WO2025042786A1 - Compositions comprising circular polyribonucleotides and uses thereof - Google Patents

Compositions comprising circular polyribonucleotides and uses thereof Download PDF

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Publication number
WO2025042786A1
WO2025042786A1 PCT/US2024/042817 US2024042817W WO2025042786A1 WO 2025042786 A1 WO2025042786 A1 WO 2025042786A1 US 2024042817 W US2024042817 W US 2024042817W WO 2025042786 A1 WO2025042786 A1 WO 2025042786A1
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complex
acid
polypeptide
circular polyribonucleotide
linker
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PCT/US2024/042817
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French (fr)
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WO2025042786A8 (en
Inventor
Avak Kahvejian
Alexandra Sophie DE BOER
Frances Marion Nicole ANASTASSACOS
Nicholas McCartney PLUGIS
Jennifer A. Nelson
Ki Young PAEK
Daniel Jean-francois CHINNAPEN
Rabih Darwiche
Shraddha SAWANT
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Flagship Pioneering Innovations Vi, Llc
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Publication of WO2025042786A1 publication Critical patent/WO2025042786A1/en
Publication of WO2025042786A8 publication Critical patent/WO2025042786A8/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Definitions

  • Circular polyribonucleotides are expressed from human genomes. Circular polyribonucleotides are produced endogenously by backsplicing, a process in which the spliceosome fuses a splice donor site in a downstream exon to a splice acceptor site in an upstream exon. Circular polyribonucleotides are produced in cells with high cell-type specificity and can exert biologically meaningful and specific functions. Circular polyribonucleotides are being developed for use in modulating biological systems, including in therapeutic applications. There remains a need to develop improved compositions and methods for the delivery of circular polyribonucleotides in vivo.
  • compositions, pharmaceutical preparations, and uses of circular polyribonucleotides complexed with one or more targeting moieties are provided.
  • the disclosure provides a complex including A and X n (L-B) z , wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20), and wherein z is an integer from 1 to 5 (1 , 2, 3, 4, or 5). In some embodiments, n is an integer from 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • n is an integer from 1 to 5 (e.g., 1 , 2, 3, 4, or 5).
  • z is an integer from 1 to 3 (e.g., 1 , 2, or 3).
  • z is 1 .
  • z is 2.
  • X n (L-B) z includes X n (L-B 1 ) z and X n (L-B 2 ) z , where B 1 is a first targeting moiety and B 2 is a second targeting moiety.
  • X n (L-B) z includes X n (L 1 -B) z and X n (L 2 -B) z , where L 1 is a first linker and L 2 is a second linker.
  • X n (L-B) z includes Xi-(L-B) z and X2-(L-B) Z , where Xi is a first moiety that binds specifically to a first region of A and X2 is a second moiety that binds specifically to a second region of A.
  • X n (L-B) z includes B 1 -L- X-L- B 2 , where B 1 is a first targeting moiety and B 2 is a second targeting moiety. In some embodiments, X n (L-B) z includes B-L 1 -X-L 2 -B, where L 1 is a first linker and L 2 is a second linker.
  • This disclosure also provides compositions, pharmaceutical preparations, and uses of circular polyribonucleotides complexed with one or more targeting moieties with one or more photoreactive crosslinking agents. In some embodiments, each X includes a photoreactive crosslinking agent.
  • the disclosure provides a complex including A and X n (L-B) z , wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A and includes a photoreactive crosslinking agent, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20), and wherein z is an integer from 1 to 5 (1 , 2, 3, 4, or 5).
  • A is a circular polyribonucleotide
  • each X is independently a moiety that binds specifically to a region of A and includes a photoreactive crosslinking agent
  • each L is independently a linker
  • each B is independently a targeting moiety
  • n is an integer from 1 to 20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1
  • n is an integer from 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, n is an integer from 1 to 5 (e.g., 1 , 2, 3, 4, or 5). In some embodiments, z is an integer from 1 to 3 (e.g., 1 , 2, or 3). In some embodiments, z is 1 . In some embodiments, z is 2. In some embodiments, X n (L-B) z includes X n (L-B 1 ) z and X n (L-B 2 ) z , where B 1 is a first targeting moiety and B 2 is a second targeting moiety.
  • X n (L-B) z includes X n (L 1 -B) z and X n (l_2-B) z , where L 1 is a first linker and L 2 is a second linker.
  • X n (L-B) z includes Xi-(L-B) z and X2-(L-B) Z , where Xi is a first moiety that binds specifically to a first region of A and X2 is a second moiety that binds specifically to a second region of A.
  • X n (L-B) z includes B 1 -L- X-L-B 2 , where B 1 is a first targeting moiety and B 2 is a second targeting moiety. In some embodiments, X n (L-B) z includes B-L 1 - X-L 2 -B, where L 1 is a first linker and L 2 is a second linker.
  • X is an oligonucleotide.
  • the oligonucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.
  • the oligonucleotide includes a photoreactive crosslinking agent.
  • the photoreactive crosslinking agent is attached to the 5’ end of the oligonucleotide.
  • the photoreactive crosslinking reagent is attached to the 3’ end of the oligonucleotide.
  • the photoreactive crosslinking agent is a photoreactive nucleotide analog. In some embodiments, the photoreactive crosslinking agent is a photoreactive nucleotide analog, optionally wherein the photoreactive nucleotide analog replaces a single nucleotide within the oligonucleotide. In some embodiments, the photoreactive nucleotide analog is located at an internal position within the oligonucleotide. In some embodiments, the 3’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog.
  • the 3’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the photoreactive nucleotide analog.
  • the 5’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog.
  • the 5’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the photoreactive nucleotide analog.
  • 1 to 50 e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the photoreactive nucleotide analog.
  • the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation.
  • the photoreactive crosslinking agent includes 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a phenylselenide, a furan, or an abasic site.
  • the carbazole is a vinylcarbazole, e.g., 3-cyanovinylcarbazole, 4- methylpyranocarbazole, or pyranocarbazole.
  • the coumarin is 7-hydroxycoumarin.
  • the oligonucleotide includes a plurality of photoreactive crosslinking agents.
  • each of the plurality of photoreactive crosslinking agents is a photoreactive nucleotide analog.
  • each photoreactive nucleotide analog replaces a single nucleotide within the oligonucleotide.
  • each photoreactive nucleotide analog is located at an internal position within the oligonucleotide.
  • the 3’ end of the oligonucleotide has at least 1 , at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog.
  • the 3’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog.
  • the 5’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog.
  • the 5’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog.
  • 1 to 50 e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog.
  • At least one of the photoreactive nucleotide analogs is attached to the 3’ end of the oligonucleotide. In some embodiments, at least one of the photoreactive nucleotide analogs is attached to the 5’ end of the oligonucleotide.
  • the oligonucleotide includes 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides between each of the photoreactive nucleotide analogs.
  • 1 to 50 e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides between each of the photoreactive nucleotide analogs.
  • each of the photoreactive nucleotide analogs crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation.
  • the oligonucleotide includes an aptamer.
  • the circular polyribonucleotide is bound to from 1 to 100, 1 and 50, 1 and 20, or 1 and 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety.
  • the oligonucleotide is from 5 to 100 (e.g., 5 to 80, 5 to 60, 5 to 40, 5 to 20, 20 to 100, 40 to 100, 60 to 100, 80 to 100, 20 to 60, or 10 to 50) nucleotides in length.
  • the circular polynucleotide includes a binding region that anneals to the oligonucleotide.
  • the circular polynucleotide includes one or more binding regions each including from 5 to 200, e.g., 6 to 200, e.g., 7 to 200, e.g., 8 to 200 (e.g., from 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 75, 8 to 50, 8 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each binding region binds to an oligonucleotide.
  • each binding region includes at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 100% complementarity to the oligonucleotide. In some embodiments, the binding region has zero or one mismatch with the oligonucleotide.
  • X is a polypeptide. In some embodiments, the polypeptide includes a photoreactive crosslinking agent. In some embodiments, the photoreactive crosslinking agent is a photoreactive amino acid analog. In some embodiments, the photoreactive crosslinking agent is a photoreactive amino acid analog, optionally wherein the photoreactive amino acid analog replaces a single amino acid within the polypeptide.
  • the photoreactive amino acid analog is located at an internal position within the polypeptide.
  • the N-terminus of the polypeptide has at least 1 , e.g., at least 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog.
  • the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids from the photoreactive amino acid analog.
  • the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog.
  • the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids from the photoreactive amino acid analog.
  • the photoreactive amino acid analog is attached to the N-terminus of the polypeptide. In some embodiments, the photoreactive nucleotide analog is attached to the C-terminus of the polypeptide. In some embodiments, the photoreactive amino acid analog crosslinks to a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. In some embodiments, the photoreactive amino acid analog is an alkyl diazirene-based, arylazide-based, benzophenone-based unnatural amino acid, or A/-e-[2-(furan-2-yl)ethoxy]carbonyl-lysine.
  • the polypeptide includes a plurality of photoreactive crosslinking agents.
  • each of the plurality of photoreactive crosslinking agents is a photoreactive amino acid analog.
  • each of the plurality of photoreactive crosslinking agents replaces a single amino acid within the polypeptide.
  • at least one photoreactive amino acid analog is located at an internal position within the polypeptide.
  • the N-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog.
  • the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids from the nearest photoreactive amino acid analog.
  • the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog.
  • the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids from the nearest photoreactive amino acid analog.
  • at least one of the photoreactive amino acid analogs is attached to the N-terminus of the polypeptide.
  • at least one of the photoreactive amino acid analogs is attached to the C-terminus of the polypeptide.
  • the polypeptide includes 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids between each of the photoreactive amino acid analogs.
  • each of the photoreactive amino acids crosslinks to a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation.
  • each photoreactive amino acid analog is an alkyl diazirene-based, arylazide-based, benzophenone-based unnatural amino acid, or A/-e-[2-(furan-2-yl)ethoxy]carbonyl-lysine.
  • the polypeptide includes an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521 -B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.
  • the RNA recognition motif is selected from a K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521 -B homology domain, a double stranded RNA binding domain
  • the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety.
  • the circular polyribonucleotide includes one or more protein binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each protein binding region binds to a polypeptide.
  • 5 to 200 e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150
  • the disclosure provides a complex that includes A and X n (L-B) z , wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically and is covalently attached to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20), and wherein z is an integer from 1 to 5 (1 , 2, 3, 4, or 5).
  • A is a circular polyribonucleotide
  • each X is independently a moiety that binds specifically and is covalently attached to a region of A
  • each L is independently a linker
  • each B is independently a targeting moiety
  • n is an integer from 1 to 20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or
  • n is an integer from 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, n is an integer from 1 to 5 (e.g., 1 , 2, 3, 4, or 5). In some embodiments, z is an integer from 1 to 3 (e.g., 1 , 2, or 3). In some embodiments, z is 1 . In some embodiments, z is 2. In some embodiments, X n (L-B) z includes X n (L-B 1 ) z and X n (L-B 2 ) z , where B 1 is a first targeting moiety and B 2 is a second targeting moiety.
  • X n (L-B) z includes X n (L 1 -B) z and X n (L 2 -B) z , where L 1 is a first linker and L 2 is a second linker.
  • X n (L-B) z includes Xi-(L-B) z and X2-(L-B) Z , where Xi is a first moiety that binds specifically and covalently attaches to a first region of A and X2 is a second moiety that binds specifically and covalently attaches to a second region of A.
  • X n (L-B) z includes B 1 - L- X-L-B 2 , where B 1 is a first targeting moiety and B 2 is a second targeting moiety. In some embodiments, X n (L-B) z includes B-L 1 -X-L 2 -B, where L 1 is a first linker and L 2 is a second linker.
  • X is an oligonucleotide.
  • the oligonucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.
  • the oligonucleotide includes an aptamer.
  • the circular polyribonucleotide is bound to from 1 to 100, 1 and 50, 1 and 20, or 1 and 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety.
  • the oligonucleotide is from 5 to 100 (e.g., 5 to 80, 5 to 60, 5 to 40, 5 to 20, 20 to 100, 40 to 100, 60 to 100, 80 to 100, 20 to 60, or 10 to 50) nucleotides in length.
  • the circular polynucleotide includes a binding region that anneals to the oligonucleotide.
  • the circular polynucleotide includes one or more binding regions each including from 5 to 200, e.g., 6 to 200, e.g., 7 to 200, e.g., 8 to 200 (e.g., from 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 75, 8 to 50, 8 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each binding region binds to an oligonucleotide.
  • each binding region binds to an oligonucleotide.
  • each binding region includes at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 100% complementarity to the oligonucleotide. In some embodiments, the binding region has zero or one mismatch with the oligonucleotide.
  • X is a polypeptide.
  • the polypeptide includes an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521 -B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.
  • the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety.
  • the circular polyribonucleotide includes one or more protein binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each protein binding region binds to a polypeptide.
  • 5 to 200 e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150
  • the targeting moiety includes a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid, or a combination thereof. In some embodiments, the targeting moiety includes a small molecule.
  • the small molecule is selected from folic acid, urea, a-mannose, high mannose, ursodeoxycholic acid, an endosomal escape agent, or lithocholic acid.
  • the targeting moiety includes a polypeptide.
  • the polypeptide is a cell-penetrating peptide.
  • the polypeptide is selected from ASSLNIA (SEQ ID NO: 19), M12, RGD, melittin, LPS-binding protein (LBP) peptide, an adipose-homing peptide, or an endolytic peptide.
  • the polypeptide is an antibody or a target-binding fragment thereof.
  • the antibody or target-binding fragment thereof is selected from a monoclonal antibody or target-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, or a tandem scFv (taFv).
  • scFv single-chain Fv molecule
  • the antibody or target- binding fragment thereof is selected from an anti-FcRn antibody, an anti-MR antibody, an anti-CD205 antibody, an anti-CD169 antibody, an anti-CD14 antibody, an anti-CD36 antibody, an anti-CD5 antibody, an anti -CD71 antibody, an anti-CD38 antibody, or an anti-prohibin antibody.
  • the polypeptide is a nanobody.
  • the nanobody is selected from an anti-transferrin nanobody, an anti-HER2 nanobody, or an anti-EGFR nanobody.
  • the targeting moiety includes a carbohydrate.
  • the carbohydrate includes a saccharide, disaccharide, or polysaccharide.
  • the carbohydrate includes mannose, galactose, or glucose.
  • the carbohydrate includes GalNAc or mannose 6-phosphate.
  • the carbohydrate includes a mono-, di-, tri-, or tetra-GalNAc. In some embodiments, the carbohydrate is tri-GalNAc.
  • the targeting moiety includes a lipid.
  • the lipid includes a fatty acid.
  • the fatty acid is a saturated, monounsaturated, or polyunsaturated fatty acid.
  • the fatty acid is a branched or unbranched chain including from 4 to 40 (e.g., 4, 5, 6, 7, 8, 9, 10, 1 1 , 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, or 40) main-chain carbon atoms.
  • the fatty acid includes squalene, stearic acid, oleic acid, palmitic acid, linoleic acid, stearic acid, lauric acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, a-tocopherol, a-tocopherol succinate, or a retinoic acid.
  • the fatty acid includes docosanoic acid.
  • the fatty acid includes docosahexanoic acid.
  • the fatty acid includes myristic acid.
  • the lipid includes a steroid or sterol selected from cholesterol, tocopherol, ursodeoxycholic acid, or lithocholic acid.
  • the steroid or sterol is cholesterol.
  • the steroid or sterol is tocopherol.
  • the lipid includes a fat-soluble vitamin selected from vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof.
  • the lipid includes a phospholipid.
  • the phospholipid is selected from phosphocholine (PC), PC-docosahexaenoic acid (PC-DHA), PC-docosanoic acid (PC-DCA), PC- eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC-retinoic acid (PC-RA), and PC-a- tocopherol succinate (PC-TS).
  • PC phosphocholine
  • PC-DHA PC-docosahexaenoic acid
  • PC-DCA PC-docosanoic acid
  • PC-EPA PC- eicosapentaenoic acid
  • PC-LA PC-lithocholic acid
  • PC-RA PC-retinoic acid
  • PC-TS PC-a- tocopherol succinate
  • the targeting moiety includes an oligonucleotide. In some embodiments, the targeting moiety includes an aptamer.
  • the linker is a bond.
  • the linker includes 1 to 250 (e.g., 1 to 225, 1 to 200, 1 to 175, 1 to 150, 1 to 125, 1 to 100, 1 to 75, 1 to 50, 1 to 25, 25 to 250, 50 to 250, 75 to 250, 100 to 250, 125 to 250, 150 to 250, 175 to 250, 200 to 250, 225 to 250, 5 to 50, or 5 to 30) backbone atoms, wherein the backbone atoms are selected from C, N, O, and S.
  • the linker includes an oligonucleotide.
  • the linker includes a polypeptide.
  • the linker includes at least one PEG unit.
  • the linker includes at least one TEG unit. In some embodiments, the TEG is a In some embodiments, the linker is an enzymatically cleavable linker. In some embodiments, the linker has a length of at least 0.1 nm.
  • the linker has a length of from 0.1 nm to 20 nm (e.g., 0.1 nm to 18 nm, 0.1 nm to 16 nm, 0.1 nm to 14 nm, 0.1 nm to 12 nm, 0.1 nm to 10 nm, 0.1 nm to 8 nm, 0.1 nm to 6 nm, 0.1 nm to 4 nm, 0.1 nm to 2 nm, 0.1 nm to 0.5 nm, 0.5 nm to 20 nm, 2 nm to 20 nm, 4 nm to 20 nm, 6 nm to 20 nm, 8 nm to 20 nm, 10 nm to 20 nm, 12 nm to 20 nm, 14 nm to 20 nm, 16 nm to 20 nm, 18 nm to 20 nm, 0.2 nm to 15 nm, or
  • the linker is further conjugated to avidin.
  • the avidin binds from 1 to 4 (e.g., 1 , 2, 3, or 4) targeting moieties.
  • the avidin binds from 1 to 4 (e.g., 1 , 2, 3, or 4) targeting moieties, wherein the targeting moieties are conjugated to at least one biotin compound.
  • X is an oligonucleotide.
  • the oligonucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.
  • the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety.
  • the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety.
  • the oligonucleotide is from 5 to 100 (e.g., 5 to 80, 5 to 60, 5 to 40, 5 to 20, 20 to 100, 40 to 100, 60 to 100, 80 to 100, 20 to 60, or 10 to 50) nucleotides in length.
  • the circular polynucleotide includes one or more binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each binding region binds to an oligonucleotide.
  • each binding region includes at least 50%, 60%, 70%, 80%, 90%, or 100% complementarity to the oligonucleotide.
  • X is a polypeptide.
  • the polypeptide includes an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521 -B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.
  • the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety.
  • the circular polyribonucleotide includes one or more protein binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each protein binding region binds to a polypeptide.
  • the circular polyribonucleotide further includes at least one coding region.
  • the at least one coding region includes an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide.
  • IRS internal ribosomal entry site
  • the polypeptide is expressed in the cell.
  • the polypeptide is expressed in the cell, optionally wherein the polypeptide expressed in the cell is functional.
  • the circular polyribonucleotide includes one or more binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each binding region binds to X.
  • the binding region is contained within or overlaps in-part with an expression sequence or with a spacer region. In some embodiments, the binding region is not contained within nor does it overlap in-part with an IRES.
  • the 3’ end of the binding region is at least 5 ribonucleotides from 5’ end of the IRES; the 3’ end of the binding region is from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides from 5’ end of the IRES; the 5’ end of the binding region is at least 5 ribonucleotides from 3’ end of the IRES; or the 5’ end of the binding region is from 5 to 200 ribonucleotides from 3’ end of the IRES.
  • 5 to 200 e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200,
  • the circular polyribonucleotide includes the following elements, arranged in the following order: (i) a first spacer region; (ii) at least one coding region including an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide; (iii) optionally a second spacer region; and (iv) a binding region including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein the binding region binds to X.
  • a first spacer region e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200,
  • the circular polyribonucleotide includes the following elements, arranged in the following order: (i) a first spacer region; (ii) a target binding region including at least one aptamer or at least ribozyme sequence; (iii) optionally a second spacer region; and (iv) a binding region including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein the binding region binds to X.
  • 5 to 200 e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to
  • the first spacer region includes from 10 to 500 (e.g., from 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to 500, 100 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 150, or 100 to 300) ribonucleotides.
  • 10 to 500 e.g., from 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to 500, 100 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 150, or 100 to 300
  • 10 to 500 e.g., from 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to
  • the second spacer region includes 10 to 500 ribonucleotides (e.g., 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to 500, 100 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 150, or 100 to 300).
  • 10 to 500 ribonucleotides e.g., 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to 500, 100 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 150, or 100 to 300).
  • the binding region is contained within or overlaps in-part with the expression sequence. In some embodiments, the binding region is contained within or overlaps the first spacer region. In some embodiments, the binding region is contained within or overlaps the second spacer region. In some embodiments, the first spacer and second spacer are adjacent to one another. In some embodiments, the binding region overlaps the first and second spacer regions. In some embodiments, the binding region is not contained within nor does it overlap in-part with the expression sequence or with the first or second spacer region.
  • the circular polyribonucleotide includes at least 1 ,000 ribonucleotides. In some embodiments, the circular polyribonucleotide includes at least 3,000 ribonucleotides. In some embodiments, the circular polyribonucleotide includes from 1 ,000 to 20,000 (e.g., from 1 ,000 to 18,000; 1 ,000 to 16,000; 1 ,000 to 14,000; 1 ,000 to 12,000; 1 ,000 to 10,000; 1 ,000 to 8,000; 1 ,000 to 6,000; 1 ,000 to 4,000; 1 ,000 to 2,000; 2,000 to 20,000; 4,000 to 20,000; 6,000 to 20,000; 8,000 to 20,000; 10,000 to 20,000; 12,000 to 20,000; 14,000 to 20,000; 16,000 to 20,000; 18,000 to 20,000; 5,000 to 10,000; or 3,000 to 12,000) ribonucleotides.
  • 1 ,000 to 20,000 e.g., from 1 ,000 to 18,000
  • the circular polyribonucleotide encodes a polypeptide.
  • the polypeptide is a eukaryotic polypeptide.
  • the polypeptide is a mammalian polypeptide.
  • the polypeptide is a mammalian polypeptide, optionally wherein the polypeptide is a human polypeptide.
  • the polypeptide is a viral, bacterial, or fungal polypeptide.
  • the polypeptide is for therapeutic use.
  • the polypeptide is an immunogen.
  • the circular polyribonucleotide includes an IRES.
  • the IRES is selected from 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, fuman poliovirus 1 , Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1 , Human Immunodeficiency Virus type 1 , Homalodisca coagulata virus- 1 , Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 , Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus (EMCV), Dros
  • the disclosure provides a pharmaceutical composition including any one of the circular polyribonucleotides described herein and one or more delivery agents.
  • the delivery agent is selected from calcium, magnesium, manganese, or strontium.
  • the delivery agent is an endosomal escape agent.
  • the endosomal escape agent includes chloroquine, amantadine, ammonium chloride, 4-bromobenzaldehyde N-(2,6- dimethylphenyl)semicarbazone (EGA), UNC-108, or any combination thereof.
  • the delivery agent is a globular protein.
  • the globular protein is albumin.
  • the delivery agent is ribonuclease inhibitor.
  • the disclosure provides a method of delivering a circular polyribonucleotide to a cell, the method including contacting the cell with any one of the complexes or pharmaceutical compositions described herein.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the circular polyribonucleotide is delivered to the cell ex-vivo.
  • the cell is administered to a subject after the delivery of the circular polyribonucleotide to the cell.
  • administration of the cell to the subject treats a disease, disorder, or condition in the subject.
  • the disclosure provides a method of delivering a circular polyribonucleotide to a subject, the method including administering to the subject any one of the complexes or pharmaceutical compositions described herein.
  • the disclosure provides a method of treating a disease, disorder, or condition in a subject, the method including administering to the subject any one of the complexes or pharmaceutical compositions described herein.
  • the disclosure provides a method of inducing an immune response in a subject, the method including administering to the subject any one of the complexes or pharmaceutical compositions described herein.
  • the complex or pharmaceutical composition is administered intramuscularly, subcutaneously, intravenously, intraperitoneally, topically, or orally.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the non-human mammal is a cow, a sheep, a goat, a pig, a dog, a horse, or a cat. In some embodiments, the subject is a bird. In some embodiments, the bird is a hen, a rooster, a turkey, or a parrot.
  • the disclosure provides a method of covalently attaching a targeting moiety to a circular polyribonucleotide.
  • the method includes forming a complex as described herein (e.g., of any of the above embodiments) and irradiating the complex with light.
  • the wavelength of the irradiated light is from 350-370 (e.g., 350, 351 , 352, 353. 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, or 370) nm.
  • the complex is irradiated for 1 to 120 (e.g., 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes.
  • the disclosure provides a method of removing the covalent attachment between the circular polyribonucleotide and the moiety that binds specifically to a region of the circular polyribonucleotide and includes one or more photoreactive crosslinking agent.
  • the method includes irradiating any one of the complexes described herein with light at a second wavelength.
