions and methods for oligonucleotide delivery
US20080020058A1 (en) 2005-02-14 2008-01-24 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US20080042973A1 (en) 2006-07-10 2008-02-21 Memsic, Inc. System for sensing yaw rate using a magnetic field sensor and portable electronic devices using the same
WO2009127060A1 (fr) 2008-04-15 2009-10-22 Protiva Biotherapeutics, Inc. Nouvelles formulations lipidiques pour l'administration d'acides nucléiques
WO2009132131A1 (fr) 2008-04-22 2009-10-29 Alnylam Pharmaceuticals, Inc. Formulation lipidique améliorée à base d'amino lipide
US20100062967A1 (en) 2004-12-27 2010-03-11 Silence Therapeutics Ag Coated lipid complexes and their use
WO2010053572A2 (fr) 2008-11-07 2010-05-14 Massachusetts Institute Of Technology Lipidoïdes aminoalcool et leurs utilisations
US20100137407A1 (en) 2007-05-09 2010-06-03 Riken Single-chain circular rna and method of producing the same
WO2010084371A1 (fr) 2009-01-26 2010-07-29 Mitoprod Nouvelles molécules d'arn interférent circulaire
US20110076335A1 (en) 2009-07-01 2011-03-31 Protiva Biotherapeutics, Inc. Novel lipid formulations for delivery of therapeutic agents to solid tumors
US20110117125A1 (en) 2008-01-02 2011-05-19 Tekmira Pharmaceuticals Corporation Compositions and methods for the delivery of nucleic acids
WO2011097480A1 (fr) 2010-02-05 2011-08-11 University Of Louisville Research Foundation, Inc. Compositions exosomales et procédés pour le traitement de maladies
US20110256175A1 (en) 2008-10-09 2011-10-20 The University Of British Columbia Amino lipids and methods for the delivery of nucleic acids
US20120011478A1 (en) 2005-02-24 2012-01-12 Texas Instruments Incorporated Merging sub-resolution assist features of a photolithographic mask
US20120027803A1 (en) 2010-06-03 2012-02-02 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US20120027796A1 (en) 2008-11-10 2012-02-02 Alnylam Pharmaceuticals, Inc. Novel lipids and compositions for the delivery of therapeutics
US20120058144A1 (en) 2008-11-10 2012-03-08 Alnylam Pharmaceuticals, Inc. Lipids and compositions for the delivery of therapeutics
US8158601B2 (en) 2009-06-10 2012-04-17 Alnylam Pharmaceuticals, Inc. Lipid formulation
US8168775B2 (en) 2008-10-20 2012-05-01 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of transthyretin
US20120149894A1 (en) 2009-08-20 2012-06-14 Mark Cameron Novel cationic lipids with various head groups for oligonucleotide delivery
US20120202871A1 (en) 2009-07-01 2012-08-09 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US20120287670A1 (en) 2010-04-09 2012-11-15 Sharp Kabushiki Kaisha Light source module and electronic apparatus provided with same
WO2012162210A1 (fr) 2011-05-26 2012-11-29 Merck Sharp & Dohme Corp. Lipides cationiques maintenus dans un anneau pour une fourniture d'oligonucléotides
US20130022649A1 (en) 2009-12-01 2013-01-24 Protiva Biotherapeutics, Inc. Snalp formulations containing antioxidants
WO2013016058A1 (fr) 2011-07-22 2013-01-31 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques contenant du bis-azote pour administration d'oligonucléotide
US20130053572A1 (en) 2010-01-22 2013-02-28 Steven L. Colletti Novel Cationic Lipids for Oligonucleotide Delivery
US20130090372A1 (en) 2010-06-04 2013-04-11 Brian W. Budzik Novel Low Molecular Weight Cationic Lipids for Oligonucleotide Delivery
US20130116307A1 (en) 2010-05-12 2013-05-09 Protiva Biotherapeutics Inc. Novel cyclic cationic lipids and methods of use
WO2013070324A1 (fr) 2011-11-07 2013-05-16 University Of Louisville Research Foundation, Inc. Compositions de microvésicules dérivées de plante comestible pour le diagnostic et le traitement d'une maladie
US20130123338A1 (en) 2010-05-12 2013-05-16 Protiva Biotherapeutics, Inc. Novel cationic lipids and methods of use thereof
US20130178541A1 (en) 2010-09-20 2013-07-11 Matthew G. Stanton Novel low molecular weight cationic lipids for oligonucleotide delivery
US20130189351A1 (en) 2010-08-31 2013-07-25 Novartis Ag Lipids suitable for liposomal delivery of protein coding rna
US20130195920A1 (en) 2011-12-07 2013-08-01 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
WO2013116126A1 (fr) 2012-02-01 2013-08-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques biodégradables de faible masse moléculaire pour la délivrance d'oligonucléotides