  • the second wavelength of the irradiated light is from 300-320 (e.g., 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311 , 312, 313, 314, 315, 316, 317, 318, 319, or 320) nm.
  • the complex is irradiated at the second wavelength for 1 to 120 (e.g., 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes.
  • the disclosure provides a method of delivering a circular polyribonucleotide to a cell.
  • the method includes contacting the cell with any one of the complexes or pharmaceutical compositions described herein.
  • the complex is irradiated prior to contacting the cell.
  • the complex is irradiated after contacting the cell.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the circular polyribonucleotide is delivered to the cell ex-vivo.
  • the complex is delivered to the cell in-vivo.
  • the cell is administered to a subject after the delivery of the circular polyribonucleotide to the cell.
  • the complex is irradiated prior to administration to the subject.
  • the complex is irradiated after administration to the subject.
  • administration of the cell to the subject treats a disease, disorder, or condition in the subject.
  • the disclosure provides a method of delivering a circular polyribonucleotide to a subject.
  • the method includes administering to the subject any one of the complexes or pharmaceutical compositions described herein.
  • the disclosure provides a method of treating a disease, disorder, or condition in a subject.
  • the method includes administering to the subject any one of the complexes or pharmaceutical compositions described herein.
  • the disclosure provides a covalent complex produced by any of the methods described herein.
  • the term “about” refers to a value that is within ⁇ 10% of a recited value.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • An "antibody fragment” refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies.
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161 ; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003).
  • Single-domain antibodies are antibody fragments including all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single- domain antibody. Antibody fragments can be made by various techniques.
  • aptamer is a non-naturally occurring or synthetic oligonucleotide that specifically binds to a target molecule.
  • an aptamer is from 20 to 500 nucleotides.
  • an aptamer binds to its target through secondary structure rather than sequence homology.
  • binding moiety refers to a moiety that specifically binds to (e.g., hybridized or hybridized and covalently bound to) a portion of the circular polyribonucleotide.
  • the binding moiety may include one or more photoreactive crosslinking agents.
  • binding region refers to a portion of the circular polyribonucleotide to which one or more moieties specifically bind.
  • the one or more moieties specifically bind to the portion of the circular polyribonucleotides by non-covalent interactions.
  • an oligonucleotide may anneal to the binding region by way of hydrogen bonds, or a polypeptide may bind to a binding region by way of hydrogen bonds, hydrophobic interactions, or Van der Waals interactions.
  • 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
  • cell-penetrating peptide refers to a peptide having from about 5 to about 30 amino acids residues which has a net positive charge, which facilitate penetration into cells across the cell membrane.
  • circular polyribonucleotide and “circular RNA” are used interchangeably and mean a polyribonucleotide molecule that has a structure having no free ends (i.e. , no free 3’ or 5’ ends), for example a polyribonucleotide molecule that forms a circular or end-less structure through covalent 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.
  • coding region refers to a region of the polynucleotide including an expression sequence encoding a polypeptide and an IRES and/or another regulatory element.
  • crosslink refers to forming a covalent bond between two molecules.
  • a photoactivatable crosslinking agent may form a crosslink between the agent and a ribonucleotide upon irradiation with light.
  • the term “delivery agent” refers to an agent that, when added to the pharmaceutical composition including the circular polyribonucleotides described herein, increases cellular delivery, functional delivery, or endosomal escape of the circular polyribonucleotide relative to the same pharmaceutical composition that lacks the agent.
  • the increase in cellular delivery, functional delivery, or endosomal escape of the circular polyribonucleotide may be measured, e.g., by assessing the amount of expression of a polypeptide encoded by the circular polyribonucleotide in comparison to a pharmaceutical composition that does not include the delivery agent.
  • nucleic acid refers to in the present specification in the context of a nucleic acid, i.e., for a nucleic acid “derived from” (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares e.g. at least 60%, 70%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived.
  • sequence identity is typically calculated for the same types of nucleic acids, i.e., for DNA sequences or for RNA sequences.
  • a DNA is “derived from” an RNA or if an RNA is “derived from” a DNA
  • the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa
  • the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by U throughout the sequence).
  • sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined.
  • nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g., in order to increase RNA stability even further and/or to prolong and/or increase protein production.
  • the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares e.g.
  • diluent means a vehicle including an inactive solvent in which a composition described herein (e.g., a composition including a circular polyribonucleotide) may be diluted or dissolved.
  • a diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof.
  • a diluent can be a liquid diluent or a solid diluent.
  • Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3- butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetra hydrofurfury I alcohol, polyethylene glycols and fatty acid esters of sorbitan, and 1 ,3-butanediol.
  • solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3- butylene glycol, di
  • Non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, or powdered sugar.
  • 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.
  • expression sequence is a nucleic acid sequence that encodes a product, e.g., a peptide or polypeptide, beginning with a start codon and ending with a stop codon.
  • An exemplary expression sequence that codes for a peptide or polypeptide can 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 polypeptide or the nucleic acid.
  • a fragment of a polypeptide or a nucleic acid sequence encoding a polypeptide 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.
  • the term “functional delivery” refers to delivery of a polyribonucleotide encoding a polypeptide into a cell, wherein the polypeptide is expressed in the cell.
  • 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.
  • 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 identity) 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 identity) 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 identity) 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 includes a 5’ cap. In some embodiments, the linear counterpart further includes a poly adenosine tail. In some embodiments, the linear counterpart further includes a 3’ UTR. In some embodiments, the linear counterpart further includes a 5’ UTR.
  • Linear RNA As used herein, the terms “linear RNA,” “linear polyribonucleotide,” and “linear polyribonucleotide molecule” are used interchangeably and mean 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.
  • Linear RNA includes RNA that has not undergone circularization (e.g., is pre-circularized) and can be used as a starting material for circularization.
  • linker refers to a covalent linkage or connection between two or more components in a conjugate (e.g., between a polyribonucleotide or oligonucleotide and a lipid moiety and a PEG, as described herein).
  • Molecules that may be used as linkers include at least two functional groups, which may be the same or different, e.g., two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid group, an amine group and a maleimide group, an amine group and an alkyne group, or an amine group and a sulfonic acid group.
  • two functional groups which may be the same or different, e.g., two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid
  • the first functional group may form a covalent linkage with a first component in the conjugate and the second functional group may form a covalent linkage with the second component in the conjugate.
  • a linker typically provides space, rigidity, and/or flexibility between the two or more components.
  • lipid or “lipid moiety” refer to organic compounds, most preferably organic biomolecules, that are selectively soluble in nonpolar solvents over water.
  • Lipid moieties specifically contemplated by the present invention include, for example, fatty acids, steroids or sterols, glycerides (e.g., monoglycerides, diglycerides, and triglycerides), phospholipids, and fat-soluble vitamins (e.g., vitamins A, D, E, and K).
  • modified ribonucleotide means a nucleotide with at least one modification to the sugar, the nucleobase, or the internucleoside linkage.
  • naked delivery is a formulation for delivery to a cell without the aid of a carrier and without covalent modification to a moiety that aids in delivery to a cell.
  • a naked delivery formulation is free from any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers.
  • naked delivery formulation of a circular polyribonucleotide is a formulation that includes a circular polyribonucleotide without covalent modification and is free from a carrier.
  • nanobody refers to a polypeptide having a single monomeric variable domain and has a molecular weight of between about 12 kDa and about 15 kDa. A nanobody is able to selectively bind to a specific antigen.
  • photoreactive crosslinking agent refers to any compound that is configured to form a covalent adduct with another molecule upon irradiation with light.
  • a photoreactive crosslinking agent may covalently attach to a ribonucleotide within the circular polyribonucleotide upon irradiation with light.
  • a photoreactive crosslinking agent may be able to form a covalent attachment to a ribonucleotide within the circular polyribonucleotide that is reversible.
  • the photoreactive crosslinking agent can be a photoreactive nucleotide analog or photoreactive amino acid analog.
  • photoreactive nucleotide analog refers to any nucleic acid analog that is configured to form a covalent adduct with another molecule upon irradiation with light.
  • the photoreactive nucleotide analog may be present within an oligonucleotide and be configured to covalently attach to a circular polyribonucleotide upon irradiation with light.
  • the photoreactive nucleotide analog can covalently attach to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation.
  • photoreactive nucleotide analogs include, but are not limited to, 4- thiouridene (4sU), 5-bromo-2’-deoxyuridine (BrdU), coumarin derivatives, 3-cyanovinylcarbazole D- threoninol (cnvD) derivatives, 3-cyanovinylcarbazole nucleoside ( CNV K) derivatives, diazirene derivatives, phenylselenide derivatives, psoralen derivatives, or pyranocarbazole ( PC X) derivatives.
  • PC X pyranocarbazole
  • the covalent attachment of the photoreactive nucleotide analog to the ribonucleotide is reversible, e.g., it may be removed when irradiated with light, e.g., of a second different wavelength.
  • photoreactive amino acid analog refers to any amino acid analog that is configured to form a covalent adduct with another molecule upon irradiation with light.
  • the photoreactive amino acid analog may be present within a polypeptide that covalently attaches to a circular polyribonucleotide upon irradiation with light.
  • the photoreactive amino acid analog can covalently attach to a nearby ribonucleotide within the circular polyribonucleotide upon photoirradiation.
  • photoreactive amino acid analogs include but are not limited to diazirene- based, aryl azide-based, benzophenone-based unnatural amino acid, or A/-e-[2-(furan-2- yl)ethoxy]carbonyl-lysine.
  • the covalent attachment of the photoreactive amino acid analog to the ribonucleotide is reversible, e.g., it may be removed when irradiated with light, e.g., a second different wavelength.
  • composition is intended to also disclose that the circular or linear polyribonucleotide included within a pharmaceutical composition can be used for the treatment of the human or animal body by therapy. It is thus meant to be equivalent to “a polyribonucleotide for use in therapy.”
  • polynucleotide 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.
  • 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 a polypeptide.
  • 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 sequences.
  • IRS internal ribosomal entry site
  • polyA refers to an untranslated, contiguous region of a nucleic acid molecule of at least 5 nucleotides in length and consisting of adenosine residues.
  • a polyA tail is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length.
  • a polyA tail is located 3’ to (e.g., downstream of) an open reading frame (e.g., an open reading frame encoding a polypeptide), and the polyA tail is 3’ to a termination element (e.g., a stop codon) such that the polyA is not translated.
  • a polyA tail is located 3’ to a termination element and a 3’ untranslated region.
  • 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, that 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).
  • 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.
  • 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-fluorouracil, 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, 5'- meth
  • nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety.
  • modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates).
  • 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-hydroxysuccinimide 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 photo-programmed 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 include 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.
  • 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.
  • a therapeutic agent can be administered to a subject who is at increased risk of developing a viral infection relative to a member of the general population in order to prevent the development of, or lessen the severity of, the disease or condition.
  • a therapeutic agent can be administered as a prophylactic, e.g., before development of any symptom or manifestation of a viral infection.
  • regulatory element is a moiety, such as a nucleic acid sequence, that modifies expression of an expression sequence within the circular or linear polyribonucleotide.
  • replication element is a sequence and/or motif useful for replication or that initiate transcription of the circular polyribonucleotide.
  • small molecule refers to a low molecular weight compound (e.g., a compound (e.g., an organic compound) having less than 1000 Da, that may regulate a biological process, with a size on the order of 1 nm.
  • a therapeutic agent is a small molecule therapeutic agent.
  • the small molecule agent is from about 300 to about 700 Da (e.g., about 325 Da, about 350 Da, about 375 Da, about 400 Da, about 425 Da, about 450 Da, about 475 Da, about 500 Da, about 525 Da, about 550 Da, about 575 Da, about 600 Da, about 625 Da, about 650 Da, or about 675 Da).
  • a “spacer” refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.
  • a “signal sequence” refers to a polypeptide sequence, e.g., from 10 to 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.
  • sequence identity is determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm. Sequences are referred to as “substantially identical” or “essentially similar” when they share at least a certain minimal percentage of sequence identity when optimally aligned (e.g., when aligned by programs such as GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps.
  • the default scoring matrix used is nwsgapdna
  • the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).
  • Sequence alignments and scores for percentage sequence identity are determined, e.g., 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 is determined by searching against databases, e.g., using algorithms such as FASTA, BLAST, etc. Sequence identity refers to the sequence identity over the entire length of the sequence.
  • 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 mollusk.
  • 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.
  • targeting moiety refers to a moiety that binds specifically to a cell or a portion thereof (e.g., an extracellular, intracellular, or membrane portion thereof) thereby promoting intracellular delivery of the moiety and any cargo bound or complexed thereto (e.g., a polyribonucleotide).
  • a targeting moiety may include a lipid, a small molecule, a carbohydrate, a polypeptide, a nucleic acid (e.g., an aptamer), or a combination thereof.
  • a targeting moiety interacts with the cellular membrane thereby promoting intracellular delivery.
  • a targeting moiety promotes endosomal delivery and/or endosomal escape. In some embodiments a targeting moiety promotes cellular delivery that is not specific to any cell type. In some embodiments, the targeting moiety binds preferentially to a particular cell type and therefore promotes cell-type specific delivery.
  • termination element is a moiety, such as a nucleic acid sequence, that terminates translation of the expression sequence in the circular or linear polyribonucleotide.
  • 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., a cell-free translation system like rabbit reticulocyte lysate.
  • translation initiation sequence is a nucleic acid sequence that initiates translation of an expression sequence in the circular or linear polyribonucleotide.
  • the terms “treat” and “treating” refer to a therapeutic 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 therapeutic treatment.
  • a ’’vector means a polynucleotide (e.g., DNA) that is synthesized (e.g., using PCR), or that is taken from a virus, plasmid, or cell of a higher organism into which a foreign DNA fragment can be or has been inserted for cloning or expression purposes.
  • a vector can be stably maintained in an organism.
  • a vector can include, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, or a multiple cloning site (MCS) .
  • the term includes linear DNA fragments (e.g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like.
  • the vectors provided herein include a multiple cloning site (MCS). In another embodiment, the vectors provided herein do not include an MCS.
  • FIG. 1 is a schematic of an exemplary circular polyribonucleotide complexed with X n (L-B) z , where X is a moiety that binds specifically to a region of the circular polyribonucleotide, L is a linker, and B is a targeting moiety, wherein n is 2, and wherein z is 1 in this schematic.
  • FIG. 2 is a schematic of a circular polyribonucleotide having an annealing or binding region in the spacer region and/or an annealing or binding region in the coding region.
  • FIG. 3 is schematic of a circular polyribonucleotide bound to an oligonucleotide covalently linked to a trivalent GalNAc moiety.
  • the oligonucleotide-linker-trivalent GalNAc is bound to either the spacer region or the coding region of the circular polyribonucleotide.
  • FIG. 4 shows the amount of luminescence measured from circular polyribonucleotides encoding a luciferase polypeptide bound to one or two targeting moieties in comparison to a circular polyribonucleotide without a targeting moiety, wherein the targeting moieties are GalNAc (A or B), cholesterol (I), tocopherol (Q), or a combination thereof.
  • GalNAc GalNAc
  • I cholesterol
  • Q tocopherol
  • FIG. 5 shows the amount of luminescence measured from circular polyribonucleotides encoding a luciferase polypeptide bound to one or two targeting moieties in comparison to a circular polyribonucleotide without a targeting moiety, wherein the targeting moieties are GalNAc (B or C), cholesterol (I or J), tocopherol (Q or R), or a combination thereof.
  • the targeting moieties are GalNAc (B or C), cholesterol (I or J), tocopherol (Q or R), or a combination thereof.
  • FIG. 6 shows the amount of luminescence measured for circular polyribonucleotides from circular polyribonucleotides encoding a luciferase polypeptide bound to a GalNAc and/or tocopherol targeting moiety in the presence of a ribonuclease inhibitor and/or various concentration of calcium chloride in comparison to a circular polyribonucleotide without a targeting moiety.
  • FIG. 7 shows the amount of luminescence measured from circular polyribonucleotides encoding a luciferase polypeptide bound to one or two targeting moieties in comparison to a circular polyribonucleotide without a targeting moiety, wherein the targeting moieties are GalNAc, cholesterol, or tocopherol in the absence of calcium chloride.
  • FIG. 8A and FIG. 8B shows the amount of luminescence measured for circular polyribonucleotides encoding a luciferase polypeptide bound to a GalNAc and tocopherol targeting moiety in the presence of or absence of BSA in a 1 :1 ratio with the circular polyribonucleotide (FIG. 8A) or 60 pM of chloroquine and/or 20 mM of calcium chloride (FIG. 8B) in comparison to in combination with lipofectamine.
  • FIG. 9A shows the amount of luminescence measured in primary adipocytes for circular polyribonucleotides encoding a luciferase polypeptide complexed with an oligomer conjugated to a cholesterol by way of a linker and/or an oligomer conjugated to a tocopherol by way of a linker in the presence or absence of calcium in comparison to a circular polyribonucleotide in the presence of lipofectamine.
  • FIG. 9B shows the amount of flux measured for circular polyribonucleotides encoding a luciferase polypeptide complexed with: (a) an oligomer conjugated to a cholesterol by way of a linker and an oligomer conjugated to a tocopherol, or (b) an oligomer conjugated to an aptamer by way of a linker 6 hours, 24 hours, and 48 hours after subcutaneous administration to mice in comparison to a circular polyribonucleotide administered with lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • FIG. 10 shows a schematic of circular polyribonucleotide complexed with a biotinylated oligomer bound to avidin having three additional biotin binding sites which may bind one or more biotinylated targeting moieties such as biotinylated lipids, biotinylated sugars, biotinylated peptides, or biotinylated antibodies.
  • biotinylated targeting moieties such as biotinylated lipids, biotinylated sugars, biotinylated peptides, or biotinylated antibodies.
  • FIG. 11 A shows the amount of luminescence measured in HEK293T cells transfected with circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to cholesterol by way of a linker in various concentrations of calcium.
  • FIG. 11 B shows the amount of flux measured in mice 72 hours after they were administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to cholesterol by way of a linker in various concentrations of calcium in comparison to a circular polyribonucleotide administered with an LNP.
  • FIG. 12A shows the amount of luminescence measured in primary mouse hepatocytes transfected with circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to tocopherol by way of a linker and an oligomer conjugated to GalNAc by way of a linker in various concentrations of calcium, chloroquine, and/or ribonuclease inhibitor in comparison to a circular polyribonucleotide in lipofectamine.
  • FIG. 12B shows the amount of flux measured in mice 5 hours after they were administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to cholesterol by way of a linker in various concentrations of calcium and/or chloroquine.
  • FIG. 13 show the polypeptide expression resulting from administering a circular polyribonucleotide encoding a SARS-CoV-2 RNA binding protein (RBD) that is complexed either to (a) an oligomer conjugated to cholesterol by way of a linker, or (b) a biotinylated oligomer bound to an avidin bound to biotinylated mannose in the presence or absence of chloroquine.
  • RBD SARS-CoV-2 RNA binding protein
  • FIG. 14A and FIG. 14B shows the amount of flux measured in mice 4-5 hours, 24 hours, and 72 hours after they were intramuscularly administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with: (a) an oligomer conjugated to a biotinylated avidin conjugated to biotinylated mannose by way of a linker, or (b) an oligomer conjugated to a tocopherol by way of a linker and an oligomer conjugated to a cholesterol by way of a linker in comparison to a circular polyribonucleotide administered with an LNP and visualized by the dorsal (FIG.
  • FIG. 15 shows the amount of flux measured in mice over a period of 30 days after they were intradermally administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to cholesterol by way of a linker or an oligomer, wherein the complex is mixed with chloroquine, calcium, albumin, and ribonuclease inhibitor, in comparison to a circular polyribonucleotide administered with an LNP and visualized by the dorsal view.
  • FIG. 16 shows the amount of flux measured in mice 6 hours, 24 hours, or 48 hours after they were subcutaneously administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with: (a) an oligomer conjugated to cholesterol by way of a linker and an oligomer and an oligomer conjugated to tocopherol by way of a linker, (b) a oligomer conjugated to an aptamer by way of a linker, and (c) within an LNP and visualized by the dorsal view.
  • FIG. 17A and FIG. 17B show the percentage of cells that internalized (FIG. 17A) and the mean uptake of cells (FIG. 17B) of the circular polyribonucleotides complexed to: (a) a biotinylated oligomer bound to an avidin bound to an antibody at various concentrations; (b) two biotinylated oligomers each bound to an avidin bound to an antibody and a biotinylated oligomer bound to an avidin conjugated to a mixture of two different antibodies; (c) two biotinylated oligomers each bound to an avidin bound to an antibody and a biotinylated oligomer bound to an avidin conjugated to a mixture of two different aptamers; (d) two biotinylated oligomers each bound to an avidin bound to an antibody and a biotinylated oligomer bound to an avidin conjugated to a mixture of an antibody and phosphatidylserine;
  • FIG. 18 shows the number copies of the circular polyribonucleotide measured to be internalized by Raw264.7 macrophages, wherein the circular polyribonucleotide is complexed with: (a) two biotinylated oligomers each of which are bound to an avidin which is bound to a mixture of antibodies, aptamers, phosphatidylserine, and/or mannose; or (b) an oligomer conjugated to tocopherol by way of a linker and an oligomer conjugated to cholesterol by way of a linker in the presence or absence of calcium.
  • FIG. 18 demonstrates that the various complexed circular polyribonucleotides were localized inside the cells.
  • FIG. 19 shows the amount of luminescence measured in Raw264.7 macrophages having been transfected with a circular polyribonucleotide complexed two biotinylated oligomers each of which are bound to an avidin which is bound to one or more targeting moieties including: an aptamer, antibody, mannose, phosphatidylserine or a mixture thereof in the presence or absence of chloroquine.
  • FIG. 20 shows the amount of luminescence measured for circular polyribonucleotides complexed with either two biotinylated oligomers in comparison to a circular polyribonucleotide complexed with to oligomers each of which independently were bound to an avidin which was bound to biotinylated antibodies.
  • FIG. 21 shows the amount of luminescence measured in HeLa cells after having been transfected with a circular polyribonucleotide encoding a luciferase polypeptide complexed with two biotinylated oligomers each bound to avidin which was bound to Trf antibodies, wherein the complex was administered in the presence or absence of calcium and/or excess transferrin.
  • FIG. 22 shows the amount of luminescence measured from circular polyribonucleotides encoding a luciferase polypeptide bound to a GalNAc targeting moiety in comparison to a circular polyribonucleotide without a targeting moiety.
  • the GalNAc targeting moiety was conjugated by way of a linker at the 5’ end (FC and FC) or 3’ end (FA and FB) an oligomer annealed to the ORF of the circular polyribonucleotide.
  • the linker was either a TEG linker (FA, FB, FC, and FD) or PEG24 linker (FE24).
  • FIG. 23 is a schematic of an exemplary circular polyribonucleotide complexed with X(L-B), where X is a moiety that binds specifically to a region of the circular polyribonucleotide and includes a photoreactive crosslinking agent, L is a linker, and B is a targeting moiety.
  • FIG. 24 is an HPLC chromatogram showing a linear, complementary oligomer (oligomimic) and a binding moiety that includes a 23-mer ribonucleotide, a 3-cyanovinylcarbazole nucleoside ( CNV K) photoreactive crosslinking agent, a linker, and a biotin targeting moiety, wherein the linear complementary oligomer and the binding moiety were bound by annealing or by annealing and irradiation.
  • CNV K 3-cyanovinylcarbazole nucleoside
  • FIG. 25A and 25B are graphs showing the amount of luminescence measured in HEK293 cells transfected with circular polyribonucleotides encoding a luciferase polypeptide complexed with a dimeric fluorescent TAT (dfTAT) peptide (eRNA) complexed by way of annealing then irradiation for 0, 1 , 5, or 30 minutes.
  • dfTAT dimeric fluorescent TAT
  • eRNA peptide
  • the circular polyribonucleotide was complexed with an oligomer bound to an E1 binding region (Chol-E1-cnvK) of the circular polyribonucleotide or an oligomer bound to an E1/E2 binding region (Chol- E2/E1-cnvK) of the circular polyribonucleotide.
  • the results are compared to no circular polyribonucleotides, the circular polyribonucleotides without a binding moiety, the circular polyribonucleotides without a binding moiety, and transfected with lipofectamine MessengerMax (lipo).
  • J Chol J including a cholesterol targeting moiety.
  • FIG. 25B shows the same data as FIG. 25A but is rearranged to emphasize the significant expression of the complex including Chol-E1-cnvK as a binding moiety after irradiation for 30 minutes.
  • FIG. 26A and 26B are graphs showing the amount of luminescence measured in HEK cells transfected with circular polyribonucleotides encoding a luciferase polypeptide (eRNA) complexed by way of annealing and irradiation for 0 or 30 minutes.
  • the circular polyribonucleotide was complexed with an oligomer including one internally located photoreactive crosslinking agent (PS1 D and CHOL-E1_CK) or an oligomer including two internally located photoreactive crosslinking agents (PS2E).
  • PS1 D and CHOL-E1_CK internally located photoreactive crosslinking agent
  • PS2E oligomer including two internally located photoreactive crosslinking agents
  • J Chol J including a cholesterol targeting moiety
  • Q Chol J including a tocopherol binding moiety.
  • FIG. 26B shows the same data from FIG. 26A but is rearranged to emphasize complexes including oligomers with significant expression after irradiation for 30 minutes.
  • compositions, pharmaceutical preparations, and uses of circular polyribonucleotides complexed with one or more targeting moieties are provided.
  • the circular polyribonucleotide complexes described herein improve the efficiency of delivery to a cell or a subject, as compared to the same circular polyribonucleotide alone.
  • the circular polyribonucleotide complexes described herein improve the efficiency of delivery to a cell or a subject, as compared to the same circular polyribonucleotide alone.
  • the photoreactive crosslinking agent is able to form a covalent adduct upon irradiation between the targeting moiety and the circular polyribonucleotide, providing increase stability of the targeting complex to more efficiently deliver the complex to a desired intracellular or extracellular location.
  • the targeting moiety may be, for example, a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid (e.g., an aptamer), or a combination thereof.
  • the circular polyribonucleotide encodes a therapeutic agent (e.g., a therapeutic polypeptide), the complex improves therapeutic efficacy. In some embodiments, the circular polyribonucleotide encodes a polypeptide, the complex improves translation efficiency.
  • a therapeutic agent e.g., a therapeutic polypeptide
  • the complex improves translation efficiency.
  • Complexes and compositions described herein may be complexed with or encapsulated by a carrier (e.g., a cell, a vesicle, a membrane-based carrier, a lipid nanoparticle (LNP), a polymer nanoparticle, a viral particle, or a microbubble).
  • a carrier e.g., a cell, a vesicle, a membrane-based carrier, a lipid nanoparticle (LNP), a polymer nanoparticle, a viral particle, or a microbubble.
  • a carrier e.g., a cell, a vesicle, a membrane-based carrier, a lipid nanoparticle (LNP), a polymer nanoparticle, a viral particle, or a microbubble.
  • LNP lipid nanoparticle
  • Complexes and compositions described herein may have no carrier.
  • Complexes and compositions described herein may be formulated as a pharmaceutical composition, e.g.,
  • the disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide and contains one or more photoactivatable crosslinking agents, a linker, and targeting moiety.
  • the circular polyribonucleotide may be complexed with a molecule of X n (L-B) z , where each X is independently a moiety that binds specifically to a region of the circular polyribonucleotide and contains one or more photoactivatable crosslinking agents, each L is independently a linker, each B is independently a targeting moiety, n is an integer, e.g., from 1 to 20, and z is an integer, e.g., from 1 to 5 (see, e.g., FIG. 23).
  • the moiety that binds the circular polyribonucleotide may be, for example, an oligonucleotide, a polypeptide, or a small molecule.
  • compositions of and methods for forming a covalent complex including A and X n (L-B) z , where is a circular polyribonucleotide, each X is independently a moiety that binds specifically and is covalently attached to a region of A, each L is independently a linker, each B is independently a targeting moiety, n is an integer, e.g., from 1 to 20, and z is an integer, e.g., from 1 to 5.
  • the moiety that binds the circular polyribonucleotide may be, for example, an oligonucleotide, a polypeptide, or a small molecule.
  • the disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide, a linker, and targeting moiety.
  • the disclosure provides a circular polyribonucleotide including from 1 to 20 moieties (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, and 20) that binds specifically to a region of the circular polyribonucleotide conjugated to a linker and a targeting moiety.
  • the circular polyribonucleotide may be complexed with X n (L-B) z , wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5; for example, as shown in FIG. 1 . In some embodiments, n is an integer from 1 to 5 (e.g., 1 , 2, 3, 4, and 5).
  • the disclosure provides a complex including a circular polyribonucleotide, one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) moieties that each binds specifically to a region of the circular polyribonucleotide and includes one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) photoreactive crosslinking agents or covalent attachments, a linker, and one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeting moieties.
  • a complex including a circular polyribonucleotide, one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) moieties that each binds specifically to a region of the circular polyribonucleotide and includes one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) photoreactive crosslinking agents or covalent attachments, a linker, and one or
  • the disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide, a linker, and two or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeting moieties.
  • the target moieties are the same targeting moiety.
  • the targeting moieties are different from one another. Examples of combinations of targeting moieties are described in Table 1.
  • the targeting moiety may be a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid (e.g., an aptamer), or a combination thereof.
  • the targeting moiety is not an aptamer.
  • the targeting moiety is biotinylated.
  • the circular polyribonucleotide may be bound to two targeting moieties (e.g., X n (L-B) z includes B 1 -L-X-L-B 2 wherein each of B 1 and B 2 is an independently selected targeting moiety selected from any targeting moiety described herein, and wherein z is 2).
  • that circular polyribonucleotide may be bound to a GalNAc targeting moiety and a tocopherol targeting moiety.
  • the two targeting moieties are a GalNAc targeting moiety and a cholesterol targeting moiety.
  • both targeting moieties are lipid targeting moieties.
  • the two lipid targeting moieties may be a tocopherol and a cholesterol targeting moiety.
  • the circular polyribonucleotide may be bound to 1 , 2, 3, 4, or 5 targeting moieties (e.g., X n (L-B) z , wherein z is 1 , 2, 3, 4, or 5, and B z is up to 5 independently selected targeting moieties selected from any targeting moiety described herein).
  • all targeting moieties are different.
  • the circular polyribonucleotide may be bound to a GalNAc targeting moiety, a tocopherol targeting moiety, a urea targeting moiety, a melittin targeting moiety, and an aptamer targeting moiety.
  • all targeting moieties are the same class of moiety, but differ within that class.
  • the circular polyribonucleotide may be bound to four lipid targeting moieties, wherein the lipid targeting moieties are cholesterol, tocopherol, vitamin A, and phosphocholine. In some embodiments, all targeting moieties are the same.
  • the circular polyribonucleotide may be bound to 1 , 2, 3, 4, or 5 GalNAc targeting moieties. In some embodiments, 2, 3, or 4 targeting moieties are the same and 1 targeting moiety is different. In some embodiments, 2 or 3 targeting moieties are the same and 1 or 2 are different. In some embodiments, 2 targeting moieties are the same and 1 , 2, or 3 are different.
  • the circular polyribonucleotide may be bound to two tocopherol targeting moieties, one cholesterol targeting moiety, and one aptamer targeting moiety.
  • the circular polyribonucleotide may be bound to two tocopherol targeting moieties and two cholesterol moieties.
  • 2 sets of 2 targeting moieties are the same and one is different.
  • a circular polyribonucleotide may be bound to two GalNAc targeting moieties, two cellulose targeting moieties, and one Transportan targeting moiety.
  • the targeting moiety targets (e.g., binds to or delivers the circular polynucleotide preferentially to) a specific cell type; for example, the polypeptide targeting moiety may preferentially target hepatic cells, muscle cells (e.g., skeletal muscle cell), blood cells, bone cells, fat cells, skin cells, nerve cells, endothelial cells, stem cells, sex cells, pancreatic cells, or cancer cells.
  • the polypeptides target cancer cells (e.g., tumor cells).
  • the polypeptide targeting moiety targets cells of a specific organ; for example, the polypeptide targeting moiety may preferentially target cells in the heart, lungs, liver, or kidneys.
  • the targeting moiety complexed to the circular polyribonucleotide is a lipid.
  • Lipid moieties include organic compounds, most preferably organic biomolecules, that are selectively soluble in nonpolar solvents over water.
  • Lipid moieties specifically contemplated by the present invention include, for example, fatty acids, steroids or sterols, glycerides (e.g., monoglycerides, diglycerides, and triglycerides), phospholipids (e.g., phosphatidylcholines), fat-soluble vitamins (e.g., vitamins A, D, E, and K), cationic lipids, ionizable lipids, or zwitterionic lipids.
  • the lipid moiety is a fatty acid.
  • a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated.
  • the fatty acid may be naturally occurring, a modified variant of a naturally occurring fatty acid, or a synthetic (e.g., non-naturally occurring fatty acid).
  • Fatty acids are known to those of skill in the art.
  • the fatty acid is a saturated fatty acid.
  • the fatty acid is an unsaturated fatty acid.
  • the fatty acid includes a branched or unbranched chain including from 4 to 40 main-chain carbon atoms (e.g., from 4 to 28 main-chain carbon atoms, from 4 to 15 main-chain carbon atoms, from 10 to 30 main-chain carbon atoms, or from 15 to 40 main-chain carbon atoms).
  • 4 to 40 main-chain carbon atoms e.g., from 4 to 28 main-chain carbon atoms, from 4 to 15 main-chain carbon atoms, from 10 to 30 main-chain carbon atoms, or from 15 to 40 main-chain carbon atoms.
  • the fatty acid includes squalene, stearic acid, oleic acid, palmitic acid, linoleic acid, stearic acid, lauric acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, a-tocopherol, a-tocopherol succinate, or a retinoic acid (e.g., all-trans-retinoic acid), or an analog or metabolite thereof.
  • the fatty acid includes one, two, three, four, or five lipid chains (e.g., aliphatic lipids selected from above).
  • the lipid moiety is a steroid or a sterol.
  • Steroid and sterols are known to those of skill in the art.
  • a steroid is an organic compound with four rings in a fused ring molecular configuration, in particular, a having a core cyclopentanoperhydrophenanthrene ring system.
  • a sterol is any steroid-based alcohol.
  • Steroids and sterols may be naturally occurring or synthetic.
  • the steroid or sterol is selected from cholesterol, ursodeoxycholic acid, lithocholic acid, or an analog or metabolite thereof.
  • the lipid moiety is a fat-soluble vitamin.
  • the fat- soluble vitamin is selected from vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof.
  • the lipid moiety is a phospholipid.
  • Phospholipids are a class of lipids whose molecule has a hydrophilic "head” containing a phosphate group, and one or more (e.g., two) hydrophobic "tails" derived from fatty acids, often joined by an alcohol residue.
  • the phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine.
  • the phospholipid is a phosphatidylcholine (e.g., phospholipids that incorporate choline as a headgroup).
  • the phosphatidylcholine is selected from phosphocholine (PC), PC- docosahexaenoic acid (PC-DHA), PC-docosanoic acid (PC-DCA), PC-eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC-retinoic acid (PC-RA), or PC-a-tocopherol succinate (PC-TS).
  • PC phosphocholine
  • PC-DHA PC- docosahexaenoic acid
  • PC-DCA PC-docosanoic acid
  • PC-EPA PC-eicosapentaenoic acid
  • PC-LA PC-lithocholic acid
  • PC-RA PC-retinoic acid
  • PC-TS PC-a-tocopherol succinate
  • any of the above-described lipid moieties may be chemically-modified (e.g., derivatized or conjugate to a linker) to allow for conjugation to a nucleic acid (e.g., an oligonucleotide or a circular polyribonucleotide).
  • a nucleic acid e.g., an oligonucleotide or a circular polyribonucleotide.
  • Any of the above-described lipid moieties may also be biotinylated to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
  • the disclosure provides small molecule targeting moieties which may form a complex with a circular polyribonucleotide.
  • Small molecule targeting moiety have an average molecular weight less than 1 kDa, corresponding to a Stokes-Einstein radius of 1 nm.
  • the small molecule may have a molecular weight of from about 300 to about 700 Da (e.g., about 325 Da, about 350 Da, about 375 Da, about 400 Da, about 425 Da, about 450 Da, about 475 Da, about 500 Da, about 525 Da, about 550 Da, about 575 Da, about 600 Da, about 625 Da, about 650 Da, or about 675 Da).
  • the small molecule may have a molecular weight of from 500 Da to 1000 Da (e.g., from about 500 Da to 900 Da, about 500 Da to 800 Da, 500 Da to 700 Da, 500 Da to 600 Da, 600 Da to 1000 Da, 700 Da to 1000 Da, 800 Da to 1000 Da, or 900 Da to 1000 Da).
  • 500 Da to 1000 Da e.g., from about 500 Da to 900 Da, about 500 Da to 800 Da, 500 Da to 700 Da, 500 Da to 600 Da, 600 Da to 1000 Da, 700 Da to 1000 Da, 800 Da to 1000 Da, or 900 Da to 1000 Da).
  • Small molecule targeting moieties upon reaching the target tissue, must cross the plasma membrane to reach the cytosol or nucleus of the cell.
  • the plasma membrane is relatively permeable to hydrophobic small molecules.
  • the transport of macromolecules requires an active uptake mechanism, which requires the small molecule have efficient interactions with the plasma membrane and subsequent endocytosis processes, such as phagocytic-, clathrin-, and caveolae-mediated endocytosis.
  • the small molecule binding targeting moieties specifically bind to a protein of a cell and the protein mediates internalization of the circular polyribonucleotide bound to the targeting small molecule moiety into the cell upon binding to the targeting small molecule moiety.
  • the small molecule targeting moiety is folic acid.
  • Folic acid may enable efficient intracellular delivery of the circular polyribonucleotide, following folic acid receptor mediated endocytosis.
  • the small molecule targeting molecule is urea. Urea may be internalized into the cell using clathrin-mediated endocytosis to deliver the attached circular polyribonucleotide.
  • the small molecule may be glutamate urea or 2-[3-(1 ,3- dicarboxypropyl)-ureido] pentanedioic acid.
  • Urea may be used as a small molecule targeting moiety to target the prostate specific membrane antigen (PSMAIn some embodiments, the small molecule is ursodexoycholic acid. In some embodiments, the small molecule is lithocholic acid. In some embodiments, glycyrrhetinic acid derivatives may be the small molecule targeting moiety. Glycyrrhetinic acid derivatives may be used for hepatocellular applications due to presence of glycyrrhetinic acid receptors. In some embodiments, a sulfonamide may be used as the small molecule targeting moiety. Sulfonamide derivatives may be used to target tumors expressing carbonic anhydrase IX.
  • a benzamide may be used as the small molecule targeting moiety. Benzamides, such as anisamide, may be used to specifically target sigma-1 receptors.
  • phenyl boronic acid may be used as the small molecule targeting moiety. Phenyl boronic acid may be used to target components of sialic acid.
  • hyaluronic acid may be used as the small molecule targeting moiety. Hyaluronic acid has been shown to have affinity toward CD44 receptors.
  • bisphosphonate may be used as the small molecule targeting moiety. Bisphosphonates may be used to effectively target bone.
  • biotin may be used as the small molecule targeting moiety.
  • Biotin-mediated drug delivery may be used for tumor targeting applications by way of the biotin receptor.
  • biotin may be conjugated to any of the above-described small molecules to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
  • a biotin-binding protein e.g., avidin, streptavidin, NeutrAvidin.
  • the disclosure provides carbohydrate targeting moieties which may form a complex with a circular polyribonucleotide.
  • the carbohydrate targeting moiety may, for example, include a saccharide, disaccharide, or polysaccharide.
  • the carbohydrate includes mannose, galactose, or glucose.
  • a carbohydrate moiety described herein includes one or more monosaccharide moieties.
  • the one or more monosaccharide moieties includes at least 3 carbon atoms (e.g., arranged in a linear, branched, or cyclic structure) and an oxygen, nitrogen, or sulfur atom, or a fragment or variant of a monosaccharide moiety including at least 3 carbon atoms (e.g., arranged in a linear, branched, or cyclic structure) and an oxygen, nitrogen, or sulfur atom.
  • Each monosaccharide moiety or fragment or variant thereof may be a tetrose, pentose, hexose, or heptose .
  • Each monosaccharide moiety or fragment or variant thereof may exist as an aldose, ketose, sugar alcohol, and, where appropriate, in the L or D form.
  • Exemplary monosaccharide moieties may be amino sugars, N-acetylamino sugars, imino sugars, deoxysugars, or sugar acids.
  • Carbohydrates may include individual monosaccharide moieties, or may further include a disaccharide, oligosaccharide (e.g., a trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide), a polysaccharide, or combinations thereof.
  • Exemplary carbohydrates include ribose, arabinose, lyxose, xylose, deoxyribose, ribulose, xylulose, glucose, galactose, mannose, gulose, idose, talose, allose, altrose, psicose, fructose, sorbose, tagatose, rhamnose, pneumose, quinovose, fucose, mannuheptulose, sedoheptulose, galactosamine, mannosamine, glucosamine, N-acetylglucosamine, N- acetylgalactosamine, N-acetylmannosamine, glucuronic acid, galacturonic acid, mannuronic acid, guluronic acid, iduronic acid, tagaturonic acid, frucuronic acid, galactosaminuronic acid, mannosaminuronic acid, glucosaminuronic acid,
  • the carbohydrate moiety may include one or more monosaccharide moieties linked by a glycosidic bond.
  • the glycosidic bond includes a 1 ⁇ 2 glycosidic bond, a 1 ⁇ 3 glycosidic bond, a 1 ⁇ 4 glycosidic bond, or a 1 ⁇ 6 glycosidic bond.
  • each glycosidic bonds may be present in the alpha or beta configuration.
  • the one or more monosaccharide moieties are linked directly by a glycosidic bond or are separated by a linker.
  • carbohydrate refers to compound including one or more monosaccharide moieties including at least 3 carbon atoms (e.g., arranged in a linear, branched, or cyclic structure) and an oxygen, nitrogen, or sulfur atom, or a fragment or variant of a monosaccharide moiety including at least 3 carbon atoms (e.g., arranged in a linear, branched, or cyclic structure) and an oxygen, nitrogen, or sulfur atom.
  • Each monosaccharide moiety or fragment or variant thereof may be a tetrose, pentose, hexose, or heptose.
  • Each monosaccharide moiety or fragment or variant thereof may exist as an aldose, ketose, sugar alcohol, and, where appropriate, in the L or D form.
  • Exemplary monosaccharide moieties may be amino sugars, N-acetylamino sugars, imino sugars, deoxysugars, or sugar acids.
  • Carbohydrates may include individual monosaccharide moieties, or may further include a disaccharide, oligosaccharide (e.g., a trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide), a polysaccharide, or combinations thereof.
  • Exemplary carbohydrates include ribose, arabinose, lyxose, xylose, deoxyribose, ribulose, xylulose, glucose, galactose, mannose, gulose, idose, talose, allose, altrose, psicose, fructose, sorbose, tagatose, rhamnose, pneumose, quinovose, fucose, mannuheptulose, sedoheptulose, galactosamine, mannosamine, glucosamine, N- acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, glucuronic acid, galacturonic acid, mannuronic acid, guluronic acid, iduronic acid, tagaturonic acid, frucuronic acid, galactosaminuronic acid, mannosaminuronic acid, glucosaminuronic acid,
  • the carbohydrate may include one or more monosaccharide moieties linked by a glycosidic bond.
  • the glycosidic bond includes a 1->2 glycosidic bond, a 1->3 glycosidic bond, a 1->4 glycosidic bond, or a 1->6 glycosidic bond.
  • each glycosidic bonds may be present in the alpha or beta configuration.
  • the one or more monosaccharide moieties are linked directly by a glycosidic bond or are separated by a linker.
  • the present disclosure features a circular polyribonucleotide complexed with a carbohydrate targeting moiety, wherein the carbohydrate targeting moiety includes an asialoglycoprotein receptor (ASGPR) binding moiety.
  • the ASGPR is a C-type lectin primarily expressed on the sinusoidal surface of hepatocytes, and includes a major (48 kDa, ASGPR-1) and a minor (40 kDa, ASGPR-2) subunit.
  • the ASGPR is involved in the binding, internalization, and subsequent clearance of glycoproteins containing an N-terminal galactose (Gal) or N-terminal N-acetylgalactosamine (GalNAc) residues from circulation, such as antibodies.
  • ASGPRs have also been shown to be involved in the clearance of low-density lipoprotein, fibronection, and certain immune cells, and may be utilized by certain viruses for hepatocyte entry (see, e.g., Yang J., et al (2006) J Viral Hepat 13:158-165 and Guy, CS et al (2011) Nat Rev Immunol 8:874-887).
  • the carbohydrate targeting moiety is mannose.
  • carbohydrate targeting moiety may be a-mannose or high-mannose.
  • the carbohydrate targeting moiety includes a mannose 6-phosphate (M6P) or analog thereof.
  • the carbohydrate targeting moiety include a plurality of M6P moieties (e.g., M6Ps), e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more M6P moieties.
  • the carbohydrate targeting moiety includes from 2 to 20 M6P moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 M6P moieties). In an embodiment, the carbohydrate targeting moiety includes from 2 to 10 M6P moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 M6P moieties). In an embodiment, the carbohydrate targeting moiety includes from 2 to 5 M6P moieties (e.g., 2, 3, 4, or 5 M6P moieties).
  • the carbohydrate targeting moiety includes a galactose (Gal), galactosamine (GalNH2), or an N-acetylgalactosamine (GalNAc) moiety, for example, a Gal, GalNH2, or GalNAc, or an analog thereof.
  • the carbohydrate targeting moiety includes a GalNAc moiety (e.g., GalNAc).
  • the carbohydrate targeting moiety includes a plurality of GalNAc moieties (e.g., GalNAcs), e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more GalNAc moieties (e.g., GalNAcs).
  • the carbohydrate targeting moiety includes from 2 to 20 GalNAcs moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 GalNAc moieties). In an embodiment, the carbohydrate targeting moiety includes from 2 to 10 GalNAc moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 GalNAc moieties). In an embodiment, the carbohydrate targeting moiety includes from 2 to 5 GalNAc moieties (e.g., 2, 3, 4, or 5 GalNAc moieties). In an embodiment, the carbohydrate targeting moiety includes 2 GalNAc moieties. In an embodiment, the carbohydrate targeting moiety includes 3 GalNAc moieties.
  • the carbohydrate targeting moiety includes 4 GalNAc moieties. In an embodiment, the carbohydrate targeting moieties includes 5 GalNAc moieties. In some embodiments, the carbohydrate targeting moiety includes a mono-, di-, tri-, or tetra-GalNAc
  • the GalNAc moiety includes a structure of Formula (I): salt thereof, wherein X is O, N(R 7 ), or S; each of R 1 , R 3 , R 4 , and R 5 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, C(O)-alkyl, C(O)-alkenyl, C(O)-alkynyl, C(O)-heteroalkyl, C(O)-haloalkyl, C(O)-aryl, C(O)- heteroaryl, C(O)-cycloalkyl, or C(O)-heterocyclyl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl is optionally
  • X is O.
  • each of R 1 , R 3 , R 4 , and R 5 are independently hydrogen or alkyl (e.g., CH 3 ).
  • R 2a is hydrogen.
  • R 2b is C(O)CH 3 .
  • each of R 6a and R 6b is hydrogen.
  • the GalNAc moiety is connected to a linker at R 2a .
  • the GalNAc moiety is connected to a linker at R 2b .
  • the GalNAc moiety is connected to a linker or at R 3 .
  • the GalNAc moiety is connected to a linker at R 4 .
  • the GalNAc moiety is connected to a linker at R 5 . In some embodiments, the GalNAc moiety is connected to a linker at R 6a or R 6b . In some embodiments, the GalNAc moiety is connected to a linker at a plurality of positions, e.g., at least two of R 1 , R2a, R2b, R 3 , R 4 , R 5 , R 6a , and R 6b .
  • the GalNAc moiety is includes a structure of Formula (l-a) (
  • each of R 3 , R 4 , and R 5 are independently hydrogen or alkyl (e.g., CH 3 ).
  • R 2a is hydrogen.
  • R 2b is C(O)CH 3 .
  • the carbohydrate targeting moiety includes a structure of Formula (II): (II), or a salt thereof, wherein each of R 1 , R2a, R2b, R 3 ,
  • R 4 , R 5 , R 6a , and R 6b and subvariables thereof are as defined for Formula (I), L is a linker, and n is an integer from 1 to 100, wherein represents an attachment point to a branching point, additional linker.
  • X is O.
  • each of R 1 , R 3 , R 4 , and R 5 are independently hydrogen or alkyl (e.g., CH 3 ).
  • R 2a is hydrogen.
  • R 2b is C(O)CH 3 .
  • each of R 6a and R 6b is hydrogen.
  • n is an integer from 1 to 50.
  • n is an integer from 1 to 25.
  • n is an integer from 1 to 10.
  • n is an integer from 1 to 5.
  • n is 1 , 2, 3, 4, or 5.
  • n is 1.
  • the carbohydrate targeting moiety includes a structure of Formula (ll-a):
  • R 1 , R 2a , R 2b , R 3 , R 4 , R 5 , R 6a , and R 6b and subvariables thereof are as defined for Formula (I), each of L 1 and L 2 is independently a linker, each of m and n is independently an integer from 1 to 100, and M is a linker, wherein represents an attachment point to a branching point, additional linker.
  • X is O (e.g., X in each of A and B is O).
  • each of R 1 , R 3 , R 4 , and R 5 are independently hydrogen or alkyl (e.g., CH 3 ) (e.g., R 1 , R 3 , R 4 , and R 5 in each of A and B is independently hydrogen or alkyl).
  • R 2a is hydrogen (e.g., R 2a in each of A and B is hydrogen).
  • R 2b is C(O) CH 3 (e.g., R 2b in each of A and B is C(O)CH 3 ).
  • each of R 6a and R 6b is hydrogen (e.g., R 6a and R 6b in each of A and B is hydrogen).