US20130274504A1 (en) 2010-10-21 2013-10-17 Steven L. Colletti Novel Low Molecular Weight Cationic Lipids For Oligonucleotide Delivery
US20130274523A1 (en) 2010-09-30 2013-10-17 John A. Bawiec, III Low molecular weight cationic lipids for oligonucleotide delivery
US20130303587A1 (en) 2010-06-30 2013-11-14 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US20130323269A1 (en) 2010-07-30 2013-12-05 Muthiah Manoharan Methods and compositions for delivery of active agents
US20130338210A1 (en) 2009-12-07 2013-12-19 Alnylam Pharmaceuticals, Inc. Compositions for nucleic acid delivery
US20140039032A1 (en) 2011-12-12 2014-02-06 Kyowa Hakko Kirin Co., Ltd. Lipid nano particles comprising cationic lipid for drug delivery system
US20140200257A1 (en) 2011-01-11 2014-07-17 Alnylam Pharmaceuticals, Inc. Pegylated lipids and their use for drug delivery
WO2014136086A1 (fr) 2013-03-08 2014-09-12 Novartis Ag Lipides et compositions lipidiques pour l'administration de principes actifs
US20140308304A1 (en) 2011-12-07 2014-10-16 Alnylam Pharmaceuticals, Inc. Lipids for the delivery of active agents
US20150005363A1 (en) 2011-12-07 2015-01-01 Alnylam Pharmaceuticals, Inc. Branched Alkyl And Cycloalkyl Terminated Biodegradable Lipids For The Delivery Of Active Agents
US20150057373A1 (en) 2012-03-27 2015-02-26 Sirna Therapeutics, Inc DIETHER BASED BIODEGRADABLE CATIONIC LIPIDS FOR siRNA DELIVERY
US20150064242A1 (en) 2012-02-24 2015-03-05 Protiva Biotherapeutics, Inc. Trialkyl cationic lipids and methods of use thereof
US20150140070A1 (en) 2013-10-22 2015-05-21 Shire Human Genetic Therapies, Inc. Lipid formulations for delivery of messenger rna
US20150141678A1 (en) 2013-11-18 2015-05-21 Arcturus Therapeutics, Inc. Ionizable cationic lipid for rna delivery
WO2015073587A2 (fr) 2013-11-18 2015-05-21 Rubius Therapeutics, Inc. Complexes membrane synthétique- récepteur
WO2015095340A1 (fr) 2013-12-19 2015-06-25 Novartis Ag Lipides et compositions lipidiques pour le largage d'agents actifs
US20150203446A1 (en) 2011-09-27 2015-07-23 Takeda Pharmaceutical Company Limited Di-aliphatic substituted pegylated lipids
US20150239926A1 (en) 2013-11-18 2015-08-27 Arcturus Therapeutics, Inc. Asymmetric ionizable cationic lipid for rna delivery
WO2015153102A1 (fr) 2014-04-01 2015-10-08 Rubius Therapeutics, Inc. Méthodes et compositions d'immunomodulation
US20150376115A1 (en) 2014-06-25 2015-12-31 Acuitas Therapeutics Inc. Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
US20160151284A1 (en) 2013-07-23 2016-06-02 Protiva Biotherapeutics, Inc. Compositions and methods for delivering messenger rna
US20160317458A1 (en) 2013-12-19 2016-11-03 Luis Brito Lipids and Lipid Compositions for the Delivery of Active Agents
WO2016183482A1 (fr) 2015-05-13 2016-11-17 Rubius Therapeutics, Inc. Agents thérapeutiques d'un complexe membrane-récepteur
US20160376224A1 (en) 2015-06-29 2016-12-29 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2017004526A1 (fr) 2015-07-02 2017-01-05 University Of Louisville Research Foundation, Inc. Compositions de microvésicules dérivées d'une plante comestible pour l'administration d'arnmi et méthodes de traitement du cancer
US20170119904A1 (en) 2015-10-28 2017-05-04 Acuitas Therapeutics, Inc. Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2017099823A1 (fr) 2015-12-10 2017-06-15 Modernatx, Inc. Compositions et procédés permettant d'administrer des agents thérapeutiques
WO2017117528A1 (fr) 2015-12-30 2017-07-06 Acuitas Therapeutics, Inc. Lipides et formulations de nanoparticules de lipides pour la libération d'acides nucléiques
US9708628B2 (en) 2011-11-18 2017-07-18 Nof Corporation Cationic lipid having improved intracellular kinetics
WO2017123646A1 (fr) 2016-01-11 2017-07-20 Rubius Therapeutics, Inc. Compositions et méthodes associées à des systèmes cellulaires thérapeutiques multimodaux pour des indications du cancer
US20170210967A1 (en) 2010-12-06 2017-07-27 Schlumberger Technology Corporation Compositions and methods for well completions
WO2017173054A1 (fr) 2016-03-30 2017-10-05 Intellia Therapeutics, Inc. Formulations de nanoparticules lipidiques pour des composés crispr/cas
WO2018009838A1 (fr) 2016-07-07 2018-01-11 Rubius Therapeutics, Inc. Compositions et procédés associés à des systèmes cellulaires thérapeutiques exprimant de l'arn exogène