  • each of m and n is independently an integer from 1 to 50.
  • each of m and n is independently an integer from 1 to 25.
  • each of m and n is independently an integer from 1 to 10.
  • each of m and n is independently an integer from 1 to 5.
  • each of m and n is independently 1 , 2, 3, 4, or 5.
  • each of m and n is independently 1.
  • each of L 1 and L 2 independently includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, each of L 1 and L 2 independently includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, each of L 1 and L 2 independently is cleavable or non-cleavable.
  • M includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, M includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, M is cleavable or non-cleavable. In some embodiments, the ASGPR moiety includes a structure of Formula (I l-b): (I l-b), or a salt thereof, wherein each of R 1 ,
  • X is O (e.g., X in each of A, B, and C is O).
  • each of R 1 , R 3 , R 4 , and R 5 are independently hydrogen or alkyl (e.g., CH 3 ) (e.g., R 1 , R 3 , R 4 , and R 5 in each of A, B, and C is independently hydrogen or alkyl).
  • R 2a is hydrogen (e.g., R 2a in each of A, B, and C is hydrogen).
  • R 2b is C(O)CH 3 (e.g., R 2b in each of A, B, and C is C(O)CH 3 ).
  • each of R 6a and R 6b is hydrogen (e.g., R 6a and R 6b in each of A, B, and
  • each of m, n, and 0 is independently an integer from 1 to 50. In some embodiments, each of m, n, and 0 is independently an integer from 1 to 25. In some embodiments, each of m, n, and 0 is independently an integer from 1 to 10. In some embodiments, each of m, n, and 0 is independently an integer from 1 to 5. In some embodiments, each of m, n, and 0 is independently 1 , 2, 3, 4, or 5. In some embodiments, each of m, n, and 0 is independently 1 .
  • each of L 1 , L 2 , and L 3 independently includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, each of L 1 , L 2 , and L 3 independently includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, each of L 1 , L 2 , and L 3 independently is cleavable or non-cleavable.
  • M includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, M includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, M is cleavable or non-cleavable. In some embodiments, the carbohydrate targeting moiety includes a structure of Formula (I l-c): (I l-c) , or a salt thereof, wherein each of R 2a , R 2b , R 3 , R 4 ,
  • R 5 and subvariables thereof are as defined for Formula (I), each of L 1 , L 2 , and L 3 is independently a linker, and M is a linker, wherein represents an attachment point to a branching point, additional linker.
  • each of R 3 , R 4 , and R 5 are independently hydrogen or alkyl (e.g., CH 3 ).
  • R 2a is hydrogen.
  • R 2b is C(O)CH 3 .
  • each of L 1 , L 2 , and L 3 independently includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, each of L 1 , L 2 , and L 3 independently includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, each of L 1 , L 2 , and L 3 independently is cleavable or non-cleavable.
  • M includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group.
  • M includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group.
  • M is cleavable or non-cleavable.
  • the carbohydrate targeting moiety includes a compound selected from:
  • the carbohydrate targeting moiety includes a linker including a cyclic moiety, such as a pyrroline ring.
  • the carbohydrate targeting moiety includes a structure of Formula (CH):
  • R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , and R 18 are each independently for each occurrence H, — CH 2 OR a , or OR b ;
  • R a and R b are each independently for each occurrence hydrogen, a hydroxyl protecting group, optionally substituted alkyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted alkenyl, optionally substituted heteroaryl, polyethyleneglycol (PEG), a phosphate, a diphosphate, a triphosphate, a phosphonate, a phosphonothioate, a phosphonodithioate, a phosphorothioate
  • R L O-oligonucleotide
  • R 30 is independently for each occurrence -linker-R L or R 31
  • R L is hydrogen or a ligand
  • R 31 is — C(O)CH(N(R 32 )2)(CH2)hN(R 32 )2
  • R 32 is independently for each occurrence H, — R L , -linker- RL or R 31
  • Z 1 is independently for each occurrence O or S
  • Z 2 is independently for each occurrence O, S, N(alkyl) or optionally substituted alkyl
  • h is independently for each occurrence 1 -20.
  • the compound of Formula (Cll) is selected from:
  • the carbohydrate targeting moiety is a compound or substructure disclosed in U.S. Patent No. 8,106,022, which is incorporated herein by reference in its entirety. In other embodiments, the carbohydrate targeting moiety is selected from:
  • one ofX or Y is a branching point or a linker, and the other of X and Y is hydrogen.
  • the ASGPR moiety includes a structure of Formula (Xll-a):
  • the carbohydrate targeting moiety is a compound or substructure disclosed in Nucleic Acids (2016) 5:e317 or WO2015/042447, each of which is incorporated herein by reference in its entirety.
  • the carbohydrate targeting moiety includes a structure of Formula (V-a): wherein n is an integer from 1 to 20.
  • the compound of Formula (V-a) is selected from:
  • the carbohydrate binding moiety includes a structure of Formula (V-b): wherein A is O or S, A’ is O, S, or
  • NH NH
  • Z is an oligomeric compound, e.g., a linker.
  • the carbohydrate targeting moiety includes
  • the carbohydrate targeting moiety is selected from:
  • the carbohydrate targeting moiety is a compound or substructure disclosed in WO 2017/156012, which is incorporated herein by reference in its entirety.
  • a hydroxyl group within a carbohydrate targeting moiety is protected, for example, with an acetyl or acetonide moiety.
  • a hydroxyl group within a carbohydrate targeting moiety is protected with an acetyl group.
  • a hydroxyl group within an ASGPR moiety is protected with acetonide group.
  • 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or more hydroxyl groups within a carbohydrate targeting moiety may be protected, e.g., with an acetyl group or an acetonide group. In some embodiments, all of the hydroxyl groups within a carbohydrate targeting moiety are protected.
  • the carbohydrate targeting moiety includes an additional active agent, such as a ligand (e.g., a steroid).
  • a ligand e.g., a steroid
  • the ligand may be covalently or non-covalently associated with the carbohydrate targeting moiety.
  • the ligand may be covalently bound to a carbohydrate, linker, or a branching point within the carbohydrate targeting moiety.
  • the carbohydrate targeting moiety includes
  • carbohydrate targeting moieties may also be biotinylated to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
  • a biotin-binding protein e.g., avidin, streptavidin, NeutrAvidin.
  • polypeptide targeting moieties which may form a complex with a circular polyribonucleotide.
  • the polypeptide targeting moieties may also be referred to as protein drug conjugates.
  • the polypeptide targeting moieties specifically bind to a protein of a cell and the protein mediates internalization of the circular polyribonucleotide bound to the targeting polypeptide moiety into the cell upon binding to the targeting polypeptide moiety.
  • the polypeptide targeting moiety may include all natural amino acids.
  • the polypeptide targeting moiety includes at least one unnatural amino acid reside; for example, the polypeptide targeting moiety may include 1 , 2, 3, 4, 5, 6, 8, 9, 10, or more unnatural amino acid residues. In some embodiments, the polypeptide targeting moiety may include all unnatural amino acid residues.
  • the polypeptide targeting moiety is a cell-penetrating peptide.
  • a cell penetrating peptide may include from 2 to 100 amino acid residues (e.g., from 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 10 to 100, 20 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, and 90 to 100 amino acid residues).
  • the polypeptide targeting moiety includes from 2 to 50 amino acids (e.g., from 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 and 20, 2 to 15, 2 to 10, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, and 45 to 50 amino acid residues).
  • Cell-penetrating peptides may be characterized as a synthetic cell-penetrating peptides, protein- derived cell-penetrating peptides, and chimeric cell-penetrating peptides. Furthermore, cell-penetrating peptides may be either linear or cyclic. Cell-penetrating peptides may also be categorized by physical properties and include cationic cell-penetrating peptides, amphipathic cell-penetrating peptides, or hydrophobic cell-penetrating peptides. The cell-penetrating peptide may be a one of the as described in Table 2. In some embodiments, the cell-penetrating peptide is melittin.
  • the cell-penetrating peptide is a TAT peptide. In some embodiments, the cell-penetrating peptide is Pep-1. In some embodiments, the cell- penetrating peptide is Transportan. In some embodiments, the cell-penetrating peptide is Penetratin. Further examples of cell-penetrating peptides may be found on the CPPsite 2.0 Database of Cell- Penetrating Peptides (webs.iiitd.edu.in/raghava/cppsite/information.php). This database contains >1500 cell-penetrating peptide sequences.
  • cell-penetrating peptides may transport hydrophilic macromolecules to a cell through energy-independent pathways.
  • Cell-penetrating peptides may enter the cell in a noninvasive way, as they usually do not disturb the structure of the plasma membranes and are considered safe and highly efficient.
  • Some cell-penetrating peptides are reported to cross the cell membrane by an energy- dependent cellular process using, for example, endocytosis or receptor-mediated uptake, whereas others use energy-independent non-endocytic translocation pathways.
  • the polypeptide targeting moiety binds to a receptor on a cell surface. In some embodiments, the polypeptide binds to the receptor with high affinity. In some embodiments, the receptor is overexpressed on the cell surface. In some embodiments, the polypeptide includes an Arg- Gly-Asp (RGD) motif. In some embodiments, the RGD motif binds integrins. In some embodiments, the RGD motif is a cyclic RGD motif. In some embodiments, the cyclic RGD motif includes the amino acid sequence of RGDFK (SEQ ID NO: 16). In some embodiments, the cyclic RGD may be an internalizing RGD conjugate having an amino acid sequence of CRGDKRGPDC (SEQ ID NO: 17).
  • the polypeptide targeting moiety targets the circular polyribonucleotide to muscle cells.
  • this polypeptide is a M12 peptide having the amino acid sequence RRQPPRSISSHP (SEQ ID NO: 18).
  • the muscle targeting polypeptide is the 7 amino acid residue peptide ASSLNIA (SEQ ID NO: 19).
  • the polypeptide is an LPS-binding protein (LBP) peptide.
  • the polypeptide is an adipose-homing peptide.
  • the peptide is an endolytic peptide.
  • the endolytic peptide may have the structure H2N-CKRKKRRQRRRG(dPEG6)GWWG(K/AzidePEG10)-amide.
  • the peptide is cyclo-F ⁇ t>RRRRQ.
  • the polypeptide is melittin. In some embodiments, the melittin targets tumor cells.
  • the polypeptide targets a specific cell type; for example, the polypeptide may preferentially target muscle cells (e.g., skeletal muscle cell), blood cells, bone cells, fat cells, skin cells, nerve cells, endothelial cells, stem cells, sex cells, pancreatic cells, or cancer cells.
  • the polypeptides target cancer cells (e.g., tumor cells).
  • the polypeptide targets cells of a specific organ; for example, the polypeptide may preferentially target cells in the heart, lungs, liver, or kidneys.
  • the polypeptide targeting moiety is an antibody, which may also then be known an antibody drug conjugate.
  • the antibody may target an overexpressed antigen or receptor on the surface of a cell.
  • the polypeptide targeting moiety is an antibody fragment.
  • the polypeptide targeting moiety is a single chain Fv molecule (scFv), a diabody, a triabody, a nanobody, a domain antibody, an antibody-like protein scaffold, a Fab fragment, a Fv fragment, a Fab’, a F(ab’)2, a tandem scFv (taFv), or an scFv-Fc.
  • the polypeptide targeting moiety is a bispecific antibody.
  • the peptide targeting moiety is an scFv.
  • the polypeptide targeting moiety is a nanobody.
  • the antibody may bind to a tumor cell-specific antigen.
  • the antibody may be an anti-CD33 antibody, an anti-CD30 antibody, an anti-HER2 antibody, an anti-CD22 antibody, an anti-EGFR antibody, an anti-nectin4 antibody, an anti HER2 antibody, an anti-AXL antibody, an anti-CD74 antibody, an anti-ALK antibody, an anti-PTK antibody, anti-MR antibody, an anti-CD205 antibody, an anti-CD169 antibody, an anti-CD14 antibody, an anti-CD36 antibody, an anti-CD5 antibody, an anti -CD71 antibody, an anti-CD38 antibody, or an anti-prohibin antibody.
  • the antibody may bind to a tumor tissue-associated antigen.
  • the antibody may be an anti-PSMA antibody, an anti-TM4SF1 antibody, or an anti-CD276 antibody.
  • the peptide targeting moiety is an anti-FcRn antibody.
  • the peptide targeting moiety is an anti-MR antibody.
  • the peptide targeting moiety is an anti-CD205.
  • the peptide targeting moiety is an anti-CD14 antibody.
  • the peptide targeting moiety is an anti-CD36 antibody.
  • the peptide targeting moiety is an anti- CD5 antibody.
  • the peptide targeting moiety is an anti-CD71 Fab.
  • the peptide targeting moiety is an anti-prohibin.
  • the peptide targeting moiety is an anti-DEC205 antibody.
  • the nanobody may bind to a tumor cell-specific antigen.
  • the nanobody may be an anti-CD33 nanobody, an anti-CD30 nanobody, an anti-HER2 nanobody, an anti- CD22 nanobody, an anti-EGFR nanobody, an anti-transferrin nanobody, an anti-nectin4 nanobody, an anti HER2 nanobody, an anti-AXL nanobody, and anti-CD74 nanobody, an anti-ALK nanobody, or an anti-PTK nanobody.
  • the nanobody may bind to a tumor tissue-associated antigen.
  • the antibody may be an anti-PSMA nanobody, an anti-TM4SF1 nanobody, or an anti- CD276 nanobody.
  • the polypeptide targeting moiety is an anti-transferrin nanobody. In some embodiments, the polypeptide targeting moiety is an anti-EGFR nanobody. In some embodiments, the polypeptide targeting moiety is an anti-HER2 nanobody.
  • polypeptide moieties may also be biotinylated to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
  • a biotin-binding protein e.g., avidin, streptavidin, NeutrAvidin.
  • a targeting moiety is an aptamer.
  • An aptamer is an isolated nucleic acid molecule that binds with high specificity and affinity to a binding moiety or target molecule, such as a protein.
  • An aptamer is a three-dimensional structure held in certain conformation(s) that provides chemical contacts to specifically bind its given target.
  • aptamers are nucleic acid-based molecules, there is a fundamental difference between aptamers and other nucleic acid molecules such as genes and mRNA. In the latter, the nucleic acid structure encodes information through its linear base sequence and thus this sequence is of importance to the function of information storage.
  • aptamer function which is based upon the specific binding of a target molecule, is not entirely dependent on a conserved linear base sequence (a non-coding sequence), but rather a particular secondary /tertiary/quaternary structure. Any coding potential that an aptamer may possess is fortuitous and is not thought to play a role in the binding of an aptamer to its cognate target.
  • Aptamers are differentiated from naturally occurring nucleic acid sequences that bind to certain proteins. These latter sequences are naturally occurring sequences embedded within the genome of the organism that bind to a specialized sub-group of proteins that are involved in the transcription, translation, and transportation of naturally occurring nucleic acids (e.g., nucleic acid-binding proteins).
  • Aptamers on the other hand are non-naturally occurring nucleic acid molecules. While aptamers can be identified that bind nucleic acid- binding proteins, in most cases such aptamers have little or no sequence identity to the sequences recognized by the nucleic acid-binding proteins in nature. More importantly, aptamers can bind virtually any protein (not just nucleic acid-binding proteins) as well as almost any partner of interest including small molecules, carbohydrates, peptides, etc. For most partners, even proteins, a naturally occurring nucleic acid sequence to which it binds does not exist.
  • aptamers are capable of specifically binding to selected partners and modulating the partner’s activity or binding interactions, e.g., through binding, aptamers may block their partner’s ability to function.
  • the functional property of specific binding to a partner is an inherent property an aptamer.
  • An aptamer can be 6-35 kDa.
  • An aptamer can be from 20 to 500 nucleotides.
  • An aptamer can bind its partner with micromolar to sub-nanomolar affinity, and may discriminate against closely related targets (e.g., aptamers may selectively bind related proteins from the same gene family). In some cases, an aptamer only binds one molecule. In some cases, an aptamer binds family members of a molecule of interest. An aptamer, in some cases, binds to multiple different molecules. Aptamers are capable of using commonly seen intermolecular interactions such as hydrogen bonding, electrostatic complementarities, hydrophobic contacts, and steric exclusion to bind with a specific partner.
  • An aptamer can include a molecular stem and loop structure formed from the ou hybridization of complementary polynucleotides that are covalently linked (e.g., a hairpin loop structure).
  • the stem includes the hybridized polynucleotides and the loop is the region that covalently links the two complementary polynucleotides.
  • An aptamer can be a linear ribonucleic acid (e.g., linear aptamer) including an aptamer or a circular polyribonucleic acid including an aptamer (e.g., a circular aptamer).
  • linear ribonucleic acid e.g., linear aptamer
  • circular polyribonucleic acid e.g., a circular aptamer
  • one or more of the aptamer targeting moieties includes aptamer K19, M2 aptamer, Ly75 aptamer, aptamer CTApt-268, Aptamer14, Aptamer NAFLD01 , TfnR-aptamer, DEC205 aptamer (GGGAGGUGUGUUAGCACACGAUUCAUAAUCAGCUACCCUCCC (SEQ ID NO: 20)),, or MA33 aptamer (GTTACCGCGGTGAAGGGTGGATGTGTCTGGA (SEQ ID NO: 21)), A01 B aptamer (CAGGAGCCGAGAACCGGTTGGTGGGTAATCCTGTTAGCGC (SEQ ID NO: 22)), HG19 aptamer (GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCC (SEQ ID NO: 23)), C2.min aptamer (GGGGGAUCAAUCCAAGGGACCCGGAAACGCUCCCUUACACCCC (SEQ ID NO:
  • aptamer targeting moieties may also be biotinylated to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
  • a biotin-binding protein e.g., avidin, streptavidin, NeutrAvidin.
  • a linker provides space, rigidity, and/or flexibility between the moiety which forms a complex with the circular polyribonucleotide and the targeting moiety described herein.
  • the disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide, two or more linkers (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), and two or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeting moieties (e.g., X-(L-B) Z , wherein z is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).
  • the linkers are the same linker.
  • the linkers are different from one another.
  • some of the linkers are the same linker and some of the linkers are different from one another.
  • a linker may be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a C-0 bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
  • a covalent bond e.g., an amide bond, a disulfide bond, a C-0 bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
  • a linker includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1- 180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)).
  • a linker includes no more than 250 non-hydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1- 30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1- 130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 non-hydrogen atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non- hydrogen atom(s)
  • the backbone of a linker includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)).
  • the linker includes from 1 to 1000 atoms (e.g., from 1 to 750 atoms, 1 to 500 atoms, 1 to 250 atoms, 1 to 100 atoms, 1 to 75 atoms, 1 to 50 atoms, 1 to 25 atoms, and 1 to 10 atoms). In some embodiments, the linker includes from 1 to 100 atoms. In some embodiments, the linker includes from 1 to 50 atoms. In some embodiments, the linker includes from 1 to 25 atoms.
  • the linker is linear and includes from 1 to 1000 atoms (e.g., from 1 to 750 atoms, 1 to 500 atoms, 1 to 250 atoms, 1 to 100 atoms, 1 to 75 atoms, 1 to 50 atoms, 1 to 25 atoms, and 1 to 10 atoms). In some embodiments, the linker is linear and includes from 1 to 100 atoms. In some embodiments, the linker is linear and includes from 1 to 50 atoms. In some embodiments, the linker is linear and includes from 1 to 25 atoms.
  • the linker is branched, and each branch includes from 1 to 1000 atoms (e.g., from 1 to 750 atoms, 1 to 500 atoms, 1 to 250 atoms, 1 to 100 atoms, 1 to 75 atoms, 1 to 50 atoms, 1 to 25 atoms, and 1 to 10 atoms). In some embodiments, the linker is branched, and each branch includes from 1 to 100 atoms. In some embodiments, the linker is branched, and each branch includes from 1 to 50 atoms. In some embodiments, the linker is branched, and each branch includes from 1 to 25 atoms.
  • the “backbone” of a linker refers to the atoms in the linker that together form the shortest path from one part of the conjugate to another part of the conjugate.
  • the atoms in the backbone of the linker are directly involved in linking one part of the conjugate to another part of the conjugate.
  • hydrogen atoms attached to carbons in the backbone of the linker are not considered as directly involved in linking one part of the conjugate to another part of the conjugate.
  • a linker includes a heteroatom, such as a nitrogen, sulfur, oxygen, phosphorus, silicon, or boron atom.
  • the linker includes a cyclic group (e.g., an aryl, heteroaryl, cycloalkyl, or heterocyclyl group).
  • a linker includes a functional group such as an amide, ketone, ester, ether, thioester, thioether, thiol, hydroxyl, amine, cyano, nitro, azide, triazole, pyrroline, p-nitrophenyl, alkene, or alkyne group.
  • a linker includes an arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alky ny larylalky ny I, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylhexyl, alkylheteroarylalkenyl, alkylhexyl
  • a linker includes a polyethylene glycol group (e.g., PEG1 , PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG10, PEG12, PEGU, PEG16, PEG18, PEG20, PEG24, PEG28, PEG32, PEG100, PEG200, PEG250, PEG500, PEG600, PEG700, PEG750, PEG800, PEG900, PEG1000, PEG2000, PEG3000, PEG4000, PEG5000, PEG6000, PEG7000, PEG8000, PEG9000, and PEG10000).
  • the linker includes at least one PEG unit.
  • the linker may include PEG2 to PEG10000 (e.g., PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG10, PEG12, PEGU, PEG16, PEG18, PEG20, PEG24, PEG28, PEG32, PEG100, PEG200, PEG250, PEG500, PEG600, PEG700, PEG750, PEG800, PEG900, PEG1000, PEG2000, PEG3000, PEG4000, PEG5000, PEG6000, PEG7000, PEG8000, PEG9000, and PEG10000).
  • PEG2 to PEG10000 e.g., PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG10, PEG12, PEGU, PEG16, PEG18, PEG20, PEG24, PEG28, PEG32, PEG100, PEG200, PEG250, PEG500, PEG600, PEG700,
  • a linker includes a triethylene glycol group (e.g., TEG1 , TEG2, TEG3, TEG4, TEG5, TEG6, TEG7, TEG8, TEG10, TEG12, TEG14, TEG16, TEG18, TEG20, TEG24, TEG28, TEG32, TEG100, TEG200, TEG250, TEG500, TEG600, TEG700, TEG750, TEG800, TEG900, TEG1000, TEG2000, TEG3000, TEG4000, TEG5000, TEG6000, TEG7000, TEG8000, TEG9000, and TEG10000).
  • the linker includes at least one TEG unit.
  • the linker may include TEG2 to TEG10000 (e.g., TEG2, TEG3, TEG4, TEG5, TEG6, TEG7, TEG8, TEG10, TEG12, TEG14, TEG16, TEG18, TEG20, TEG24, TEG28, TEG32, TEG100, TEG200, TEG250, TEG500, TEG600, TEG700, TEG750, TEG800, TEG900, TEG1000, TEG2000, TEG3000, TEG4000, TEG5000, TEG6000, TEG7000, TEG8000, TEG9000, and TEG10000).
  • TEG2 to TEG10000 e.g., TEG2, TEG3, TEG4, TEG5, TEG6, TEG7, TEG8, TEG10, TEG12, TEG14, TEG16, TEG18, TEG20, TEG24, TEG28, TEG32, TEG100, TEG200, TEG250, TEG500, TEG600, TEG
  • the composition includes at least one linker that connects the targeting moiety to the moiety that binds or conjugates to the circular polyribonucleotide.
  • the moiety that binds or conjugates to the circular polyribonucleotide is connected to one or more targeting moieties, through a linker as described herein.
  • the linker may be monovalent or multivalent, e.g., bivalent, trivalent, tetravalent, or pentavalent.
  • the linker includes a structure selected from: wherein q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of ( ) ( ) ( ) ( ) (
  • the linker includes: wherein L 5A , L 5B and L 5C represent a targeting moiety, e.g., as described herein.
  • molecules that may be used to make linkers include at least two functional groups, e.g., two carboxylic acid groups.
  • two arms of a linker may contain two dicarboxylic acids.
  • dicarboxylic acid molecules may be used as linkers (e.g., a dicarboxylic acid linker).
  • n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • dicarboxylic acids molecules that may be used to form linkers include, but are not limited to,
  • dicarboxylic acid molecules such as the ones described herein, may be further functionalized to contain one or more additional functional groups.
  • Dicarboxylic acids may be further functionalized, for example, to provide an attachment point to one or more targeting moieties and a moiety which forms a complex with the circular polyribonucleotide.
  • the linking group when the targeting moiety is attached to a moiety which forms a complex with the targeting circular polyribonucleotide, may include a moiety including a carboxylic acid moiety and an amino moiety that are spaced by from 1 to 25 atoms.
  • Examples of such linking groups include, but are not limited to, wherein n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • a linking group may include a moiety including a carboxylic acid moiety and an amino moiety, such as the ones described herein, may be further functionalized to contain one or more additional functional groups. Such linking groups may be further functionalized, for example, to provide an attachment point to a targeting moiety.
  • the linking group may include a moiety including two or amino moieties (e.g., a diamino moiety) that are spaced by from 1 to 25 atoms.
  • Examples of such linking groups include, but are not limited to, wherein n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • a linking group may include a diamino moiety, such as the ones described herein, may be further functionalized to contain one or more additional functional groups. Such diamino linking groups may be further functionalized, for example, to provide an attachment point to a targeting moiety.
  • a molecule containing an azide group may be used to form a linker, in which the azide group may undergo cycloaddition with an alkyne to form a 1 ,2,3-triazole linkage.
  • a molecule containing an alkyne group may be used to form a linker, in which the alkyne group may undergo cycloaddition with an azide to form a 1 ,2,3-triazole linkage.
  • a molecule containing a maleimide group may be used to form a linker, in which the maleimide group may react with a cysteine to form a C-S linkage.
  • a molecule containing one or more haloalkyl groups may be used to form a linker, in which the haloalky I group may form a covalent linkage, e.g., C-N and C-0 linkages, with a targeting moiety.
  • the linker may be an oligonucleotide, which includes a string of nucleic acids.
  • the targeting moiety described herein include a string of nucleic acids, which is in turn linked a moiety which complexes with the circular polyribonucleotide.
  • the linker can have any sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence specifically chosen for its molecular or biochemical properties.
  • the linker includes 20 one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof.
  • the linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof.
  • the string of nucleic acids includes from 1 to 50 nucleic acid residues (e.g., from 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, 5 to 50, 10 to 50, 20 to 50, 30 to 50, or 40 to 50 residues).
  • the string of nucleic acids includes from 5 to 30 nucleic acid residues (e.g., from 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 30, 15 to 30, 20 to 30, or 25 to 30 residues).
  • the linker includes one or more guanines, for example from 1-10 guanines (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 guanine residues).
  • the linker is a polypeptide.
  • the linker may include at least one amino acid.
  • the polypeptide linker may include from two to 100 amino acid residues (e.g., from 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 amino acid residues).
  • the linker is a single amino acid (e.g., any naturally occurring amino acid such as Cys).
  • the linker includes a series of glycine residue to form a polyglycine linker.
  • the linker includes an amino acid sequence of (G ly) n, wherein n may be from 2 to 20 residues (e.g., from 2 to 15, 2 to 10, 2 to 5, 5 to 20, 10 to 20, or 15 to 20 glycine residues).
  • polyglycine linkers include but are not limited to GGG, GGGA (SEQ ID NO: 27), GGGG (SEQ ID NO: 28), GGGAG (SEQ ID NO: 29), GGGAGG (SEQ ID NO: 30), GGGAGGG (SEQ ID NO: 31), GGAG (SEQ ID NO: 32),GGSG (SEQ ID NO: 33), AGGG (SEQ ID NO: 34), SGGG (SEQ ID NO: 35), GGAGGA (SEQ ID NO: 36), GGSGGS (SEQ ID NO: 37), GGAGGAGGA (SEQ ID NO: 38), GGSGGSGGS (SEQ ID NO: 39), GGAGGAGGAGGA (SEQ ID NO: 40), GGSGGSGGSGGS (SEQ ID NO: 41), GGAGGGAG (SEQ ID NO: 42), GGSGGGSG (SEQ ID NO: 43), GGAGGGAGGGAG (SEQ ID NO: 44), GGSGGGSGGGSG (SEQ ID NO: 45), GGGGA
  • the polypeptide linker is a glycine-rich polypeptide such as a polypeptide having the sequence [Gly-Gly- Gly-Gly-Ser] n (SEQ ID NO: 49) where n is 1 , 2, 3, 4, 5 or 6 is used.
  • the polypeptide linker is a serine-rich polypeptide linker.
  • Serine rich peptide linkers include those of the formula [X-X-X-X-Gly] y (SEQ ID NO: 50), where up to two of the X are Thr, and the remaining X are Ser, and y is 1 to 5 (e.g., Ser-Ser-Ser-Ser-Gly (SEQ ID NO: 51), where y is greater than 1).
  • the linker is a single amino acid (e.g., any amino acid, such as Gly or Cys).
  • Amino acid linkers may be selected for flexibility (e.g., flexible or rigid) or may be selected on the basis of charge (e.g., positive, negative, or neutral).
  • Flexible linkers typically include those with Gly resides (e.g., [Gly-Gly-Gly-Gly-Ser] n where n is 1 , 2, 3, 4, 5 or 6).
  • Other linkers include rigid linkers (e.g., PAPAP (SEQ ID NO: 52) and (PT) n P (SEQ ID NO: 53), where n is 2, 3, 4, 5, 6, or 7) and a-helical linkers (e.g., A(EAAAK) n A (SEQ ID NO: 54), where n is 1 , 2, 3, 4, or 5).
  • linkers are succinyl, Lys, Glu, and Asp, or a dipeptide such as Gly-Lys.
  • the linker is succinyl
  • one carboxyl group thereof may form an amide bond with an amino group of the amino acid residue
  • the other carboxyl group thereof may, for example, form an ester bond with a hydroxyl group of opioid moiety.
  • the linker is Lys, Glu, or Asp
  • the carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the amino group thereof may, for example, form an amide bond with a carboxyl group of the opioid moiety.
  • a further linker may be inserted between the ⁇ -amino group of Lys and the opioid moiety.
  • the further linker is succinyl acid, which can form an amide bond with the ⁇ - amino group of Lys and an ester bond with a hydroxyl group present in the opioid moiety.
  • the further linker is Glu or Asp (e.g., which forms an amide bond with the ⁇ -amino group of Lys and an ester or amide bond with a hydroxyl or amino group present in the opioid moiety).
  • the peptide linker is a branched polypeptide.
  • the linker is an enzymatically cleavable linker.
  • a cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together.
  • the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
  • a first reference condition which can, e.g., be selected to mimic or represent intracellular conditions
  • a second reference condition which can, e.g., be selected to mimic or represent conditions found in the blood or serum.
  • Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
  • redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g.,
  • a cleavable linkage group such as a disulfide bond can be susceptible to pH.
  • the pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 -7.3.
  • Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0.
  • Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
  • a linker can include a cleavable linking group that is cleavable by a particular enzyme.
  • the type of cleavable linking group incorporated into a linker can depend on the cell to be targeted.
  • a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group.
  • Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich.
  • Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.
  • Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
  • the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • a degradative agent or condition
  • the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • the evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals.
  • useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
  • a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation.
  • An example of reductively cleavable linking group is a disulphide linking group (-S-S-).
  • a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular RNA moiety and particular targeting agent one can look to methods described herein.
  • a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell.
  • the candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions.
  • candidate compounds are cleaved by at most about 10% in the blood.
  • useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).
  • the rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
  • a cleavable linker in another embodiment, includes a phosphate-based cleavable linking group.
  • a phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group.
  • An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells.
  • phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)- O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S- P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(O)(Rk)-S-, -O- P(
  • Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S- P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O- P(S)(H)-O-, -S-P(O)(H)-O, -S-P(O)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-, -O-P(S)(H)-S-.
  • a preferred embodiment is -O- P(O)(OH)-O-.
  • a cleavable linker in another embodiment, includes an acid cleavable linking group.
  • An acid cleavable linking group is a linking group that is cleaved under acidic conditions.
  • acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid.
  • specific low pH organelles such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups.
  • Acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids.
  • a preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl.
  • a cleavable linker in another embodiment, includes an ester-based cleavable linking group.
  • An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells.
  • Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups.
  • Ester cleavable linking groups have the general formula -C(O)O-, or - OC(O)-. These candidates can be evaluated using methods analogous to those described above.
  • a cleavable linker includes a peptide-based cleavable linking group.
  • a peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells.
  • Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.
  • Peptide-based cleavable groups do not include the amide group (-C(O)NH-).
  • the amide group can be formed between any alkylene, alkenylene or alkynelene.
  • a peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins.
  • the peptide-based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group.
  • Peptide-based cleavable linking groups have the general formula - NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
  • the linker further includes a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
  • the biotin-binding protein may be avidin.
  • the avidin binds a biotinylated targeting moiety, as shown for example in FIG. 10.
  • the avidin binds 1 , 2, 3, 4, or more biotinylated targeting moieties.
  • the biotinylated targeting moiety includes a lipid, small molecule, carbohydrate, polypeptide, and/or aptamer.
  • two or more of the biotinylated targeting moieties are the same targeting moiety.
  • the disclosure provides a complex that includes a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide, a photoreactive crosslinking agent, a linker, and targeting moiety.
  • the photoreactive crosslinking agent provides a covalent attachment between the circular polyribonucleotide and the moiety that attaches to a region of the circular polyribonucleotide upon irradiation with light, e.g., at a first wavelength.
  • the photoreactive crosslinking agent is reversibly attached.
  • the covalent attachment may be removed upon irradiation with light, e.g., at a second wavelength that is different from the first wavelength.
  • the photoreactive crosslinking agent is attached to the end of the moiety.
  • the photoreactive crosslinking agent is located at an internal position within the moiety.
  • the disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide and includes one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) photoreactive crosslinking agents, a linker, and a targeting moiety.
  • the photoreactive crosslinking agents are the same photoreactive crosslinking agent.
  • the photoreactive crosslinking agents are different from one another.
  • each of the photoreactive crosslinking agents is located at an internal position within the moiety.
  • at least one of the photoreactive crosslinking agents is attached to an end of the moiety.
  • at least one of the photoreactive crosslinking agents is attached to a second end of the moiety.
  • the photoreactive crosslinking agent may be a photoreactive nucleotide analog or a photoreactive amino acid analog.
  • the moiety that specifically binds a region of the circular polyribonucleotide and includes one or more photoreactive crosslinking agents is an oligonucleotide. Oligonucleotide binding moieties are described in greater detail in a later section of the Detailed Description. In some embodiments, the moiety that specifically binds a region of the circular polyribonucleotide and includes one or more photoreactive crosslinking agent is an oligonucleotide, wherein each of the one or more photoreactive crosslinking agents is a photoreactive nucleotide analog.
  • each of the one or more photoreactive nucleotide analogs replaces a single nucleotide within the oligonucleotide.
  • Each of the one or more photoreactive nucleotide analogs may include a nucleotide or nucleoside modified to contain a photoreactive group.
  • the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the opposing ribonucleotide within the circular polyribonucleotide upon photoirradiation.
  • the disclosure provides photoreactive nucleotide analogs included within the oligonucleotide.
  • Photoreactive nucleotide analogs include, for example, 4-thiouridene (4sU), 5-bromo-2’-deoxyuridine (BrdU), coumarin derivatives, D-threoninol ( CNV D) derivatives, 3- cyanovinylcarbazole nucleoside ( CNV K) derivatives, diazirene derivatives, phenylselenide derivatives, psoralen derivatives, or pyranovinylcarbazole ( PC X) derivatives.
  • the photoreactive crosslinking agent includes 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a pheylselenide, a furan, or an abasic site.
  • NrdU 5-bromo-2’-deoxyuridine
  • carbazole a carbazole
  • a psoralen a coumarin
  • 4’-thiouridine a diazirine
  • a pheylselenide a furan, or an abasic site.
  • the carbazole is 3-cyanovinylcarbazole, 4-methylpyranocarbazole, or pyranocarbazole.
  • the coumarin is 7-hydroxycoumarin.
  • the photoreactive nucleotide analog is one disclosed in U.S. Pat. No. 8,481 ,714, the disclosure of which is hereby incorporated by reference in its entirety.
  • the photoreactive crosslinking agent includes a compound of Formula (I): wherein, in the formula (I), R a represents a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen, wherein, in the formula (I), Ri and R2 each independently represent a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen, and wherein, in the formula (I), Rb represents a sugar, a polysaccharide, a polyether, a polyol, a polypeptide chain, or a water-soluble synthetic polymer.
  • R a represents a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen
  • Ri and R2 each independently represent a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group
  • the photoreactive crosslinking agent is the compound according to Formula (I), wherein R& is represented by any one of Formulae (II) - (V):
  • the photoreactive crosslinking agent is the compound of any one of
  • n is represented as any number 2 to 5 (e.g., 2, 3, 4, or 5).
  • the photoreactive crosslinking agent includes a compound of Formula wherein, in Formula (XII), R 1 represents a hydrogen or methyl group, wherein, in Formula (XII), R2 represents a hydrogen or methoxy group, wherein in Formula (XII), R3 represents a hydrogen, a methyl group, or a methoxy group, wherein in Formula (XII), R4 represents an aminomethyl group, an azidomethyl group, or a hydrogen group, wherein in Formula (XII), R5 represents a hydrogen or methyl group.
  • the photoreactive crosslinking agent is the compound of any one of Formulae (XIII) - (XVII):
  • the photoreactive nucleotide analog may be 4sU.
  • the structure of 4sU mimics the structure of uridine analogs but gains photoreactive properties by replacing the carbonyl oxygen at position 4 of the ring with a sulfur atom.
  • photoreactive 4sU within an oligonucleotide covalently binds to a nearby cytosine nucleobase through a [2+2] cycloaddition reaction.
  • the photoreactive nucleotide analog may be BrdU.
  • BrdU Upon irradiation with light having wavelength of 308 nm, BrdU generates 2’-deoxyuridin-5-yl radicals.
  • the photoreactive nucleotide analog may be a coumarin, a coumarin analog, or a derivative thereof.
  • Coumarin and derivatives thereof located within the oligonucleotide covalently bind to a nearby thymine nucleobase through a [2+2] cycloaddition reaction upon irradiation with light having a wavelength of 250 nm, resulting in the formation of a syn-cycloaddition photo-adduct. Irradiating the syn-cycloaddition photo-adduct with light having a wavelength of 254 nm reverses the reaction.
  • coumarin derivative that is a photoreactive nucleotide analog is 7- hydroxycoumarin (see, e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 , which is hereby incorporated by reference in its entirety).
  • the photoreactive nucleotide analog may be CNV K or derivatives thereof.
  • CNV K and derivatives thereof within an oligonucleotide have a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby oligonucleotide.
  • CNV K or a derivative thereof covalently binds with a nearby pyrimidine-containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct.
  • Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents the photo-induced DNA damage at shorter wavelengths.
  • n- CNV K can form a covalent bond with a nearby pyrimidine nucleobase at locations other than the 1 base downstream position of an opposing nucleotide depending on the linker length (see, e.g., Fujimoto et al. Photochem. Photobiol. Sci. 19, 776-782, 2020, which is hereby incorporated by reference in its entirety).
  • the photoreactive nucleotide analog may be CNV D.
  • CNV D and derivatives thereof within an oligonucleotide have a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby oligonucleotide.
  • CNV D is a derivative of CNV K that has the same chemical structure of CNV K except for a D- threoninol linker in the place of the deoxyribose sugar which acts to increase the flexibility as compared to C NV K, increasing the photo-induced crosslinking reaction rate due to minimization of entropic losses during hybridization (see, e.g., Sakamoto et al. Org. Lett. 17, 936-939, 2015, which is hereby incorporated by reference in its entirety).
  • CNV D or a derivative thereof covalently binds with a nearby pyrimidine-containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct.
  • Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents photo-induced DNA damage at shorter wavelengths.
  • the photoreactive nucleotide analog may be a diazirene derivative.
  • a diazirene derivative Upon irradiation with light having a wavelength of 365 nm, a diazirene derivative becomes a reactive carbene intermediate.
  • the reactive carbene intermediate can react with all nucleobases, including adenine, cytosine, guanine, thymine, and uracil, forming a covalent attachment with a nearby nucleobase.
  • diazirene derivative is 3-phenyl-3-trifluoromethyl- which is attached to the sugar ring through an acetal linkage (see, e.g., Nakamoto et al. J. Org. Chem. 79, 2463-2472, 2014, which is hereby incorporated by reference in its entirety).
  • the photoreactive nucleotide analog may be a phenyl selenide analog.
  • phenyl selenide Upon irradiation with light having a wavelength of 350 nm, phenyl selenide becomes a radical intermediate which has the capability of alkylating the N1 or N6 position of nearby purine nucleobases, including adenine.
  • An example of a phenyl selenide analog is phenyl selenide-modified 2’-deoxythymidine (see, e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 , which is hereby incorporated by reference in its entirety).
  • the photoreactive nucleotide analog may be a psoralen analog or a derivative thereof.
  • Psoralen and derivatives thereof are tricyclic compounds that may intercalate in any AT or AU region of hybridized DNA and RNA sequences, respectively.
  • Psoralen and derivatives thereof react through their furan or pyrone photoreactive site to covalently bind the C5-C6 double bond of pyrimidine residues. This forms a covalent attachment between two nearby pyrimidine nucleobases through a [2+2] cycloaddition reaction upon irradiation with light having a wavelength of 365 nm, resulting in the formation of a cyclobutene photo-adduct.
  • the resulting cyclobutene photo-adduct may be in the syn- or cis- configuration. Pyrimidine residues are present among cytosine, thymine, and uracil bases. Irradiating the cyclobutene photo-adduct with light having a wavelength of 254 nm reverses the reaction.
  • An example of a psoralen derivative is 4’-aminomethyltrioxsalen (see, e.g., Velema et al. JACS Au 3, 316-332, 2023, which is hereby incorporated by reference in its entirety).
  • the photoreactive nucleotide analog may be pyranocarbazole and derivatives thereof, such as pyranocarbozole nucleoside ( PC X).
  • the photoreactive pyranocarbozole and derivatives thereof within an oligonucleotide include a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby oligonucleotide.
  • PC X covalently binds with a nearby, pyrimidine-containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct.
  • Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents the photo-induced DNA damage at shorter wavelengths.
  • PC X with a D-threoninol linker in the place of the deoxyribose sugar ( PCX D) which acts to increase the flexibility as compared to PC X, increasing the photo-induced crosslinking reaction rate due to minimization of entropic losses during hybridization (see, e.g., Fujimoto et al. RSC Adv. 9, 30693-30697, 2019, which is hereby incorporated by reference in its entirety).
  • Another example of a pyranocarbozole derivative is pc X that includes a methyl group at the 4-position of PC X.
  • MEP K 4-methylpyranocarbozole nucleoside
  • the oligonucleotide may include another photoreactive nucleotide analog which allows for the oligonucleotide binding moiety to covalently attach to the polyribonucleotide described herein.
  • another photoreactive nucleotide analog which allows for the oligonucleotide binding moiety to covalently attach to the polyribonucleotide described herein.
  • photoreactive nucleotide analogs see, e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 ; Tavakoli et al., RSC Adv. 12, 6484-6507, 2022; and Velema et al. JACS Au 3, 316-332, 2023.
  • the moiety that specifically binds a region of the circular polyribonucleotide is a polypeptide.
  • the moiety includes one or more photoreactive amino acid analogs.
  • the photoreactive amino acid analog replaces an amino acid within the polypeptide.
  • the one or more photoreactive amino acid analogs may include an amino acid modified to contain a photoreactive group.
  • Photoreactive nucleotide analogs include, for example, aryl azide-based, benzophenone-based, or diazirene-based unnatural amino acids or y I) et h oxy] carbonyl-lysine.
  • the photoreactive amino acid analog may be an aryl azide-based unnatural amino acid.
  • aryl azide-based unnatural amino acids Upon irradiation with light having a wavelength of 250-350 nm, aryl azide-based unnatural amino acids generate a reactive nitrene intermediate.
  • the reactive nitrene intermediate can covalently bond with a nearby nucleobase through C-H and X-H insertions.
  • An example of an arylazide- based unnatural amino acid is tetrafluorophenylazide (see, e.g., Smith et al Future Med. Chem. 7, 159- 183, 2015, which is hereby incorporated by reference in its entirety).
  • the photoreactive amino acid analog may be a benzophenone-based unnatural amino acid.
  • Benzophenone-based unnatural amino acids can be easily prepared and are further beneficial due to their inertness to solvents.
  • benzophenone-based unnatural amino acids Upon irradiation with light having a wavelength of 350- 365 nm, benzophenone-based unnatural amino acids generate a reactive diradical intermediate, containing reactive triplet carbonyl states.
  • the reactive diradical intermediate can covalently bind to a nearby nucleobase through C-H insertion.
  • Benzophenone-based unnatural amino acids are activated with light of a longer wavelength, reducing the risk of damage to biomolecules; however, irradiation times are longer, risking the potential for non-specific interactions.
  • the photoreactive amino acid analog may be a diazirene-based unnatural amino acid.
  • diazirine-based unnatural amino acids Upon irradiation with light having a wavelength of 350-380 nm, diazirine-based unnatural amino acids generate a reactive carbene intermediate.
  • the reactive carbene intermediate can react with all nucleobases, including adenine, cytosine, guanine, thymine, and uracil, forming a covalent attachment with a nearby nucleobase.
  • alkyl diazirene-based amino acid is a pyrrolysine analog that bears an alkyl diazirene reactive group attached by a short, flexible C2 linker (see, e.g., Dziuba et al. ChemBioChem 2020, 21, 88-93, which is hereby incorporated by reference in its entirety).
  • the photoreactive amino acid analog may be yl)ethoxy]carbonyl-lysine )ethoxy]carbonyl-lysine is genetically encodable and can be irradiated with red light to covalently bind the amino acid to a nearby nucleobase (see, e.g., Moritz et al. Angew. Chem. 2013, 52, 4690-4693, , which is hereby incorporated by reference in its entirety).
  • the disclosure also provides circular polyribonucleotide that are complexed with (e.g., hybridized or bound to) one or more moieties that specifically bind a region of the circular polyribonucleotide, wherein each of the one or more moieties that specifically bind a region of the circular polyribonucleotide is conjugated to (e.g., directly, chemically-covalently conjugated) to one or more targeting moieties.
  • the moieties that specifically bind a region of the circular polyribonucleotide are the same moiety that specifically binds a region of the circular polyribonucleotide.
  • the moieties that specifically bind a region of the circular polyribonucleotide are different from one another.
  • the moieties that specifically bind to the region of the circular polyribonucleotide may be one or more polypeptide binding moieties and one or more oligonucleotide binding moieties.
  • the moieties that specifically bind to the region of the circular polyribonucleotide may be two or more polypeptide binding moieties that are different polypeptides from one another.
  • the moieties that specifically bind to the region of the circular polyribonucleotide may be one or more oligonucleotide binding moieties that have different sequences from one another.
  • the disclosure also provides circular polyribonucleotide complexed with (e.g., hybridized or hybridized and covalently attached to) a moiety that specifically binds a region of the circular polyribonucleotide.
  • the moiety that specifically binds a region of the circular polyribonucleotide includes one or more photoreactive crosslinking agents.
  • the binding moiety may be, for example, a polypeptide or an oligonucleotide.
  • the complex includes from 1 to 20 moieties (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, to 20) that each bind specifically to a region of the circular polyribonucleotide.
  • the moieties that specifically bind a region of the circular polyribonucleotide are the same moiety that specifically binds a region of the circular polyribonucleotide. In some embodiments, the moieties that specifically bind a region of the circular polyribonucleotide are different from one another.
  • the moieties that specifically bind to the region of the circular polyribonucleotide may be one or more polypeptide binding moieties and/or one or more oligonucleotide binding moieties.
  • the moieties that specifically bind to the region of the circular polyribonucleotide may be two or more polypeptide binding moieties that are different polypeptides from one another.
  • the moieties that specifically bind to the region of the circular polyribonucleotide may be one or more oligonucleotide binding moieties that have different sequences from one another.
  • Each moiety that specifically binds a region of the circular polyribonucleotide may also include a linker covalently connecting the moieties that specifically bind a region of the circular polyribonucleotide and the targeting moiety.
  • the moiety that specifically binds a region of the circular polyribonucleotide may be, for example an oligonucleotide or a polypeptide.
  • the moiety binds a region of the circular polyribonucleotide that includes one or more of an IRES, a spacer sequence, or an expression sequence. In some embodiments, the moiety binds to one of the binding regions as shown in FIG. 2. In some embodiments, the moiety binds a region in an IRES. In some embodiments, the moiety does not bind a region in an IRES. In some embodiments, the moiety binds a region in a spacer sequence. In some embodiments, the moiety does not bind a region in a spacer sequence. In some embodiments, the moiety binds at least part of a spacer sequence.
  • the moiety binds at least part of two spacer sequences. In some embodiments, the moiety binds a region in a coding region. In some embodiments, the moiety does not bind a region in a coding region. In some embodiments, the moiety binds a region in a spacer sequence and/or a coding region but does not bind a region in an IRES. In some embodiments, the binding region is sufficiently distant in sequence and space from the expression sequence so that it does not interfere with or minimizes interference with translation efficiency.
  • the binding region may be from about 10 nucleotides to about 200 nucleotides (e.g., from about 10 nucleotides to 170 nucleotides, 10 nucleotides to 150 nucleotides, 10 nucleotides to 130 nucleotides, 10 nucleotides to 100 nucleotides, 10 nucleotides to 70 nucleotides, 10 nucleotides to 50 nucleotides, 10 nucleotides to 30 nucleotides, 30 nucleotides to 200 nucleotides, 50 nucleotides to 200 nucleotides, 70 nucleotides to 200 nucleotides, 100 nucleotides to 200 nucleotides, 130 nucleotides to 200 nucleotides, 150 nucleotides to 200 nucleotides, 170 nucleotides to 200 nucleotides, or 50 nucleotides to 100 nucleotides) from the 5’ or 3’ end of the
  • the moiety that specifically binds a region of the circular polyribonucleotide an oligonucleotide.