US9867888B2 (en) 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2018081480A1 (fr) 2016-10-26 2018-05-03 Acuitas Therapeutics, Inc. Formulations de nanoparticules lipidiques
WO2018102740A1 (fr) 2016-12-02 2018-06-07 Rubius Therapeutics, Inc. Compositions et méthodes se rapportant à des systèmes cellulaires destinés à pénétrer dans des tumeurs solides
US20180191722A1 (en) 2017-01-05 2018-07-05 Mastercard International Incorporated Systems and Methods for Use in Managing Access to User Profiles, and Content Blocks Included Therein
WO2018151829A1 (fr) 2017-02-17 2018-08-23 Rubius Therapeutics, Inc. Cellules érythroïdes fonctionnalisées
US10086013B2 (en) 2011-10-27 2018-10-02 Massachusetts Institute Of Technology Amino acid-, peptide- and polypeptide-lipids, isomers, compositions, and uses thereof
WO2018208728A1 (fr) 2017-05-08 2018-11-15 Flagship Pioneering, Inc. Compositions pour faciliter la fusion membranaire et leurs utilisations
WO2019051289A1 (fr) 2017-09-08 2019-03-14 Generation Bio Co. Formulations de nanoparticules lipidiques de vecteurs d'adn exempts de capside non viraux
WO2019067910A1 (fr) 2017-09-29 2019-04-04 Intellia Therapeutics, Inc. Polynucléotides, compositions et procédés pour l'édition génomique
WO2019067992A1 (fr) 2017-09-29 2019-04-04 Intellia Therapeutics, Inc. Formulations
WO2019118919A1 (fr) 2017-12-15 2019-06-20 Flagship Pioneering, Inc. Compositions comprenant des polyribonucléotides circulaires et leurs utilisations
US20190240349A1 (en) 2015-06-19 2019-08-08 Massachusetts Institute Of Technology Alkenyl substituted 2,5-piperazinediones, compositions, and uses thereof
WO2019217941A1 (fr) 2018-05-11 2019-11-14 Beam Therapeutics Inc. Procédés de suppression de mutations pathogènes à l'aide de systèmes d'éditeur de base programmables
WO2019236673A1 (fr) 2018-06-06 2019-12-12 Massachusetts Institute Of Technology Arn circulaire pour la traduction dans des cellules eucaryotes
WO2020023595A1 (fr) 2018-07-24 2020-01-30 Mayo Foundation For Medical Education And Research Arn technique circularisé et procédés
WO2020041784A1 (fr) 2018-08-24 2020-02-27 Flagship Pioneering Innovations Vi, Llc. Procédés de fabrication de paquets de messagers de plante
WO2020047124A1 (fr) 2018-08-28 2020-03-05 Flagship Pioneering, Inc. Procédés et compositions pour moduler un génome
WO2020061457A1 (fr) 2018-09-20 2020-03-26 Modernatx, Inc. Préparation de nanoparticules lipidiques et leurs méthodes d'administration
WO2020081938A1 (fr) 2018-10-18 2020-04-23 Acuitas Therapeutics, Inc. Lipides pour l'administration de nanoparticules lipidiques d'agents actifs
WO2020106946A1 (fr) 2018-11-21 2020-05-28 Translate Bio, Inc. Traitement de la fibrose kystique par administration d'arnm nébulisé codant pour la cftr
WO2020180752A1 (fr) 2019-03-01 2020-09-10 Flagship Pioneering Innovations Vi, Llc Polyribonucléotides et leurs utilisations cosmétiques
WO2020180751A1 (fr) 2019-03-01 2020-09-10 Flagship Pioneering Innovations Vi, Llc Compositions, procédés, et kits pour l'administration de polyribonucléotides
WO2020219876A1 (fr) 2019-04-25 2020-10-29 Intellia Therapeutics, Inc. Lipides aminés ionisables et nanoparticules lipidiques
US11000547B2 (en) 2015-06-05 2021-05-11 Dana-Farber Cancer Institute, Inc. Compositions related to rna in circularized form
WO2021113777A2 (fr) 2019-12-04 2021-06-10 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2021189059A2 (fr) * 2020-03-20 2021-09-23 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2021226597A2 (fr) 2020-05-08 2021-11-11 Orna Therapeutics, Inc. Compositions d'arn circulaire et méthodes
WO2021236855A1 (fr) * 2020-05-19 2021-11-25 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2021263124A2 (fr) 2020-06-25 2021-12-30 The Board Of Trustees Of The Leland Stanford Junior University Éléments génétiques commandant la traduction d'arn circulaire et procédés d'utilisation
WO2022037692A1 (fr) * 2020-08-21 2022-02-24 Peking University Vaccins à arn circulaires et leurs procédés d'utilisation
WO2022204460A1 (fr) 2021-03-26 2022-09-29 Flagship Pioneering Innovations Vii, Llc Compositions et procédés de production de polyribonucléotides circulaires
WO2022204464A1 (fr) 2021-03-26 2022-09-29 Flagship Pioneering Innovations Vii, Llc Production de polyribonucléotides circulaires dans un système eucaryote