  • the oligonucleotide may be, for example, a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.
  • each oligonucleotide is from about 10-500 nucleic acids in length (e.g., about 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 20-50, 20-100, 20-200 20-300, 20-400, 30-50, 30-100, 30-200, 30-300, 30-400, 50-100, 50-200, 50-300, or 50- 400 ribonucleotides in length).
  • each oligonucleotide has at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%) complementarity to a portion of the sequence of the circular polyribonucleotide.
  • each oligonucleotide has from 20% to 90% (e.g., from 20-90%, 20-90%, 20-90% 20-90%, 30%-90%, 40%-90%, or 50%-90% complementarity to a portion of the sequence of the circular polyribonucleotide.
  • Each oligonucleotide may be DNA, RNA, a synthetic nucleic acid, or a hybrid.
  • Each oligonucleotide may include one or more nucleic acid modifications as described herein.
  • the circular polyribonucleotide is hybridized to one, two, three, four, five, six, seven, eight, nine, ten or more oligonucleotides, each oligonucleotide being covalently conjugated to one or more targeting moieties. In some embodiments, the circular polyribonucleotide is hybridized to one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more oligonucleotides. In some embodiments, the circular polyribonucleotide is hybridized to 10, 20, 30, 40, or 50 or more oligonucleotides.
  • the circular polyribonucleotide is hybridized to 1-10, 2-10, 2-20, 2-50, 5-10, 5-20, 5-50, 10-20, 20-30, 30- 40, 40-50, 1-50, 20-50, 30-60 or 50-100 targeting moieties.
  • each oligonucleotide is hybridized to (e.g., shares at least partial complementarity sufficient for binding with) a sequence with a binding region of the circular polyribonucleotide.
  • the circular polyribonucleotide includes either a coding region (e.g., including an expression sequence)
  • the binding region is sufficiently distant in sequence and space from the coding region or target binding region so that it does not interfere with or minimizes interference with translation efficiency.
  • the binding region includes from 1 to 100 ribonucleotides (e.g., from 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 ribonucleotides).
  • the binding region may include from 10 to 90 ribonucleotides (e.g., from 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 80, 30 to 80, 40 to 80, 50 to 80, 60 to 80, or 70 to 80 ribonucleotides).
  • the oligonucleotide has from 50% to 100% (e.g., from 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%) complementarity to a portion of the binding region of the circular polyribonucleotide.
  • the oligonucleotide may have from 80% to 100% (e.g., from 85% to 100%, 90% to 100%, 95% to 100%, 80% to 95%, 80% to 90%, or 80% to 85%) and complementarity to a portion of the binding region of the circular polyribonucleotide.
  • the moiety that specifically binds a region of the circular polyribonucleotide and includes a photoreactive crosslinking agent is an oligonucleotide.
  • the oligonucleotide may be, for example, a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.
  • each oligonucleotide includes a photoreactive crosslinking agent.
  • the photoreactive crosslinking agent is attached to the 5’ end of the oligonucleotide.
  • the photoreactive crosslinking agent is attached to the 3’ end of the oligonucleotide.
  • the photoreactive crosslinking agent is a photoreactive nucleotide analog.
  • the photoreactive nucleotide analog replaces a single nucleotide within the oligonucleotide.
  • the photoreactive nucleotide analog is located at an internal position within the oligonucleotide.
  • the 3’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog.
  • the 3’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the photoreactive nucleotide analog.
  • the 5’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog.
  • the 5’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the photoreactive nucleotide analog.
  • nucleotides e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides
  • each oligonucleotide includes one, two, three, four, five, six, seven, eight, nine, ten or more photoreactive crosslinking agents.
  • each photoreactive crosslinking agent is a photoreactive nucleotide analog.
  • each of the photoreactive nucleotide analogs replaces a single nucleotide within the oligonucleotide.
  • each photoreactive nucleotide analog is located at an internal position within the oligonucleotide.
  • the 3’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 3’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the nearest photoreactive nucleotide analog.
  • 1 to 50 e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the
  • the 5’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the nearest photoreactive nucleotide analog.
  • 1 to 50 e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the
  • the photoreactive crosslinking agents is attached to the 5’ end of the oligonucleotide. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the 3’ end of the oligonucleotide. In some embodiments, the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) between each of the photoreactive nucleotide analogs.
  • 1 to 50 e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) between each of
  • the oligonucleotide may have one photoreactive nucleotide analog attached to the 5’ end and a second photoreactive nucleotide analog 8 nucleotides away from the 5’ end.
  • the oligonucleotide may have one photoreactive nucleotide analog attached to the 5’ end, a second photoreactive nucleotide analog 10 nucleotides away from the 5’ end, and a third photoreactive nucleotide analog 15 nucleotides away from the 3’ end and 8 nucleotides away from the second photoreactive nucleotide analog.
  • each oligonucleotide is from about 5-500 nucleotides in length (e.g., about 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 5-200, 5-300, 5-400, 20-50, 20-100, 20-20020-300, 20-400, 30-50, 30-100, 30-200, 30-300, 30-400, 50-100, 50-200, 50-300, or 50-400 nucleotides in length).
  • each oligonucleotide has at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%) complementarity to a portion of the sequence of the circular polyribonucleotide.
  • each oligonucleotide has from 20% to 90% (e.g., from 20-90%, 20-90%, 20-90% 20-90%, 30%-90%, 40%-90%, or 50%-90%) complementarity to a portion of the sequence of the circular polyribonucleotide.
  • Each oligonucleotide may be DNA, RNA, a synthetic nucleic acid, or a hybrid.
  • Each oligonucleotide may include one or more nucleic acid modifications as described herein.
  • the oligonucleotide includes an aptamer. In some embodiments, the aptamer specifically binds to the linker or targeting moiety.
  • the circular polyribonucleotide is annealed and covalently bound to one, two, three, four, five, six, seven, eight, nine, ten or more oligonucleotides, each oligonucleotide being covalently conjugated to one or more targeting moieties. In some embodiments, the circular polyribonucleotide is annealed and covalently bound to one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more oligonucleotides.
  • the circular polyribonucleotide is annealed and covalently bound to 10, 20, 30, 40, or 50 or more oligonucleotides. In some embodiments, the circular polyribonucleotide is annealed and covalently bound to 1-10, 2-10, 2-20, 2-50, 5-10, 5-20, 5-50, 10-20, 20-30, 30-40, 40-50, 1- 50, 20-50, 30-60 or 50-100 targeting moieties.
  • each oligonucleotide is annealed (e.g., shares at least partial complementarity sufficient for binding with) and covalently bound to (e.g., photo-crosslinked) a sequence with a binding region of the circular polyribonucleotide.
  • the circular polyribonucleotide includes either a coding region (e.g., including an expression sequence)
  • the binding region is sufficiently distant in sequence and space from the coding region or target binding region so that it does not interfere with or minimizes interference with translation efficiency.
  • the binding region includes from 1 to 100 ribonucleotides (e.g., from 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 ribonucleotides).
  • the binding region may include from 10 to 90 ribonucleotides (e.g., from 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 80, 30 to 80, 40 to 80, 50 to 80, 60 to 80, or 70 to 80 ribonucleotides).
  • the oligonucleotide has from 50% to 100% (e.g., from 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%) complementarity to a portion of the binding region of the circular polyribonucleotide.
  • the oligonucleotide may have from 80% to 100% (e.g., from 85% to 100%, 90% to 100%, 95% to 100%, 80% to 95%, 80% to 90%, or 80% to 85%) and complementarity to a portion of the binding region of the circular polyribonucleotide.
  • the binding region has zero or one mismatch with the oligonucleotide.
  • the moiety that specifically binds a region of the circular polyribonucleotide and a targeting moiety by way of a linker is polypeptide.
  • the circular polyribonucleotide is bound to at least one polypeptide, wherein each polypeptide is complexed to at least one targeting moiety.
  • the circular polyribonucleotide is bound to from 1 to 50 polypeptides (e.g., from 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 50, 10 to 50, 20 to 50, 30 to 50, or 40 to 50 polypeptides), wherein each polypeptide is complexed to at least one targeting moiety.
  • the polypeptide may include an RNA binding protein domain.
  • the RNA binding protein domain may a be an RNA recognition motif (RRM), K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521-B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.
  • RRM RNA recognition motif
  • the circular polyribonucleotide is bound to at least one polypeptide, wherein each polypeptide is complexed to at least one targeting moiety.
  • the moiety that specifically binds a region of the circular polyribonucleotide and includes one or more photoreactive crosslinking agents is a polypeptide.
  • the circular polyribonucleotide is bound to at least one polypeptide, wherein each polypeptide is complexed to at least one targeting moiety.
  • the circular polyribonucleotide is bound to from 1 to 50 polypeptides (e.g., from 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 50, 10 to 50, 20 to 50, 30 to 50, or 40 to 50 polypeptides), wherein each polypeptide is complexed to at least one targeting moiety.
  • each polypeptide includes a photoreactive crosslinking agent.
  • the photoreactive crosslinking agent is attached to the N-terminus of the polypeptide.
  • the photoreactive crosslinking agent is attached to the C-terminus of the polypeptide.
  • the photoreactive crosslinking agent is a photoreactive amino acid analog.
  • the photoreactive amino acid analog replaces a single amino acid within the polypeptide.
  • the photoreactive amino acid analog is located at an internal position within the polypeptide.
  • the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the photoreactive amino acid analog.
  • 1 to 50 e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the photoreactive amino acid analog.
  • the N-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the photoreactive amino acid analog.
  • 1 to 50 e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the photoreactive amino acid analog.
  • each polypeptide includes one, two, three, four, five, six, seven, eight, nine, ten or more photoreactive crosslinking agents.
  • each photoreactive crosslinking agent is a photoreactive amino acid analog.
  • each of the photoreactive amino acid analogs replaces a single amino acid within the polypeptide.
  • each photoreactive amino acid analog is located at an internal position within the polypeptide.
  • the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog.
  • the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the nearest photoreactive amino acid analog.
  • the N-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog.
  • the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the nearest photoreactive amino acid analog.
  • at least one of the photoreactive crosslinking agents is attached to the N-terminus of the polypeptide.
  • at least one of the photoreactive crosslinking agents is attached to the C-terminus of the polypeptide.
  • the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) between each of the photoreactive amino acid analogs.
  • a polypeptide may have one photoreactive amino acid analog attached to the N-terminus and a second photoreactive amino acid analog 8 amino acids away from the N-terminus.
  • the polypeptide may have one photoreactive amino acid analog attached to the N-terminus, a second photoreactive amino acid analog 10 amino acids away from the N-terminus, and a third photoreactive amino acid analog 15 amino acids away from the C- terminus and 8 amino acids away from the second photoreactive amino acid analog.
  • the RNA binding protein domain may be an RNA recognition motif.
  • RRMs are the most common and well-studied RNA-binding domain.
  • the Protein Data Bank includes over 500 known structures which have an RRM.
  • RRMs average 90 amino acids in size and adopt a ⁇ 1 ⁇ 1 ⁇ 2 ⁇ 3 ⁇ 2 ⁇ 4 topology forming two ⁇ helices against an antiparallel ⁇ sheet, which houses the conserved RNA-binding ribonucleoprotein 1 and ribonucleoprotein 2 motifs in the central ⁇ 1 and ⁇ 3 strands.
  • RRMs interact with from 2 to 8 nucleotides (e.g., 2, 3, 4, 5, 6, 7, and 8 nucleotides) in single-stranded RNA commonly. This interaction may occur through several sequential stacking interactions and hydrogen bonds with ribonucleoprotein motifs, often with nanomolar affinities.
  • each RRM may have its own sequence preferences, often for degenerate sequences such as GU-rich tracts.
  • the RBP includes multiple ribonucleoprotein motifs. The combination of consecutive ribonucleoprotein motifs in an RBP dramatically increases binding affinity and specificity.
  • the polypeptide has a K homology domain.
  • the K homology (KH) domain was first discovered in heterogeneous nuclear ribonucleoprotein K (hnRNPK).
  • the KH domain includes about 70 amino acid residues, and typically recognizes about 4 nucleotides in single stranded RNA.
  • KH domains may adopt either a type I ⁇ 1 ⁇ 1 ⁇ 2 ⁇ 2 ⁇ ′ ⁇ ′ topology, in eukaryotes, or the reverse type II ⁇ ′ ⁇ ′ ⁇ 1 ⁇ 1 ⁇ 2 ⁇ 2 topology, in prokaryotes, with a conserved “GXXG” RNA-binding motif (SEQ ID NO: 56) located between the ⁇ 1 and ⁇ 2 helices. RNA binding may occur in a hydrophobic pocket of the KH domain and may include several hydrogen bonds coordinated by the “GXXG” motif (SEQ ID NO: 56). Proteins having a KH domain and RNA have few stacking interactions. Furthermore, proteins having a KH domain usually have weak micromolar RNA affinities.
  • the polypeptide includes a zinc finger motif.
  • Zinc finger motifs may be found in a large family of proteins that average 30 amino acids in size and form a simple ⁇ topology in which residues in the ⁇ hairpin turn and ⁇ helix are coordinated by a Zn2+ ion.
  • Zinc finger motif polypeptides may bind DNA but have been additionally shown to bind RNA.
  • Zinc finger motifs include subtypes that interact with RNA and include amino acid sequences of CCHC (SEQ ID NO: 57), CCCH (SEQ ID NO: 58), CCCC (SEQ ID NO: 59), and CCHH (SEQ ID NO: 60) subtypes, where C and H refer to the interspersed cysteine and histidine residues that coordinate the zinc atom, respectively.
  • the zinc finger motif subtypes display a range of sequence and structural specificities.
  • the CCHC (SEQ ID NO: 57) subtype recognizes stem-loop elements in RNA through contacts with bases in the loop and the phosphate backbone of the stem.
  • CCCH SEQ ID NO: 58
  • CCCC SEQ ID NO: 59
  • the CCCH SEQ ID NO: 58
  • CCCC SEQ ID NO: 59
  • the CCCH SEQ ID NO: 58
  • CCCC SEQ ID NO: 59
  • the versatile and abundant CCHH SEQ ID NO: 60
  • designer zinc finger motifs may be use for directed binding of RNA sequences.
  • the RNA binding protein includes a Pumilio homology domain.
  • the Pumilio family of proteins occurs in most eukaryotes and is defined by the Pumilio homology domain.
  • the Pumilio homology domain is very large, consisting of eight ⁇ -helical repeats of a highly conserved 36- amino acid sequence that forms a concave RNA-binding surface. Each repeat recognizes one unpaired RNA base through hydrogen bonds and a stabilizing stacking interaction, where the full domain recognizes up to 8 nucleotides in single-stranded RNA with low-nanomolar affinity. Wild-type Pumilio homology domain repeats do not specifically recognize cytosine; however, protein engineering has produced repeats that do. This protein engineering combined with the PUF domain’s predictable base recognition code allow modular design of Pumilio proteins that recognize from 8 nucleotides to 10- nucleotides sequences containing all RNA bases.
  • the polypeptide includes a pentatricopeptide repeat domain.
  • Pentatricopeptide repeats include about 35 amino acid residues in length and form two antiparallel ⁇ helices.2–30 repeats form a solenoid-shaped scaffold that binds specific single-stranded RNA sequences with nanomolar affinity. Two residues in each repeat determine base-specific binding through hydrogen bonds, enabling the development of pentatricopeptide repeat domains designed to bind specified single-stranded RNA sequences.
  • the polypeptide includes a pseudouridine synthase and archaeosine transglycosylase (PUA) domain.
  • PUA pseudouridine synthase and archaeosine transglycosylase
  • PUA domains include a range from 67 to 94 amino acids residues (e.g., 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 94 amino acid residues), with a ⁇ 1 ⁇ 1 ⁇ 2 ⁇ 3 ⁇ 4 ⁇ 5 ⁇ 2 ⁇ 6 architecture that forms a pseudobarrel encased by two ⁇ helices.
  • PUA domains may contact double-stranded RNA, and its adjacent loops or overhangs through extensive hydrogen bonds may contact all parts of the RNA.
  • the polypeptide includes a THUMP domain.
  • THUMP domain Named for thiouridine synthase, methyltransferase, and pseudouridine synthase, the THUMP domain is found in numerous tRNA-modifying enzymes. THUMP domains are always found in proximity to RNA-modifying domains and often in proximity to an N-terminal ferredoxin-like domain.
  • THUMP domains include about 100 amino acids residues and display a ⁇ 1 ⁇ 2 ⁇ 1 ⁇ 3 ⁇ 2 ⁇ 2 topology that forms parallel ⁇ helices flanking a ⁇ sheet.
  • the polypeptide includes a YT521-B homology (YTH) domain.
  • the YTH domain is found in the YTH family of proteins that identify N6-methyladenosine (m6A) marks in RNA.
  • the YTH domain ranges from 100 to 150 amino acids residues in length and forms a six-stranded ⁇ barrel surrounded by four or five ⁇ helices.
  • the polypeptide includes a double-stranded RNA binding domain or motif.
  • the double-stranded RNA binding domain or motif may include from 65 to 70 amino acids residues. Double-stranded RNA binding domains specifically recognize and bind double-stranded RNA and are found in proteins with roles in viral protection, RNAi, and cellular transport.
  • Double-stranded RNA binding domains often appear as tandem repeats or in combination with other functional RNA-binding domains, such as RNA-editing or helicase domains.
  • the double-stranded RNA binding domain is made up of an ⁇ 1 ⁇ 1 ⁇ 2 ⁇ 3 ⁇ 2 fold that forms an antiparallel ⁇ sheet flanked by ⁇ helices on one face.
  • Double-stranded RNA binding domains specifically recognize the structure of an A-form RNA helix, spanning up to 16 base pairs with hydrogen-bond contacts to the phosphodiester backbone and 2′ OH. In some cases, double- stranded RNA binding domains have demonstrated base-specific contacts, such as to bases in adjacent loops.
  • the polypeptide includes a helicase domain.
  • Helicase domains are found in all forms of life in helicase proteins, which unwind both DNA and double-stranded RNA.
  • Helicases include six superfamilies, of which superfamily 1 and superfamily 2 contain all the eukaryotic RNA helicases.
  • RNA-binding helicases include the Upf1-like family in superfamily 1 and the DEAD-box, DEAH, RIG-I-like, Ski2-like, and NS3 families in superfamily 2. The remaining superfamilies, 3–6, contain bacterial and viral helicases that form multimeric rings.
  • Helicase domains are very large, containing 350– 400 amino acid residues.
  • the helicase domain is composed of two “recombinase A (recA)-like” subdomains, each of which contains an ATP-catalytic core, a nucleic-acid- binding region, and subdomains that coordinate the two. Within families of helicases these subdomains are quite conserved. Helicase monomers in the ring-forming superfamilies of helicases are similarly quite large and composed of multiple subdomains. Bound RNA is surrounded by recA-like domains or, in the case of multimeric helicases, RNA is pulled through the center of the ring. Contacts with RNA are dominated by hydrogen bonds to phosphate and sugar moieties, but contacts with bases have only occasionally been observed.
  • recA recombinase A
  • the polypeptide may include a cold shock domain.
  • the cold shock domain is found in a large family of proteins associated with cold adaptation found in all domains of life. Cold shock domains are composed of about 70 amino acids residues or more and five antiparallel ⁇ strands that form a common ⁇ barrel structure known as an oligosaccharide/oligonucleotide-binding fold.
  • Cold shock domains contain the conserved ribonucleoprotein 1 and ribonucleoprotein 2 motifs common to RNA recognition motifs, which bind single-stranded RNA. Cold shock domains contact 3 or 4 nucleotides through sequential stacking interactions and hydrogen bonds with bases, achieving nanomolar affinities. Cold shock domain containing proteins vary greatly in the types of sequences they recognize.
  • the polypeptide includes an S1 RNA-binding domain.
  • the S1 domain includes about 70 amino acid residues, which form a 5-stranded antiparallel ⁇ barrel in the same oligonucleotide binding fold family as the cold shock domain.
  • S1 domains are additionally found in several exoribonucleases and eukaryotic translation initiation factors and in combination with other RNA-binding domains such as the KH domain or cold shock domains. Despite their abundance, very little structural information is available for S1 domains in complex with RNA. S1 domains interact with both single- stranded RNA and double-stranded RNA in the context of the RNA-binding channel of exoribonucleases.
  • the polypeptide includes a Sm-RNA binding motif.
  • the Sm RNA-binding motif is found in Sm and like-Sm proteins in eukaryotes and archaea and in Hfq protein in prokaryotes.
  • the Sm motif consists of about 70 residues with an a1 p1 p2p3p4p5 topology that forms a curved antiparallel p sheet.
  • Sm-containing proteins readily multimerize through interactions between strands p4 and p5 in two Sm motifs. For example, Sm-Sm interactions link the seven human Sm proteins that make up the protein core of small nuclear ribonucleoproteins. The Sm multimers bind RNA with nanomolar affinity.
  • Two Sm motifs form a 6-nucleotide binding surface that binds specific bases, often uridines, through hydrogen bonds and stacking interactions.
  • the polypeptide includes a La motif.
  • La motif includes about 90 amino acid residues and is found in eukaryotic La and La-related proteins (LARPs).
  • the La motif consists of five a helices and three p strands that form a small antiparallel p sheet against a modified winged-helix fold. The winged-helix structure itself is common to several other RNA-binding proteins.
  • La motifs are always found adjacent to at least one RNA recognition motif, where the combination of these two domains likely evolved as a unit. In La proteins, the dual La motif and RNA recognition motif region tightly binds the UUU-OH elements at the 3' ends of polymerase-l Il-transcribed small RNAs.
  • Binding occurs in a cleft between the La motif and the RNA recognition motif rather than the traditional RNA-binding surfaces of either the RNA recognition motif or the La motif winged-helix fold.
  • Several uracil bases stack with highly conserved aromatic residues in the La motif, and hydrogen bonds from both the La motif and RNA- recognition motif coordinate bases, phosphates, and the terminating 2' OH. These contacts result in low- nanomolar affinities of the La motif for 3'-terminal UUU-OH elements.
  • the polypeptide includes a Piwi-Argonaute-Zwille or PIWI RNA-binding domains.
  • These RNA binding domains define the Argonaute family of proteins found in eukaryotes. These domains are found on opposite sides of the Argonaute protein, both domains facilitate binding of small interfering RNA and microRNA guides to mRNA targets.
  • the Piwi-Argonaute-Zwille domain occurs in Dicer proteins in addition to Argonaute proteins.
  • Crystal structures of the Piwi-Argonaute-Zwille domain display a six-stranded p barrel topped with two a helices and flanked on the opposite side by a special appendage containing a p hairpin and short a helix.
  • a binding pocket formed between this appendage and the p barrel binds the 2 nucleotide 3' overhang in guide RNAs with low-micromolar affinity. Binding is coordinated mostly by conserved tyrosine residues that form hydrogen bonds with the phosphate backbone and sugar hydroxyls of the two terminal nucleotides.
  • the PIWI RNA-binding domain tertiary structure forms an RNase H-like fold consisting of a five-stranded p sheet flanked by a helices on both faces.
  • the PIWI domain has endonucleolytic activity in some cases, but primarily stabilizes the gRNA- mRNA duplex seed region through hydrogen bonds with the gRNA backbone of nucleotides 3-5 and the 5' overhang base.
  • the polypeptide includes an intrinsically disordered region.
  • Intrinsically disordered regions are unstructured and often consist of repeats of arginine/serine residues, arginine/glycine, arginine- or lysine-rich patches, or short linear motifs of amino acids. Despite their lack of structure, intrinsically disordered regions have been found to dominate the composition in over 20% of RNA binding proteins. Intrinsically disordered regions may be the sole RNA-binding domain in an RNA binding protein and may actually drive the majority of protein-RNA interactions in the cell.
  • Intrinsically disordered regions are conserved, often occur multiple times in one RNA binding protein, and can coordinate RNA binding in concert with other domains. Intrinsically disordered regions have been shown to drive higher affinity to RNA in RNA binding proteins that contain ordered RNA-binding domains and can themselves transition to an ordered state once bound to RNA. Intrinsically disordered regions show little RNA sequence dependence, however, suggesting that these regions’ high affinity for RNA is predominantly driven by electrostatic attraction to the phosphodiester backbone.
  • the polypeptide may include another domain which allows for the protein to complex with the polyribonucleotide described herein.
  • the disclosure provides circular polyribonucleotide that are complexed with one or more moieties that specifically bind a region of the circular polyribonucleotide, wherein each of the one or more moieties that specifically bind a region of the circular polyribonucleotide is conjugated to (e.g., directly, chemically- covalently conjugated) to one or more targeting moieties.
  • the circular polyribonucleotide includes at least one expression sequence (e.g., coding region) that encodes a polypeptide.
  • the polypeptide when expressed in the cell is functional.
  • the moiety that specifically bind a region of the circular polyribonucleotide binds a region of the circular polyribonucleotide includes or more expression sequences.
  • the encoded polypeptide may have 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 include a fluorescent tag or marker, an antigen, a peptide therapeutic, a synthetic or analog peptide from a naturally-bioactive peptide, an agonist or antagonist peptide, an anti-microbial peptide, a pore-forming peptide, a bicyclic peptide, a targeting or cytotoxic peptide, a degradation or self-destruction peptide, and degradation or self-destruction peptides.
  • Peptides useful in the invention described herein also include antigen-binding peptides, e.g., antigen binding antibody or antibody-like fragments, such as single chain antibodies, nanobodies (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21 (7): 1076-113).
  • antigen binding peptides may bind a cytosolic antigen, a nuclear antigen, an intra- organellar antigen.
  • the polypeptide may be produced in substantial amounts.
  • the polypeptide may be any proteinaceous molecule that can be produced.
  • a polypeptide can be a polypeptide that can be secreted from a cell, or localized to the cytoplasm, nucleus or membrane compartment of a cell.
  • Some polypeptides include, but are not limited to, at least a portion of a viral envelope protein, metabolic regulatory enzymes (e.g., that regulate lipid or steroid production), an antigen, a toleragen, a cytokine, a toxin, enzymes whose absence is associated with a disease, and polypeptides that are not active in an animal until cleaved (e.g., in the gut of an animal), and a hormone.
  • the circular polyribonucleotide includes an expression sequence encoding a protein e.g., a therapeutic protein.
  • therapeutic proteins that can be expressed from the circular polyribonucleotide disclosed herein have antioxidant activity, binding, cargo receptor activity, catalytic activity, molecular carrier activity, molecular function regulator, molecular transducer activity, nutrient reservoir activity, protein tag, structural molecule activity, toxin activity, transcription regulator activity, translation regulator activity, or transporter activity.
  • therapeutic proteins may include, but are not limited to, an enzyme replacement protein, a protein for supplementation, a protein vaccination, antigens (e.g.
  • tumor antigens viral, bacterial
  • hormones cytokines
  • antibodies immunotherapy (e.g. cancer)
  • cellular reprogramming/transdifferentiation factor transcription factors
  • transcription factors chimeric antigen receptor
  • immune effector e.g., influences susceptibility to an immune response/signal
  • a regulated death effector protein e.g., an inducer of apoptosis or necrosis
  • a non-lytic inhibitor of a tumor e.g., an inhibitor of an oncoprotein
  • an epigenetic modifying agent epigenetic enzyme, a transcription factor, a DNA or protein modification enzyme, a DNA-intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor for an enzyme, a protein synthesis effector or inhibitor, a nuclease, a protein fragment or domain, a ligand or a receptor, and a CRISPR system or component thereof
  • exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include human proteins, for instance, receptor binding protein, hormone, growth factor, growth factor receptor modulator, and regenerative protein (e.g., proteins implicated in proliferation and differentiation, e.g., therapeutic protein, for wound healing).
  • exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include EGF (epithelial growth factor).
  • exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include enzymes, for instance, oxidoreductase enzymes, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzyme, and desaturases.
  • exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include an intracellular protein or cytosolic protein.
  • the circular polyribonucleotide expresses a NanoLuc® luciferase (nLuc).
  • exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include a secretary protein, for instance, a secretary enzyme.
  • the circular polyribonucleotide expresses a secretary protein that can have a short half-life therapeutic in the blood, or can be a protein with a subcellular localization signal, or protein with secretory signal peptide.
  • the circular polyribonucleotide expresses a Gaussia Luciferase (gLuc).
  • the circular polyribonucleotide expresses a non-human protein, for instance, a fluorescent protein, an energy- transfer acceptor, or a protein-tag like Flag, Myc, or His.
  • exemplary proteins that can be expressed from the circular polyribonucleotide include a GFP.
  • the circular polyribonucleotide expresses tagged proteins, .e.g., fusion proteins or engineered proteins containing a protein tag, e.g., chitin binding protein (CBP), maltose binding protein (MBP), Fc tag, glutathione-S- transferase (GST), AviTag, Calmodulin-tag, polyglutamate tag; E-tag, FLAG-tag), HA-tag, His-tag, Myc- tag, NE-tag, S-tag, SBP-tag, Softag 1 , Softag 3, Spot-tag, Strep-tag; TC tag, Ty tag, V5 tag ; VSV-tag; or Xpress tag.
  • a protein tag e.g., chitin binding protein (CBP), maltose binding protein (MBP), Fc tag, glutathione-S- transferase (GST), AviTag, Calmodulin-tag, polyglutamate tag; E-tag, FLAG-tag),
  • the circular polyribonucleotide encodes the expression of 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, IgE, IgG, or 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.
  • the polyribonucleotide described herein (e.g., the polyribonucleotide cargo 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 linked operatively 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 exists.
  • one regulatory element can increase amounts of products expressed for multiple expression sequences attached in tandem. Hence, one regulatory element can enhance the expression of one or more expression sequences. Multiple regulatory elements are well-known to persons of ordinary skill in the art.
  • 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 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. Further examples of regulatory elements are described, e.g., in paragraphs [0154] - [0161] of International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
  • the 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).
  • the IRES is located between a heterologous promoter and the 5’ end of a coding sequence.
  • the moiety that specifically bind a region of the circular polyribonucleotide binds a region of the circular polyribonucleotide includes an IRES.
  • 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 derived from the DNA of an organism including, but not limited to, a virus, a mammal, or a Drosophila.
  • viral DNA may be derived 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
  • Drosophila DNA from which an IRES element is derived includes, but is not limited to, 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, fuman poliovirus 1 , Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1 , Human Immunodeficiency Virus type 1 , Homalodisca coagulata virus- 1 , Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 , Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis
  • EMCV Encephal
  • 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. In some embodiments, the IRES sequence has more than 90% sequence identify with one of the foregoing IRES sequences.
  • the IRES sequence may have the sequence of wild-type CVB3 IRES sequence having the nucleic acid sequence of: GCAAA (SEQ ID NO: 96).
  • the IRES sequence may be a CVB3 IRES sequence having the nucleic acid sequence of : GCAA (SEQ ID NO: 97).
  • the IRES sequence may have a modified sequence in comparison to the wild-type IRES sequence.
  • the last nucleotide of the wild-type IRES when the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be 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: GCAAC (SEQ ID NO: 98).
  • the IRES sequence is an encephalomyocarditis virus (EMCV) IRES.
  • the ECMV IRES may have the nucleic acid sequence of: ( )
  • the IRES sequence is an Enterovirus 71 (EV71) IRES.
  • the IRES sequence is a wild-type EV71 sequence having the nucleic acid sequence of: AAGCG (SEQ ID NO: 100).
  • the terminal guanosine residue of the EV71 IRES sequence is modified to a cytosine residue.
  • the modified EV71 IRES may have the nucleic acid sequence of: AAGCC (SEQ ID NO: 101).
  • 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. In some embodiments, 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 includes an IRES (e.g., an IRES operably linked to a coding region).
  • the polyribonucleotide may include any IRES as described in Fan et al. Nature Communications 13(1):3751 -3765, 2022 doi: 10.1038/s41467-022-31327- y; 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.
  • a polypeptide expressed from a circular polyribonucleotide disclosed herein includes a secreted protein, for example, a protein that naturally includes a signal sequence, or one that does not usually encode a signal sequence but is modified to contain one.
  • the polypeptide encoded by the circular polyribonucleotide includes a secretion signal.
  • the secretion signal may be the naturally encoded secretion signal for a secreted protein.
  • the secretion signal may be a modified secretion signal for a secreted protein.
  • the polypeptide encoded by the circular polyribonucleotide does not include a secretion signal.
  • a circular polyribonucleotide encodes multiple copies of the same polypeptide (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more). In some embodiments, at least one copy of the polypeptide 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., a plurality of different polypeptides or a plurality of polypeptides having less than 100% sequence identity), 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., when expressed endogenously.
  • the signal sequence is heterologous to the polypeptide, 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.
  • a polypeptide encoded by a polyribonucleotide may include a signal sequence that directs the polypeptide to the secretory pathway.
  • the signal sequence may direct the polypeptide to reside in certain organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes).
  • the signal sequence directs the polypeptide 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.
  • a polypeptide encoded by a polyribonucleotide includes either a secretion signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence.
  • a circular 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
  • stagger element refers to 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.
  • 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 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 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.
  • the circular polyribonucleotide includes a stagger element.
  • 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 polyribonucleotide.
  • the stagger element may include, but is not limited to a 2A-like, or CHYSEL (SEQ ID NO: 62) (cis-acting hydrolase element) sequence.
  • the stagger element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP (SEQ ID NO: 63), where X1 is absent or G or H, X 2 is absent or D or G, X 3 is D or V or I or S or M, and X 5 is any amino acid.
  • stagger elements includes GDVESNPGP (SEQ ID NO: 65), GDIEENPGP (SEQ ID NO: 66), VEPNPGP (SEQ ID NO: 67), IETNPGP (SEQ ID NO: 68), GDIESNPGP (SEQ ID NO: 69), GDVELNPGP (SEQ ID NO: 70), GDIETNPGP (SEQ ID NO: 71), GDVENPGP (SEQ ID NO: 72), GDVEENPGP (SEQ ID NO: 73), GDVEQNPGP (SEQ ID NO: 74), IESNPGP (SEQ ID NO: 75), GDIELNPGP (SEQ ID NO: 76), HDIETNPGP (SEQ ID NO: 77), HDVETNPGP (SEQ ID NO: 78), HDVEMNPGP (SEQ ID NO: 79), GDMESNPGP (SEQ ID NO: 80), GDVETNPGP (SEQ ID NO: 81), GDIEQNPGP (SEQ ID NO: 81), GD
  • the stagger element described herein cleaves an expression product, such as between G and P of the consensus sequence described herein.
  • the circular polyribonucleotide includes at least one stagger element to cleave the expression product.
  • the circular polyribonucleotide includes a stagger element adjacent to at least one expression sequence.
  • the circular polyribonucleotide includes a stagger element after each expression sequence.
  • the circular polyribonucleotide includes a stagger element 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 / 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.
  • the stagger element is present in the circular polyribonucleotide in other forms.
  • a stagger element includes a termination element of a first expression sequence in the circular 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 polyribonucleotide.
  • the first expression sequence and the expression sequence succeeding the first expression sequence are two separate expression sequences in the circular 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 polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the succeeding sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular 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 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 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 polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the first expression sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide including a stagger element upstream of a second translation initiation sequence of a second expression sequence in the corresponding circular 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 polyribonucleotide than a distance between the first stagger element and the first translation initiation in the circular 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 polyribonucleotide includes more than one expression sequence.
  • a plurality of polypeptides encoded by a circular ribonucleotide may be separated by an IRES between each polypeptide (e.g., each polypeptide is operably linked to a separate IRES).
  • a circular polyribonucleotide may include a first IRES operably 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 polypeptides.
  • the IRES may be different between different polypeptides.
  • the plurality of polypeptides may be separated by a 2A self-cleaving peptide.
  • a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, a 2A, and a second polypeptide.
  • the plurality of polypeptides 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 polypeptide, a protease cleavage site (e.g., a furin cleavage site), and a second polypeptide.
  • a circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first polypeptide, a protease cleavage site (e.g., a furin cleavage site), a 2A, and a second polypeptide.
  • 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 polypeptides encoded by the circular ribonucleotide may be separated by both IRES and 2A sequences.
  • an IRES may be between one polypeptide and a second polypeptide while a 2A peptide may be between the second polypeptide and the third polypeptide.
  • the selection of a particular IRES or 2A self-cleaving peptide may be used to control the expression level of a polypeptide 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 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 polyribonucleotide includes from 2 to 10 cleavage sequences. In some embodiments, the circular polyribonucleotide includes from 2 to 5 cleavage sequences.
  • the multiple cleavage sequences are between multiple expression sequences; for example, a circular polyribonucleotide may include three expression sequences two cleavage sequences such that there is a cleavage sequence in between each expression sequence.
  • the circular polyribonucleotide includes a cleavage sequence, such as in an immolating circRNA or cleavable circRNA or self-cleaving circRNA.
  • the circular polyribonucleotide includes two or more cleavage sequences, leading to separation of the circular polyribonucleotide into multiple products, e.g., miRNAs, linear RNAs, smaller circular 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 polypeptides, e.g., where the two or more polypeptides are encoded by a single open-reading frame (ORF).
  • ORF open-reading frame
  • two or more polypeptides 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 polypeptides 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 single expression product including the amino acid sequence of two or more polypeptides is cleaved upon expression, such that the two or more polypeptides are separated following expression.
  • exemplary 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 (e.g., furin), urokinase-type plasminogen activator, matriptase, a cysteine protease, an aspartic protease, or a cathepsin protease.
  • the protease e.g
  • a circular polyribonucleotide described herein is an immolating circular polyribonucleotide, a cleavable circular polyribonucleotide, or a self-cleaving circular polyribonucleotide.
  • a circular polyribonucleotide can deliver cellular components including, for example, RNA, long non-coding RNA (IncRNA), long intergenic non-coding RNA (lincRNA), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), small interfering RNA (siRNA), or small hairpin RNA (shRNA).
  • IncRNA long non-coding RNA
  • lincRNA long intergenic non-coding RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • small nucleolar RNA snoRNA
  • ncRNA
  • a circular polyribonucleotide includes miRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites; (iii) degradable linkers; (iv) chemical linkers; and/or (v) spacer 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 sequences.
  • self-cleavable elements include hammerhead, splicing element, hairpin, hepatitis delta virus (HDV), Varkud Satellite (VS), and glmS ribozymes.
  • the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes at least one translation initiation sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence operably linked to an expression sequence.
  • the polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, e.g., a start codon.
  • the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence.
  • the 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 is present on one or both sides of each expression sequence, leading to separation of the expression products.
  • the polyribonucleotide includes at least one translation initiation sequence adjacent to an expression sequence.
  • the translation initiation sequence provides conformational flexibility to the polyribonucleotide.
  • the translation initiation sequence is within a substantially single stranded region of the 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 polyribonucleotide may include more than 1 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.
  • the polyribonucleotide may initiate at a codon which is not the first start codon, e.g., AUG.
  • Translation of the 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 (SEQ ID NO: 93), CAG, CTG.
  • RAN repeat-associated non-AUG
  • the polyribonucleotide described herein (e.g., the polyribonucleotide cargo 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. 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.
  • a circular polyribonucleotide includes untranslated regions (UTRs).
  • 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 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.
  • the intron is a full-length human intron, e.g., ZKSCAN1.
  • a circular polyribonucleotide includes a polyA tail. Exemplary polyA tails 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 polyA tail.
  • a circular 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 marker of an immune or inflammatory response) of the circular polyribonucleotide.
  • immunogenicity e.g., the level of one or more marker 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 polyribonucleotide lacks a 3’-UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a polyA tail and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a termination element and is competent for protein expression from its one or more expression sequences.
  • the circular 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 polyribonucleotide lacks a cap and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular 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 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 a therapeutic, 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 a therapeutic mRNA or protein.
  • 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 a therapeutic mRNA or protein.
  • a circular polyribonucleotide lacks a 5’-UTR. In some embodiments, the circular polyribonucleotide lacks a 3’-UTR. In some embodiments, the circular polyribonucleotide lacks a polyA tail. In some embodiments, the circular polyribonucleotide lacks a termination element. In some embodiments, the circular polyribonucleotide lacks an internal ribosomal entry site. In some embodiments, the circular 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.
  • the circular polyribonucleotide lacks a 5’ cap.
  • the polyribonucleotide described herein includes one or more spacer sequences.
  • the moiety that specifically bind a region of the circular polyribonucleotide binds a region of the circular polyribonucleotide includes one or more spacers.
  • a spacer refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacers may be present in between any of the nucleic acid elements described herein. Spacer may also be present within a nucleic acid element described herein.
  • a nucleic acid includes any two or more of the following elements: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment; a spacer region may be present between any one or more of the elements. Any of elements (A), (B), (C), (D), (E), (F), or (G) may be separated by a spacer sequence, as described herein. For example, there may be a spacer between (A) and (B), between (B) and (C), between (C) and (D), between (D) and (E), between (E) and (F), or between (F) and (G).
  • a spacer sequences may be used to separate an IRES from adjacent structural elements to martini the structure and function of the IRES or the adjacent element.
  • a spacer can be specifically engineered depending on the IRES.
  • an RNA folding computer software such as RNAFold, can be utilized to guide designs of the various elements of the vector, including the spacers.
  • the spacer may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length.
  • each spacer region is at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length.
  • Each spacer region may be, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length.
  • the first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a polyA tail (or, e.g., polyA sequence).
  • the first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a polyA-C tail (or, e.g., polyA-C sequence).
  • the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a polyA-G tail (or, e.g., polyA-G sequence).
  • the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a polyA-U tail (or, e.g., polyA-U sequence). In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a random sequence.
  • the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 30 nucleotides in length. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the spacer sequence is from 20 to 50 nucleotides in length.
  • the spacer sequence is 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.
  • the spacer sequences can be polyA tails, polyA-C tails, polyC tails, or poly-U tails. In some embodiments, the spacer sequences can be polyA-T, polyA-C, polyA-G, or a random sequence.
  • a polyribonucleotide e.g., circular polyribonucleotide as described herein 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 circular polyribonucleotide includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, polyA tail, 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. WO2019/1 18919, 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 / 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 circular 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 circular polyribonucleotide may enhance immune evasion.
  • the circular 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 circular 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 circular polyribonucleotide may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages.
  • Specific examples of circular polyribonucleotide include, but are not limited to, circular polyribonucleotide including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages.
  • Circular 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 circular 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 circular polyribonucleo
  • the modified nucleotides which may be incorporated into the polyribonucleotide, can be modified on the internucleoside linkage (e.g., phosphate backbone).
  • 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 circular 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-(1 - thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine (a-thio-cytidine), 5'-0-(1-thiophosphate)- guanosine, 5'-0-(1-thiophosphate)-uridine, or 5'-0-(1-thiophosphate)-pseudouridine).
  • alpha-thio-nucleoside e.g., 5'-0-(1 - thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine (a-thio-cytidine), 5'-0-(1-thiophosphate)- guanosine, 5'-0-(1-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 circular polyribonucleotide may include one or more cytotoxic nucleosides.
  • cytotoxic nucleosides may be incorporated into circular 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, 1-(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-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1
  • Additional examples include fludarabine phosphate, N4-behenoyl-1 -beta-D- arabinofuranosylcytosine, N4-octadecyl-1 -beta-D-arabinofuranosylcytosine, N4- palmitoyl-1-(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 circular polyribonucleotide includes a pseudouridine.
  • the circular polyribonucleotide includes an inosine, which may aid in the immune system characterizing the circular polyribonucleotide as endogenous versus viral RNAs.
  • 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 circular polyribonucleotide.
  • nucleotide analogs or other modification(s) may be located at any position(s) of the circular polyribonucleotide, such that the function of the circular polyribonucleotide is not substantially decreased.
  • a modification may also be a non-coding region modification.
  • the circular 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%).
  • any intervening percentage e.g.
  • RNA circle can include a DNA sequence of a naturally occurring original 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 RNA may be cyclized or concatenated to create a circRNA described herein.
  • 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 circRNA includes an active ribozyme sequence capable of ligating the 5' end of the linear polyribonucleotide to the 3' end of the linear polyribonucleotide.
  • 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 in order to cyclize or concatenate the linear polyribonucleotide.
  • 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.
  • 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.
  • linear polyribonucleotides may be cyclized or concatenated by self-splicing.
  • the linear polyribonucleotides may include loop E sequence to self-ligate.
  • the linear polyribonucleotides may include a self-circularizing intron, e.g., a 5' and 3’ slice junction, or a self-circularizing catalytic intron such as a Group I, Group II or Group III Introns.
  • Nonlimiting examples of 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.
  • a linear polyribonucleotide 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.
  • the one or more linear polyribonucleotides may be 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.
  • the linear polyribonucleotide 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, thereby 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 ligated 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 circular polyribonucleotide is purified, e.g., free ribonucleic acids, linear or nicked RNA, DNA, proteins, etc. are removed.
  • the circular polyribonucleotides may be purified by any known method commonly used in the art. Examples of nonlimiting purification methods include, column chromatography, gel excision, size exclusion, etc.
  • a circular polyribonucleotide described herein may be formulated in composition, e.g., a composition for delivery to a cell, a plant, an invertebrate animal, a non-human vertebrate animal, or a human subject, e.g., an agricultural, veterinary, or pharmaceutical composition.
  • the circular polyribonucleotide is formulated in a pharmaceutical composition.
  • a composition includes a circular polyribonucleotide and a diluent, a carrier, an adjuvant, or a combination thereof.
  • a composition in a particular embodiment, includes a circular polyribonucleotide described herein and a carrier or a diluent free of any carrier. In some embodiments, a composition including a circular polyribonucleotide with a diluent free of any carrier is used for naked delivery of the circular polyribonucleotide to a subject.
  • 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).
  • Non-limiting 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 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.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient 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.
  • the reference criterion for the amount of circular polyribonucleotide molecules present in the 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 pharmaceutical preparation.
  • the reference criterion for the amount of linear polyribonucleotide molecules present in the 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, 700 pg/ml
  • the reference criterion for the amount of linear polyribonucleotide molecules present in the 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 pharmaceutical preparation.
  • the reference criterion for the amount of nicked polyribonucleotide molecules present in the 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 pharmaceutical preparation.
  • the reference criterion for the amount of combined nicked and linear polyribonucleotide molecules present in the 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 pharmaceutical preparation.
  • a pharmaceutical preparation is an intermediate pharmaceutical preparation of a final circular polyribonucleotide drug product.
  • a pharmaceutical preparation is a drug substance or active pharmaceutical ingredient (API).
  • API active pharmaceutical ingredient
  • a pharmaceutical preparation is a drug product for administration to a subject.
  • a preparation of circular polyribonucleotides is (before, during or after the reduction of linear RNA) 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.
  • 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).
  • Delivery Agents A composition or pharmaceutical composition provided herein may include one or more delivery agents, wherein the delivery agents may increase cellular delivery, functional delivery, or endosomal escape of the circular polyribonucleotide. The delivery agent may be used in place of or in combination with lipofectamine to increase transfection of a circular polyribonucleotide into cell. In some embodiments, the circular polyribonucleotide is administered to a subject without the use of lipofectamine.
  • the circular polyribonucleotide described herein may be formulated in a composition, wherein the composition includes a ribonuclease inhibitor.
  • the ribonuclease inhibitor is selected from RNseOUT TM recombinant ribonuclease inhibitor, RNasin TM ribonuclease inhibitor, and SUPERase-In TM RNase inhibitor.
  • the ribonuclease inhibitor may be in any amount suitable for delivery of the composition; for example, the ribonuclease inhibitor may be in an amount of from 0.05 U/mL to 1 U/mL, where one unit is defined as the amount of ribonuclease inhibitor required to inhibit the activity of 5 ng of ribonuclease A by 50%, and activity is measured by the inhibition of hydrolysis of cytidine 2 ⁇ ,3 ⁇ -cyclic monophosphate by ribonuclease A.
  • the circular polyribonucleotide described herein may be formulated in a composition, wherein the composition includes a calcium cation, magnesium cation, manganese cation, strontium cation, or any combination thereof.
  • the composition may include calcium chloride, calcium acetate, calcium carbonate, calcium citrate, calcium gluconate, calcium lactate, and calcium phosphate.
  • the calcium, manganese, or strontium cation may be in any amount suitable for delivery of the composition.
  • the calcium, manganese, or strontium cation may be included at a concentration of from 0.01 mM to 500 mM (e.g., 0.01 mM to 400 mM, 0.01 mM to 300 mM, 0.01 mM to 200 mM, 0.01 mM to 100 mM, 0.01 mM to 1 mM, 0.01 mM to 0.1 mM, 0.1 mM to 500 mM, 1 mM to 500 mM, 10 mM to 500 mM, 100 mM to 500 mM, 200 mM to 500 mM, 300 mM to 500 mM, or 400 mM to 500 mM).
  • 0.01 mM to 500 mM e.g., 0.01 mM to 400 mM, 0.01 mM to 300 mM, 0.01 mM to 200 mM, 0.01 mM to 100 mM, 0.01 mM to 1 mM, 0.01
  • the calcium, manganese, or strontium cation may be included at a concentration of from 0.1 mM to 100 mM (e.g., 0.1 mM to 90 mM, 0.1 mM to 70 mM, 0.1 mM to 50 mM, 0.1 mM to 30 mM, 0.1 mM to 10 mM, 0.1 mM to 1 mM, 1 mM to 100 mM, 10 mM to 100 mM, 30 mM to 100 mM, 50 mM to 100 mM, 70 mM to 100 mM, or 90 mM to 100 mM).
  • 0.1 mM to 100 mM e.g., 0.1 mM to 90 mM, 0.1 mM to 70 mM, 0.1 mM to 50 mM, 0.1 mM to 30 mM, 0.1 mM to 10 mM, 0.1 mM to 1 mM, 1 mM
  • the circular polyribonucleotide described herein may be formulated in a composition, wherein the composition includes an endosomal escape agent.
  • the endosomal escape agent includes chloroquine, amantadine, ammonium chloride, 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), UNC-108, or any combination thereof.