WO2022204466A1 (fr) 2021-03-26 2022-09-29 Flagship Pioneering Innovations Vii, Llc Production de polyribonucléotides circulaires dans un système procaryote
WO2022245901A1 (fr) * 2021-05-18 2022-11-24 Flagship Pioneering Innovations Vi, Llc Procédés d'enrichissement de polyribonucléotides circulaires
WO2022247943A1 (fr) 2021-05-28 2022-12-01 Shanghai Circode Biomed Co., Ltd. Constructions et procédés de préparation d'arn circulaires et leur utilisation
WO2022261490A2 (fr) 2021-06-10 2022-12-15 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2022271965A2 (fr) 2021-06-25 2022-12-29 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procédés pour une traduction de protéines améliorée à partir d'arn circulaires recombinants

Patent Citations (125)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001813A1 (fr) 1990-07-25 1992-02-06 Syngene, Inc. Extension circulaire pour generer plusieurs copies complementaires d'un acide nucleique
US5426180A (en) 1991-03-27 1995-06-20 Research Corporation Technologies, Inc. Methods of making single-stranded circular oligonucleotides
US5712128A (en) 1992-01-13 1998-01-27 Duke University Enzymatic RNA molecules
US5773244A (en) 1993-05-19 1998-06-30 Regents Of The University Of California Methods of making circular RNA
US5885613A (en) 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US5766903A (en) 1995-08-23 1998-06-16 University Technology Corporation Circular RNA and uses thereof
US7115583B2 (en) 1996-06-28 2006-10-03 Avi Biopharma, Inc. Microbubble compositions and methods for oligonucleotide delivery
US6287591B1 (en) 1997-05-14 2001-09-11 Inex Pharmaceuticals Corp. Charged therapeutic agents encapsulated in lipid particles containing four lipid components
US20030082768A1 (en) 1998-04-17 2003-05-01 Whitehead Institute For Biomedical Research Use of a ribozyme to join nucleic acids and peptides
US6693086B1 (en) 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
US6210931B1 (en) 1998-11-30 2001-04-03 The United States Of America As Represented By The Secretary Of Agriculture Ribozyme-mediated synthesis of circular RNA
US20030077829A1 (en) 2001-04-30 2003-04-24 Protiva Biotherapeutics Inc.. Lipid-based formulations
US20050175682A1 (en) 2003-09-15 2005-08-11 Protiva Biotherapeutics, Inc. Polyethyleneglycol-modified lipid compounds and uses thereof
US20060008378A1 (en) 2004-04-30 2006-01-12 Kunihiro Imai Sterilization method
US20100062967A1 (en) 2004-12-27 2010-03-11 Silence Therapeutics Ag Coated lipid complexes and their use
US20080020058A1 (en) 2005-02-14 2008-01-24 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US20120011478A1 (en) 2005-02-24 2012-01-12 Texas Instruments Incorporated Merging sub-resolution assist features of a photolithographic mask
US20080042973A1 (en) 2006-07-10 2008-02-21 Memsic, Inc. System for sensing yaw rate using a magnetic field sensor and portable electronic devices using the same
US20100137407A1 (en) 2007-05-09 2010-06-03 Riken Single-chain circular rna and method of producing the same
US20110117125A1 (en) 2008-01-02 2011-05-19 Tekmira Pharmaceuticals Corporation Compositions and methods for the delivery of nucleic acids
WO2009127060A1 (fr) 2008-04-15 2009-10-22 Protiva Biotherapeutics, Inc. Nouvelles formulations lipidiques pour l'administration d'acides nucléiques
US20100130588A1 (en) 2008-04-15 2010-05-27 Protiva Biotherapeutics, Inc. Novel lipid formulations for nucleic acid delivery
WO2009132131A1 (fr) 2008-04-22 2009-10-29 Alnylam Pharmaceuticals, Inc. Formulation lipidique améliorée à base d'amino lipide
US20110256175A1 (en) 2008-10-09 2011-10-20 The University Of British Columbia Amino lipids and methods for the delivery of nucleic acids
US8168775B2 (en) 2008-10-20 2012-05-01 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of transthyretin
WO2010053572A2 (fr) 2008-11-07 2010-05-14 Massachusetts Institute Of Technology Lipidoïdes aminoalcool et leurs utilisations
US20120027796A1 (en) 2008-11-10 2012-02-02 Alnylam Pharmaceuticals, Inc. Novel lipids and compositions for the delivery of therapeutics
US20120058144A1 (en) 2008-11-10 2012-03-08 Alnylam Pharmaceuticals, Inc. Lipids and compositions for the delivery of therapeutics
WO2010084371A1 (fr) 2009-01-26 2010-07-29 Mitoprod Nouvelles molécules d'arn interférent circulaire
US8158601B2 (en) 2009-06-10 2012-04-17 Alnylam Pharmaceuticals, Inc. Lipid formulation
US20110076335A1 (en) 2009-07-01 2011-03-31 Protiva Biotherapeutics, Inc. Novel lipid formulations for delivery of therapeutic agents to solid tumors
US20120202871A1 (en) 2009-07-01 2012-08-09 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US20120149894A1 (en) 2009-08-20 2012-06-14 Mark Cameron Novel cationic lipids with various head groups for oligonucleotide delivery
US20130022649A1 (en) 2009-12-01 2013-01-24 Protiva Biotherapeutics, Inc. Snalp formulations containing antioxidants
US20130338210A1 (en) 2009-12-07 2013-12-19 Alnylam Pharmaceuticals, Inc. Compositions for nucleic acid delivery
US20130053572A1 (en) 2010-01-22 2013-02-28 Steven L. Colletti Novel Cationic Lipids for Oligonucleotide Delivery
WO2011097480A1 (fr) 2010-02-05 2011-08-11 University Of Louisville Research Foundation, Inc. Compositions exosomales et procédés pour le traitement de maladies
US20120287670A1 (en) 2010-04-09 2012-11-15 Sharp Kabushiki Kaisha Light source module and electronic apparatus provided with same
US20130123338A1 (en) 2010-05-12 2013-05-16 Protiva Biotherapeutics, Inc. Novel cationic lipids and methods of use thereof
US20130116307A1 (en) 2010-05-12 2013-05-09 Protiva Biotherapeutics Inc. Novel cyclic cationic lipids and methods of use
US20120027803A1 (en) 2010-06-03 2012-02-02 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US20130090372A1 (en) 2010-06-04 2013-04-11 Brian W. Budzik Novel Low Molecular Weight Cationic Lipids for Oligonucleotide Delivery
US20130303587A1 (en) 2010-06-30 2013-11-14 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US20130323269A1 (en) 2010-07-30 2013-12-05 Muthiah Manoharan Methods and compositions for delivery of active agents
US20130189351A1 (en) 2010-08-31 2013-07-25 Novartis Ag Lipids suitable for liposomal delivery of protein coding rna
US20130178541A1 (en) 2010-09-20 2013-07-11 Matthew G. Stanton Novel low molecular weight cationic lipids for oligonucleotide delivery
US20130274523A1 (en) 2010-09-30 2013-10-17 John A. Bawiec, III Low molecular weight cationic lipids for oligonucleotide delivery
US20130274504A1 (en) 2010-10-21 2013-10-17 Steven L. Colletti Novel Low Molecular Weight Cationic Lipids For Oligonucleotide Delivery
US20170210967A1 (en) 2010-12-06 2017-07-27 Schlumberger Technology Corporation Compositions and methods for well completions
US20140200257A1 (en) 2011-01-11 2014-07-17 Alnylam Pharmaceuticals, Inc. Pegylated lipids and their use for drug delivery
WO2012162210A1 (fr) 2011-05-26 2012-11-29 Merck Sharp & Dohme Corp. Lipides cationiques maintenus dans un anneau pour une fourniture d'oligonucléotides
WO2013016058A1 (fr) 2011-07-22 2013-01-31 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques contenant du bis-azote pour administration d'oligonucléotide
US20150203446A1 (en) 2011-09-27 2015-07-23 Takeda Pharmaceutical Company Limited Di-aliphatic substituted pegylated lipids
US10086013B2 (en) 2011-10-27 2018-10-02 Massachusetts Institute Of Technology Amino acid-, peptide- and polypeptide-lipids, isomers, compositions, and uses thereof
WO2013070324A1 (fr) 2011-11-07 2013-05-16 University Of Louisville Research Foundation, Inc. Compositions de microvésicules dérivées de plante comestible pour le diagnostic et le traitement d'une maladie
US9708628B2 (en) 2011-11-18 2017-07-18 Nof Corporation Cationic lipid having improved intracellular kinetics
US20140308304A1 (en) 2011-12-07 2014-10-16 Alnylam Pharmaceuticals, Inc. Lipids for the delivery of active agents
US20150005363A1 (en) 2011-12-07 2015-01-01 Alnylam Pharmaceuticals, Inc. Branched Alkyl And Cycloalkyl Terminated Biodegradable Lipids For The Delivery Of Active Agents
US20130195920A1 (en) 2011-12-07 2013-08-01 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US20140039032A1 (en) 2011-12-12 2014-02-06 Kyowa Hakko Kirin Co., Ltd. Lipid nano particles comprising cationic lipid for drug delivery system
WO2013116126A1 (fr) 2012-02-01 2013-08-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques biodégradables de faible masse moléculaire pour la délivrance d'oligonucléotides
US20150064242A1 (en) 2012-02-24 2015-03-05 Protiva Biotherapeutics, Inc. Trialkyl cationic lipids and methods of use thereof
US20150057373A1 (en) 2012-03-27 2015-02-26 Sirna Therapeutics, Inc DIETHER BASED BIODEGRADABLE CATIONIC LIPIDS FOR siRNA DELIVERY
WO2014136086A1 (fr) 2013-03-08 2014-09-12 Novartis Ag Lipides et compositions lipidiques pour l'administration de principes actifs
US20160151284A1 (en) 2013-07-23 2016-06-02 Protiva Biotherapeutics, Inc. Compositions and methods for delivering messenger rna
US20150140070A1 (en) 2013-10-22 2015-05-21 Shire Human Genetic Therapies, Inc. Lipid formulations for delivery of messenger rna
WO2015073587A2 (fr) 2013-11-18 2015-05-21 Rubius Therapeutics, Inc. Complexes membrane synthétique- récepteur
US20150141678A1 (en) 2013-11-18 2015-05-21 Arcturus Therapeutics, Inc. Ionizable cationic lipid for rna delivery
US20150239926A1 (en) 2013-11-18 2015-08-27 Arcturus Therapeutics, Inc. Asymmetric ionizable cationic lipid for rna delivery
US9644180B2 (en) 2013-11-18 2017-05-09 Rubius Therapeutics, Inc. Synthetic membrane-receiver complexes
US20160311759A1 (en) 2013-12-19 2016-10-27 Luis Brito Lipids and Lipid Compositions for the Delivery of Active Agents
US20160317458A1 (en) 2013-12-19 2016-11-03 Luis Brito Lipids and Lipid Compositions for the Delivery of Active Agents
WO2015095340A1 (fr) 2013-12-19 2015-06-25 Novartis Ag Lipides et compositions lipidiques pour le largage d'agents actifs
WO2015153102A1 (fr) 2014-04-01 2015-10-08 Rubius Therapeutics, Inc. Méthodes et compositions d'immunomodulation
US20150376115A1 (en) 2014-06-25 2015-12-31 Acuitas Therapeutics Inc. Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2016183482A1 (fr) 2015-05-13 2016-11-17 Rubius Therapeutics, Inc. Agents thérapeutiques d'un complexe membrane-récepteur
US11000547B2 (en) 2015-06-05 2021-05-11 Dana-Farber Cancer Institute, Inc. Compositions related to rna in circularized form
US20190240349A1 (en) 2015-06-19 2019-08-08 Massachusetts Institute Of Technology Alkenyl substituted 2,5-piperazinediones, compositions, and uses thereof
US10221127B2 (en) 2015-06-29 2019-03-05 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US20160376224A1 (en) 2015-06-29 2016-12-29 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2017004526A1 (fr) 2015-07-02 2017-01-05 University Of Louisville Research Foundation, Inc. Compositions de microvésicules dérivées d'une plante comestible pour l'administration d'arnmi et méthodes de traitement du cancer
US9867888B2 (en) 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US20170119904A1 (en) 2015-10-28 2017-05-04 Acuitas Therapeutics, Inc. Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
US20180028664A1 (en) 2015-12-10 2018-02-01 Modernatx, Inc. Compositions and methods for delivery of agents
WO2017099823A1 (fr) 2015-12-10 2017-06-15 Modernatx, Inc. Compositions et procédés permettant d'administrer des agents thérapeutiques
WO2017117528A1 (fr) 2015-12-30 2017-07-06 Acuitas Therapeutics, Inc. Lipides et formulations de nanoparticules de lipides pour la libération d'acides nucléiques
WO2017123646A1 (fr) 2016-01-11 2017-07-20 Rubius Therapeutics, Inc. Compositions et méthodes associées à des systèmes cellulaires thérapeutiques multimodaux pour des indications du cancer
WO2017123644A1 (fr) 2016-01-11 2017-07-20 Rubius Therapeutics, Inc. Compositions et procédés associés à des systèmes cellulaires thérapeutiques multimodaux pour indications immunitaires
WO2017173054A1 (fr) 2016-03-30 2017-10-05 Intellia Therapeutics, Inc. Formulations de nanoparticules lipidiques pour des composés crispr/cas
US20190136231A1 (en) 2016-03-30 2019-05-09 Intellia Therapeutics, Inc. Lipid nanoparticle formulations for crispr/cas components
WO2018009838A1 (fr) 2016-07-07 2018-01-11 Rubius Therapeutics, Inc. Compositions et procédés associés à des systèmes cellulaires thérapeutiques exprimant de l'arn exogène
WO2018081480A1 (fr) 2016-10-26 2018-05-03 Acuitas Therapeutics, Inc. Formulations de nanoparticules lipidiques
WO2018102740A1 (fr) 2016-12-02 2018-06-07 Rubius Therapeutics, Inc. Compositions et méthodes se rapportant à des systèmes cellulaires destinés à pénétrer dans des tumeurs solides
US20180191722A1 (en) 2017-01-05 2018-07-05 Mastercard International Incorporated Systems and Methods for Use in Managing Access to User Profiles, and Content Blocks Included Therein
WO2018151829A1 (fr) 2017-02-17 2018-08-23 Rubius Therapeutics, Inc. Cellules érythroïdes fonctionnalisées
WO2018208728A1 (fr) 2017-05-08 2018-11-15 Flagship Pioneering, Inc. Compositions pour faciliter la fusion membranaire et leurs utilisations
WO2019051289A1 (fr) 2017-09-08 2019-03-14 Generation Bio Co. Formulations de nanoparticules lipidiques de vecteurs d'adn exempts de capside non viraux
WO2019051289A9 (fr) 2017-09-08 2019-05-23 Generation Bio Co. Formulations de nanoparticules lipidiques de vecteurs d'adn exempts de capside non viraux
WO2019067910A1 (fr) 2017-09-29 2019-04-04 Intellia Therapeutics, Inc. Polynucléotides, compositions et procédés pour l'édition génomique
WO2019067992A1 (fr) 2017-09-29 2019-04-04 Intellia Therapeutics, Inc. Formulations
WO2019118919A1 (fr) 2017-12-15 2019-06-20 Flagship Pioneering, Inc. Compositions comprenant des polyribonucléotides circulaires et leurs utilisations
WO2019217941A1 (fr) 2018-05-11 2019-11-14 Beam Therapeutics Inc. Procédés de suppression de mutations pathogènes à l'aide de systèmes d'éditeur de base programmables
WO2019236673A1 (fr) 2018-06-06 2019-12-12 Massachusetts Institute Of Technology Arn circulaire pour la traduction dans des cellules eucaryotes
WO2020023595A1 (fr) 2018-07-24 2020-01-30 Mayo Foundation For Medical Education And Research Arn technique circularisé et procédés
WO2020041784A1 (fr) 2018-08-24 2020-02-27 Flagship Pioneering Innovations Vi, Llc. Procédés de fabrication de paquets de messagers de plante
WO2020047124A1 (fr) 2018-08-28 2020-03-05 Flagship Pioneering, Inc. Procédés et compositions pour moduler un génome
WO2020061457A1 (fr) 2018-09-20 2020-03-26 Modernatx, Inc. Préparation de nanoparticules lipidiques et leurs méthodes d'administration
WO2020081938A1 (fr) 2018-10-18 2020-04-23 Acuitas Therapeutics, Inc. Lipides pour l'administration de nanoparticules lipidiques d'agents actifs
WO2020106946A1 (fr) 2018-11-21 2020-05-28 Translate Bio, Inc. Traitement de la fibrose kystique par administration d'arnm nébulisé codant pour la cftr
WO2020180752A1 (fr) 2019-03-01 2020-09-10 Flagship Pioneering Innovations Vi, Llc Polyribonucléotides et leurs utilisations cosmétiques
WO2020180751A1 (fr) 2019-03-01 2020-09-10 Flagship Pioneering Innovations Vi, Llc Compositions, procédés, et kits pour l'administration de polyribonucléotides
WO2020219876A1 (fr) 2019-04-25 2020-10-29 Intellia Therapeutics, Inc. Lipides aminés ionisables et nanoparticules lipidiques
WO2021113777A2 (fr) 2019-12-04 2021-06-10 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2021189059A2 (fr) * 2020-03-20 2021-09-23 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2021226597A2 (fr) 2020-05-08 2021-11-11 Orna Therapeutics, Inc. Compositions d'arn circulaire et méthodes
WO2021236855A1 (fr) * 2020-05-19 2021-11-25 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2021263124A2 (fr) 2020-06-25 2021-12-30 The Board Of Trustees Of The Leland Stanford Junior University Éléments génétiques commandant la traduction d'arn circulaire et procédés d'utilisation
WO2022037692A1 (fr) * 2020-08-21 2022-02-24 Peking University Vaccins à arn circulaires et leurs procédés d'utilisation
WO2022204460A1 (fr) 2021-03-26 2022-09-29 Flagship Pioneering Innovations Vii, Llc Compositions et procédés de production de polyribonucléotides circulaires
WO2022204464A1 (fr) 2021-03-26 2022-09-29 Flagship Pioneering Innovations Vii, Llc Production de polyribonucléotides circulaires dans un système eucaryote
WO2022204466A1 (fr) 2021-03-26 2022-09-29 Flagship Pioneering Innovations Vii, Llc Production de polyribonucléotides circulaires dans un système procaryote
WO2022245901A1 (fr) * 2021-05-18 2022-11-24 Flagship Pioneering Innovations Vi, Llc Procédés d'enrichissement de polyribonucléotides circulaires
WO2022247943A1 (fr) 2021-05-28 2022-12-01 Shanghai Circode Biomed Co., Ltd. Constructions et procédés de préparation d'arn circulaires et leur utilisation
WO2022261490A2 (fr) 2021-06-10 2022-12-15 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2022271965A2 (fr) 2021-06-25 2022-12-29 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procédés pour une traduction de protéines améliorée à partir d'arn circulaires recombinants

Non-Patent Citations (41)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT WILLIAMS & WILKINS
"Sustained and Controlled Release Drug Delivery Systems", 1978, MARCEL DEKKER, INC.
ALTSTEIN, PEPTIDES, vol. 25, 2004, pages 1373 - 1376
BAI YU ET AL: "Research progress on circular RNA vaccines", FRONTIERS IN IMMUNOLOGY, vol. 13, 12 January 2023 (2023-01-12), Lausanne, CH, XP093176727, ISSN: 1664-3224, DOI: 10.3389/fimmu.2022.1091797 *
BARANICK ET AL., PNAS, vol. 105, no. 12, 2008, pages 4733 - 4738
BEERI, R ET AL., CIRCULATION, vol. 106, no. 14, 1 October 2002 (2002-10-01), pages 1756 - 59
BETZ KMALYSHEV DALAVERGNE TWELTE WDIEDERICHS KDWYER TJORDOUKHANIAN PROMESBERG FEMARX A, NAT. CHEM. BIOL., vol. 8, no. 7, July 2012 (2012-07-01), pages 612 - 4
BEZ, M ET AL., NAT PROTOC, vol. 14, no. 4, April 2019 (2019-04-01), pages 1015 - 26
CHEN ET AL., MOL. CELL, vol. 81, no. 20, 2021, pages 4300 - 4318
CHEN ET AL., NATURE BIOTECHNOLOGY, vol. 41, 2023, pages 262 - 272
CHEN ET AL., SCIENCE, vol. 268, 1995, pages 415 - 417
DOROKHOV ET AL., PNAS, vol. 99, no. 8, 2002, pages 5301 - 5306
FAN ET AL., NATURE COMMUNICATION, vol. 13, no. 1, 2022, pages 3751 - 3765
GORI ET AL.: "Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy", HUMAN GENE THERAPY, vol. 26, no. 7, July 2015 (2015-07-01), pages 443 - 451, XP055501239, DOI: 10.1089/hum.2015.074
HA ET AL., ACTA PHARMACEUTICA SINICA B, vol. 6, July 2016 (2016-07-01), pages 287 - 296, Retrieved from the Internet <URL:https://doi.org/10.1016/j.apsb.2016.02.001>
HENIKOFFHENIKOFF, PNAS, vol. 89, 1992, pages 915 - 919
HERNOT, S ET AL., ADV DRUG DELIV RΕV, vol. 60, no. 10, 30 June 2008 (2008-06-30), pages 1153 - 66
HOU ET AL.: "Lipid nanoparticles for mRNA delivery", NAT REV MATER, 2021
HUANG ET AL., NATURE COMMUNICATIONS, vol. 8, 2017, pages 423
JAYARAMAN ET AL., ANGEW CHEM INT ED ENGL, vol. 51, no. 34, 2012, pages 8529 - 8533
JOPLING ET AL., ONCOGENE, vol. 20, 2001, pages 2664 - 2670
KHUDYAKOVFIELDS: "Artificial DNA: Methods and Applications", 2002, CRC PRESS
KIMOTO ET AL., CHEM COMMUN (CAMB, vol. 53, 2017, pages 12309
LANG ET AL., MOLECULAR BIOLOGY OF THE CELL, vol. 13, no. 5, 2002, pages 1792 - 1801
LEWISPAN: "RNA modifications and structures cooperate to guide RNA-protein interactions", NAT REVIEWS MOL CELL BIOL, vol. 18, 2017, pages 202 - 210, XP055451248
LI ET AL., NANOMATERIALS, vol. 7, 2017, pages 122
MIKI ET AL., NATURE COMMUNICATIONS, vol. 21, 2021, pages 3412
MULLERAPPEL, RNA BIOL, vol. 14, no. 8, 2017, pages 1018 - 1027
PETKOVIC ET AL., NUCLEIC ACIDS RES, vol. 43, 2015, pages 2454 - 65
PETZ ET AL., NUCLEIC ACIDS RESEARCH, vol. 35, no. 8, 2007, pages 2473 - 2482
ROZENSKI, JCRAIN, PMCCLOSKEY, J: "The RNA Modification Database: 1999 update", NUCL ACIDS RES, vol. 27, 1999, pages 196 - 197
RYCHAK, J.J. ET AL., ADV DRUG DELIV RΕV, vol. 72, June 2014 (2014-06-01), pages 82 - 93
SHI ET AL., PROC NATL ACAD SCI USA, vol. 111, no. 28, 2014, pages 10131 - 10136
SHI ET AL., PROC NATL ACAD SCI USA, vol. 111, no. 28, 2014, pages 10131 - 136
SPUCHNAVARRO, JOURNAL OF DRUG DELIVERY, vol. 2011, 2011, pages 12
TEMPLETON ET AL., NATURE BIOTECH, vol. 15, 1997, pages 647 - 52
WANG ET AL., NUCLEIC ACIDS RESEARCH, vol. 33, no. 7, 2005, pages 2248 - 2258
WESSELHOEFT R. ALEXANDER ET AL: "RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo", MOLECULAR CELL, vol. 74, no. 3, 19 March 2019 (2019-03-19), AMSTERDAM, NL, pages 508 - 520.e4, XP093042911, ISSN: 1097-2765, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S1097276519301054/pdfft?md5=cd5840a429058421a8111aeaf3141574&pid=1-s2.0-S1097276519301054-main.pdf> DOI: 10.1016/j.molcel.2019.02.015 *
YU, Z ET AL.: "RNA editing by ADAR1 marks dsRNA as ''self", CELL RES, vol. 25, 2015, pages 1283 - 1284
ZHAO: "Synthetic Biology: Tools and Applications", 2013, ACADEMIC PRESS
ZURIS ET AL.: "Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo", NAT BIOTECHNOL, vol. 33, no. 1, 30 October 2014 (2014-10-30), pages 73 - 80, XP093037310, DOI: 10.1038/nbt.3081

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