  • the endosomal escape agent may be in any amount suitable for delivery of the composition.
  • the chloroquine concentration in the composition may be from 10 ⁇ M to 1 M (e.g., 10 ⁇ M to 10 mM, 10 ⁇ M to 1 mM, 10 ⁇ M to 100 ⁇ M, 100 ⁇ M to 1 M, 1 mM to 1 M, or 10 mM to 1 M) .
  • the endosomal escape agent concentration is from 10 ⁇ M to 100 ⁇ M (e.g., 10 ⁇ M to 90 ⁇ M, 10 ⁇ M to 70 ⁇ M, 10 ⁇ M to 50 ⁇ M, 10 ⁇ M to 30 ⁇ M, 30 ⁇ M to 100 ⁇ M, 50 ⁇ M to 100 ⁇ M, or 70 ⁇ M to 100 ⁇ M).
  • the circular polyribonucleotide described herein may be formulated in a composition, wherein the composition includes globular protein.
  • the globular protein is albumin.
  • the composition includes human serum albumin.
  • the composition includes bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the BSA may be in any amount suitable for delivery of the composition.
  • the composition includes one or more delivery agents described herein (e.g., in any amount described herein).
  • the composition may include endosomal escape agent and a calcium, strontium, or manganese cation (e.g., in any amounts described herein).
  • the composition may include a calcium, strontium, or manganese cation and a ribonuclease inhibitor (e.g., in any amounts described herein).
  • the composition may include a ribonuclease inhibitor and endosomal escape agent (e.g., in any amounts described herein).
  • the composition includes albumin and endosomal escape agent (e.g., in any amounts described herein).
  • the composition includes albumin and endosomal escape agent (e.g., in any amounts described herein).
  • the composition includes albumin and endosomal escape agent (e.g., in any amounts described herein).
  • the composition includes albumin and a ribonuclease inhibitor (e.g., in any amounts described herein).
  • the composition may include calcium chloride, chloroquine, and a ribonuclease inhibitor (e.g., in any amounts described herein).
  • the composition may include a calcium, manganese, or strontium cation, endosomal escape agent, a ribonuclease inhibitor, and albumin (e.g., in any amounts described herein).
  • Preservatives A composition or pharmaceutical composition provided herein can include material for a single administration, or can include material for multiple administrations (e.g., a “multidose” kit).
  • the polyribonucleotide can be present in either linear or circular form.
  • the composition or pharmaceutical composition can include one or more preservatives such as thiomersal or 2-phenoxyethanol.
  • Preservatives can be used to prevent microbial contamination during use. 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%.
  • the preservative e.g., benzalkonium chloride
  • the preservative 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.
  • Polyribonucleotides can be susceptible to RNase that can be abundant in ambient environment.
  • Compositions provided herein can include reagents that inhibit RNase activity, thereby preserving the polyribonucleotide from degradation.
  • the composition or pharmaceutical composition includes any RNase inhibitor known to one skilled in the art.
  • the polyribonucleotide, and cell-penetrating agent and/or pharmaceutically acceptable 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. Salts
  • a composition or pharmaceutical composition provided herein includes one or more salts.
  • a physiological salt such as sodium salt can be included a composition provided herein.
  • Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, or the like.
  • the 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 or pharmaceutical composition provided herein can include one or more buffers, such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (e.g., with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers, in some cases, are included in the 5-20 mM range.
  • a composition or pharmaceutical composition provided herein can have a pH from about 5.0 to about 8.5, from about 6.0 to about 8.0, from about 6.5 to about 7.5, or from about 7.0 to about 7.8.
  • the composition or pharmaceutical composition can have a pH of about 7.
  • the polyribonucleotide can be present in either linear or circular form.
  • a composition or pharmaceutical composition provided herein can include one or more detergents and/or surfactants, depending on the intended administration route, e.g., polyoxyethylene sorbitan esters surfactants (commonly referred to as “Tweens”), e.g., polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, e.g., octoxynol-9 (Triton X-100, or t- octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphati and others.
  • 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).
  • HLB hydrophile/lipophile balance
  • 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.
  • Diluents In some embodiments, a composition of the disclosure includes a circular polyribonucleotide and a diluent.
  • a composition of the disclosure includes a linear polyribonucleotide and a diluent.
  • a diluent can be a non-carrier excipient.
  • a non-carrier excipient serves as a vehicle or medium for a composition, such as a circular polyribonucleotide as described herein.
  • a non-carrier excipient serves as a vehicle or medium for a composition, such as a linear 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.
  • PBS phosphate buffered saline
  • 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 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 circular polyribonucleotide may be delivered as a naked delivery formulation, such as including a diluent.
  • a naked delivery formulation delivers a circular polyribonucleotide, to a cell without the aid of a carrier and without modification or partial or complete encapsulation of the circular polyribonucleotide, capped polyribonucleotide, or complex thereof.
  • a naked delivery formulation is a formulation that is free from a carrier and wherein the circular 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 circular polyribonucleotide.
  • a circular 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 protein, small molecule, a particle, a polymer, or a biopolymer.
  • a circular polyribonucleotide without covalent modification that binds a moiety that aids in delivery to a cell does not contain a modified phosphate group.
  • a circular 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 of: 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, 1,2-Dioleoyl-3-Trimethylammonium- Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2- (o
  • a naked delivery formulation includes a non-carrier excipient.
  • 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 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.
  • a naked delivery formulation includes a diluent.
  • a diluent may be a liquid diluent or a solid diluent.
  • a diluent is an RNA solubilizing agent, a buffer, or an isotonic agent.
  • 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.
  • MES 2-(N-morpholino)ethanesulfonic acid
  • Bis-Tris 2-[(2-amino-2-o
  • a composition of the disclosure includes a circular polyribonucleotide and a carrier.
  • a composition of the disclosure includes a linear polyribonucleotide and a carrier.
  • a composition includes a circular polyribonucleotide as described herein in a vesicle or other membrane-based carrier.
  • a composition includes a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier.
  • a composition includes the circular polyribonucleotide in or via a cell, vesicle or other membrane-based carrier. In other embodiments, a composition includes the linear polyribonucleotide in or via a cell, vesicle or other membrane-based carrier. In one embodiment, a composition includes the circular polyribonucleotide in liposomes or other similar vesicles. In one embodiment, a composition includes the linear 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 a relatively 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
  • a composition of the disclosure includes a circular polyribonucleotide and lipid nanoparticles, for example lipid nanoparticles described herein.
  • a composition of the disclosure includes a linear polyribonucleotide and lipid nanoparticles.
  • Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a circular polyribonucleotide molecule as described herein.
  • Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a linear polyribonucleotide molecule as described herein.
  • Nanostructured lipid carriers 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 are an important 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.
  • carriers include carbohydrate carriers (e.g., an anhydride- modified phytoglycogen or glycogen-type material), protein carriers (e.g., a protein covalently linked to the circular polyribonucleotide or a protein covalently linked to the linear polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or transfection reagent).
  • carbohydrate carriers e.g., an anhydride- modified phytoglycogen or glycogen-type material
  • protein carriers e.g., a protein covalently linked to the circular polyribonucleotide or a protein covalently linked to the linear polyribonucleotide
  • cationic carriers e.g., a cationic lipopolymer or transfection reagent.
  • Non-limiting examples of 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, 1,2- Dioleoyl-3-Trimethylammonium-Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride
  • Non-limiting examples of protein carriers include human serum albumin (HSA), low-density lipoprotein (LDL), high- density lipoprotein (HDL), or globulin.
  • Exosomes can also be used as drug delivery vehicles for a circular RNA composition or preparation described herein.
  • Exosomes can be used as drug delivery vehicles for a linear polyribonucleotide composition or preparation described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; doi.org/10.1016/j.apsb.2016.02.001.
  • Ex vivo differentiated red blood cells can also be used as a carrier for a circular RNA composition or preparation described herein.
  • Ex vivo differentiated red blood cells can also be used as a carrier for a linear polyribonucleotide composition or preparation described herein. See, e.g., International Patent Publication Nos. WO2015/073587; WO2017/123646; WO2017/123644; WO2018/102740; WO2016/183482; WO2015/153102; WO2018/151829; WO2018/009838; Shi et al.2014. Proc Natl Acad Sci USA.111(28): 10131–10136; US Patent 9,644,180; Huang et al.2017. Nature Communications 8: 423; Shi et al.2014. Proc Natl Acad Sci USA.111(28): 10131–10136.
  • Fusosome compositions can also be used as carriers to deliver a circular polyribonucleotide molecule described herein.
  • Fusosome compositions e.g., as described in WO2018/208728, can also be used as carriers to deliver a linear polyribonucleotide molecule described herein.
  • Virosomes and virus-like particles can also be used as carriers to deliver a circular polyribonucleotide molecule described herein to targeted cells.
  • Virosomes and virus-like particles can also be used as carriers to deliver a linear polyribonucleotide molecule described herein to targeted cells.
  • Plant nanovesicles and plant messenger packs can also be used as carriers to deliver the circular RNA composition or preparation described herein.
  • Plant nanovesicles and plant messenger packs can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein.
  • Lipid reconstructed plant messenger packs e.g., as described in International Patent Publication Nos.
  • WO2021/041301, WO2023/069498, or WO2023/122080 can also be used as carriers to deliver the circular RNA composition or preparation described herein.
  • Lipid reconstructed plant messenger packs LPMPs
  • LNMPs Lipid reconstructed natural messenger packs
  • WO2024/102434 Lipid reconstructed natural messenger packs
  • LNMPs can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein.
  • Bacteria- derived lipid compositions e.g., as described in International Patent Publication Nos. WO2023/096858 can also be used as carriers to deliver the circular RNA composition or preparation described herein.
  • Bacteria-derived lipid compositions can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein.
  • Microbubbles can also be used as carriers to deliver a circular polyribonucleotide molecule described herein.
  • Microbubbles can also be used as carriers to deliver a linear polyribonucleotide molecule described herein. See, e.g., US7115583; Beeri, R.
  • microbubbles are albumin-coated perfluorocarbon microbubbles.
  • the carrier including the circular 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 circular polyribonucleotide into a cell. Deposition of the circular polyribonucleotide into certain cell types may favor different particle sizes. For example, the particle size may be optimized for deposition of the circular polyribonucleotide into antigen presenting cells. The particle size may be optimized for deposition of the circular polyribonucleotide into dendritic 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).
  • LNPs lipid nanoparticles
  • 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 WO2019217941; 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 WO2019217941; incorporated herein by reference in its entirety
  • sterols e.g., cholesterol
  • Lipids that can be used in nanoparticle formations include, for example those described in Table 4 of WO2019217941, which is incorporated by reference—e.g., a lipid- containing nanoparticle can include one or more of the lipids in Table 4 of WO2019217941.
  • Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941, incorporated by reference.
  • conjugated lipids when present, can include one or more of PEG- diacylglycerol (DAG) (such as l-(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-glycerol (PEG
  • sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in WO2009/127060 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)) described herein includes,
  • an LNP including Formula (i) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • an LNP including Formula (ii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • an LNP including Formula (iii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • an LNP including Formula (ix) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • 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
  • R 2 taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X 2 form a 4-, 5-, or 6-membered ring
  • 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
  • an LNP including Formula (xii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • an LNP including Formula (xi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • 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) composition described herein to cells.
  • an LNP including a formulation of Formula (xvi) is used to deliver a polyribonucleotide (e.g., a circular 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)) described herein is made by one of the following reactions:
  • an LNP including Formula (xxi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • the LNP of Formula (xxi) is an LNP described by WO2021113777 (e.g., a lipid of Formula (1) such as a lipid of Table 1 of WO2021113777).
  • n is independently an integer from 2-15
  • L 1 and L 3 are each independently -OC(O)-* or - C(O)O-*, wherein “*” indicates the attachment point to R 1 or R 3
  • R 1 and R 3 are each independently a linear or branched C 9 -C 20 alkyl or C 9 -C 20 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, (heter
  • an LNP including Formula (xxii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • the LNP of Formula (xxii) is an LNP described by WO2021113777 (e.g., a lipid of Formula (2) such as a lipid of Table 2 of WO2021113777). wherein each n is independently an integer from 1-15; R 1 and R2 are each independently selected from a group consisting of:
  • R 3 is selected from a group consisting of:
  • an LNP including Formula (xxiii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
  • the LNP of Formula (xxiii) is an LNP described by WO2021113777 (e.g., a lipid of Formula (3) such as a lipid of wherein X is selected from -O-, -S-, or -OC(O)-*, wherein * indicates the attachment point to R 1 ; R 1 is selected from a group consisting of: and R 2 is selected from a group consisting of:
  • a composition described herein e.g., a nucleic acid (e.g., a circular 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 US9,867,888 (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 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 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-lH- cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020/106946 (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 from 40 to 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA (e.g., a circular 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 WO2019051289, incorporated herein by reference.
  • Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016/0311759; I of US20150376115 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-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 US2017/0119904; I or II of WO2017/117528; A of US2012/0149894; A of US2015/0057373; A of WO2013/116126; A of US2013/0090372; A of US2013/0274523; A of US2013/0274504; A of US2013/0053572; A of W02013/016058; A of W02012/162210; I of US2008/042973
  • Exemplary lipids further include a lipid of any one of Tables 1-16 of WO2021/113777. Further exemplary lipids are described in International Patent Publication Nos. WO2023/183616, WO2023/091490, WO2023/091787, and WO2024/049979, which are incorporated herein by reference in their entirety,
  • the ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28,3 l- tetraen-l9-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3- nonyldocosa-l3, l6-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (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 stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • non-cationic lipids are described in WO2017/099823 or US patent publication US2018/0028664, the contents of which is incorporated herein by reference in their entirety.
  • 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. In some embodiments, 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 sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof.
  • Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53-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)-buty1 ether.
  • the component providing membrane integrity such as a sterol
  • the component providing membrane integrity can include 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle.
  • 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.
  • 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 l-(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-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl- methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero
  • exemplary PEG-lipid conjugates are described, for example, in US5,885,6l3, US6,287,59l, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, the contents of all of which are incorporated herein by reference in their entirety.
  • a PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-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 (l-[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)-2000].
  • the PEG-lipid includes PEG-DMG, 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000].
  • 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.
  • 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 WO2019051289A9, 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.
  • PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle.
  • 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% non-cationic 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 composition
  • 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 / non-cationic- lipid / sterol / 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.
  • one or more additional compounds can also be included. Those compounds can be administered separately, or the additional compounds can be included in the lipid nanoparticles of the invention.
  • the lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first.
  • additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.
  • the LNPs include biodegradable, ionizable lipids.
  • the LNPs include (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate, also called 3- ((4,4- bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,l2Z)-octadeca- 9,l2-dienoate) or another ionizable lipid.
  • the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.
  • the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about l mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.
  • a LNP may, in some instances, be relatively homogenous.
  • a polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles.
  • a small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.
  • a LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.
  • the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about 0 mV to about +20 mV, from
  • the efficiency of encapsulation of a protein and/or nucleic acid describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided.
  • the encapsulation efficiency is desirably high (e.g., close to 100%).
  • the encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents.
  • An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution.
  • Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution.
  • the encapsulation efficiency of a protein and/or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the encapsulation efficiency may be at least 80%.
  • the encapsulation efficiency may be at least 90%.
  • the encapsulation efficiency may be at least 95%.
  • a LNP may optionally include one or more coatings.
  • a LNP may be formulated in a capsule, film, or table having a coating.
  • a capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density. Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020/061457, WO2021/113777, and WO2021226597, each of which is incorporated herein by reference in its entirety. Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021).
  • in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio).
  • LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems).
  • LNPs are formulated using 2,2 ⁇ dilinoleyl ⁇ 4 ⁇ dimethylaminoethyl ⁇ [1,3] ⁇ dioxolane (DLin ⁇ KC2 ⁇ DMA) or dilinoleylmethyl ⁇ 4 ⁇ dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.
  • LNP formulations optimized for the delivery of CRISPR-Cas systems e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA
  • WO2019067992 and WO2019067910 are described in WO2019067992 and WO2019067910, both incorporated by reference, and are useful for delivery of circular polyribonucleotides and linear polyribonucleotides described herein.
  • Additional specific LNP formulations useful for delivery of nucleic acids are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO.
  • a polyribonucleotide encoding at least a portion of a polypeptide described herein is formulated in an LNP, wherein: (a) the LNPs include a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, and (b) the LNPs have a mean particle size of from 80 nm to 160 nm.
  • Exemplary dosing of polyribonucleotide (e.g., a circular polyribonucleotide) LNP may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg/kg (RNA).
  • a dose of a polyribonucleotide composition described herein is from 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg.
  • Kits the disclosure provides a kit.
  • the kit includes (a) a circular polyribonucleotide complexed with a targeting moiety or a pharmaceutical composition described herein, and optionally (b) informational material.
  • the informational material may be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the pharmaceutical composition or circular polyribonucleotide for the methods described herein.
  • the kit may include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, a flavoring agent (e.g., a bitter antagonist or a sweetener), a fragrance, a dye or coloring agent, for example, to tint or color one or more components in the kit, or other cosmetic ingredient, and/or a second agent for treating a condition or disorder described herein.
  • the other ingredients may be included in the kit, but in different compositions or containers than a pharmaceutical composition or circular polyribonucleotide described herein.
  • a dosage form of a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein may be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein be substantially pure and/or sterile.
  • a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • a pharmaceutical composition or nucleic acid molecule e.g., a circular polyribonucleotide
  • a suitable solvent e.g., sterile water or buffer
  • the kit may include one or more containers for the composition containing a dosage form described herein.
  • the kit contains separate containers, dividers or compartments for the composition and informational material.
  • the pharmaceutical composition or circular polyribonucleotide may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or packet.
  • the separate elements of the kit are contained within a single, undivided container.
  • the dosage form of a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms of a pharmaceutical composition or circular polyribonucleotide described herein.
  • the subject is a non-human mammal is 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 mollusk.
  • an arthropod e.g., insects, arachnids, crustaceans
  • a nematode e.g., an annelid, a helminth, or a mollusk.
  • the disclosure provides a method of modifying a subject by providing to the subject a composition or formulation described herein.
  • the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject.
  • the composition or formulation is or includes or a eukaryotic or prokaryotic cell including a nucleic acid described herein.
  • the disclosure provides a method of treating a disease or condition in a subject in need thereof by providing to the subject a composition or formulation described herein.
  • the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject.
  • the composition or formulation is or includes a eukaryotic or prokaryotic cell including a nucleic acid described herein.
  • the polyribonucleotide is provided in an amount and for a duration sufficient to treat a disease or condition in a subject, e.g., in need thereof.
  • Methods of Making includes a method of forming covalent attachments between one or more binding moieties and the circular polyribonucleotide upon irradiation with light by way of one or more photoreactive crosslinking agents included within each of the one or more binding moieties, wherein each of the one or more binding moieties is conjugated (e.g., directly, chemically-covalently conjugated, etc.) to one or more targeting moieties.
  • the complex is irradiating with light at the wavelength for 1 to 120 (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes.
  • the method of covalently attaching one or more binding moieties, each including one or more photoreactive crosslinking agents, to the circular polyribonucleotide by irradiating any of the complexes described herein with light further includes a method of removing the covalent attachments between one or more binding moieties, each including one or more photoreactive crosslinking agents, and the circular polyribonucleotide includes irradiating the complex with light at a second wavelength.
  • the second wavelength of the irradiated light for removing the covalent attachment is 300-320 (e.g., 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320) nm.
  • the complex is irradiating with light at the second wavelength for 1 to 120 (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes.
  • the complex is irradiated prior to administration to a cell or tissue.
  • a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety includes parenterally administering the circular polyribonucleotide or composition thereof to a subject, wherein the circular polyribonucleotide is a translation incompetent circular polyribonucleotide.
  • a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety includes parenterally administering the circular polyribonucleotide or composition thereof to a subject, wherein the complex is irradiated prior to administration to the subject.
  • a method of in vivo delivery as described herein is a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety or composition thereof to a cell or tissue of a subject including parenterally administering to the cell or tissue the circular polyribonucleotide complexed with a targeting moiety or composition thereof, wherein the circular polyribonucleotide is a translation incompetent circular polyribonucleotide.
  • a method of in vivo delivery as described herein is a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety or composition thereof to a cell or tissue of a subject including parenterally administering to the cell or tissue the circular polyribonucleotide complexed with a targeting moiety or composition thereof, wherein the complex is irradiated prior to administration to the cell or tissue.
  • a method of in vivo delivery as described herein is a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety or composition thereof to a cell or tissue of a subject including parenterally administering to the cell or tissue the circular polyribonucleotide complexed with a targeting moiety or composition thereof, wherein the complex is irradiated after administration to the cell or tissue.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is neonatal administration. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is postnatal administration. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is oral. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intravenous injection.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration. In some embodiments, the compositions are parenterally administered and include a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is prenatal administration and includes a carrier.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is neonatal administration and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is postnatal administration and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is oral and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by injection and includes a carrier.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration and includes a carrier.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraperitoneal injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intradermal injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection and includes a diluent.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is oral and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intravenous injection and includes a parenterally acceptable diluent.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration and includes a parenterally acceptable diluent.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration and includes a parenterally acceptable diluent.
  • the compositions are parenterally administered and lack a carrier.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is prenatal administration and lacks a carrier.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is neonatal administration and lacks a carrier.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraarterial injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraperitoneal injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intradermal injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection and lacks a carrier.
  • the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration and lacks a carrier.
  • a use of a circular polyribonucleotide complexed with a targeting moiety in the manufacture of a parenteral composition is for delivering to a cell or tissue of a subject.
  • the parenteral composition is formulated for intravenous, intramuscular, ophthalmical or topical administration.
  • the parenteral composition is a pharmaceutical composition further including a pharmaceutically acceptable excipient.
  • the parenteral composition includes a carrier.
  • the parenteral composition includes a parenterally acceptable diluent and is free of any carrier.
  • the circular polyribonucleotide complexed with a targeting moiety in the manufacture of a paternal composition is irradiated with light prior to delivery.
  • the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition forms a complex with the target and the circular polyribonucleotide or the target is detectable at least 5 days after delivery.
  • the circular polyribonucleotide complexed with a targeting moiety is detectable at least 7, 8, 9, or 10 days after delivery.
  • the circular polyribonucleotide complexed with a targeting moiety is detectable 7 days after delivery.
  • the circular polyribonucleotide complexed with a targeting moiety is detectable 8 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety is detectable 9 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety is detectable 10 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition is present at least five days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present at least 6, 7, 8, 9, or 10 days after delivery.
  • the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 6 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 7 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 8 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 9 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 10 days after delivery.
  • the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition is an unmodified circular polyribonucleotide. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition has a quasi-double-stranded secondary structure. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition lacks a poly-A sequence, lacks a replication element, lacks a free 3’ end, or lacks an RNA polymerase recognition motif, or any combination thereof. In some embodiments, the circular polyribonucleotide of the parenteral composition lacks a poly-A sequence.
  • the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition lacks a replication element. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition lacks a free 3’ end. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition lacks an RNA polymerase recognition motif. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition is a translation incompetent circular polyribonucleotide.
  • the circular polyribonucleotide complexed with a targeting moiety further includes an expression sequence.
  • the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition includes a termination element or an IRES, or the combination thereof.
  • the polypeptide, when expressed in the cell is functional. Upon delivery of the circular polyribonucleotide to a cell by the targeting moiety complexed to the circular polyribonucleotide, the polypeptide encoded by the circular polyribonucleotide is expressed and folded resulting in a polypeptide capable of performing a function and/or therapeutic use.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a livestock cell. In some embodiments, the tissue is a connective tissue. In some embodiments, the tissue is a muscle tissue. In some embodiments, the tissue is a nervous tissue. In some embodiments, the tissue is an epithelial tissue. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a pet. In some embodiments, the subject is a live-stock animal.
  • the parenteral nucleic acid delivery system as disclosed herein (e.g., the nucleic acid compositions in the methods of delivery as described above) is used as a medicament or a pharmaceutical.
  • a parenteral nucleic acid delivery system as disclosed herein can be used in a method of treatment of a human or animal body by therapy.
  • a parenteral nucleic acid delivery system as disclosed herein can be used in the manufacture of a medicament or a pharmaceutical.
  • a parenteral nucleic acid delivery system as disclosed herein can be used in the manufacture of a medicament or a pharmaceutical for treating a human or animal body by therapy.
  • a method of dosing to produce a level of circular complexed with a targeting moiety in a cell after providing the cell with at least two doses or compositions of circular polyribonucleotide is disclosed herein.
  • a method of dosing to produce a level of circular polyribonucleotide complexed with a targeting moiety in a subject e.g., a mammal, e.g., a human
  • the composition includes a circular polyribonucleotide complexed with a targeting moiety as described herein.
  • the methods described herein may include administering a first dose of the pharmaceutical composition in an amount sufficient to produce a serum concentration of at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL, or more) of a polypeptide in the subject.
  • a subsequent dose is administered before the serum concentration drops below 500 ng/mL of a polypeptide in the subject.
  • multiple doses are provided to produce a level of the composition or express a level of the polypeptide in a cell, tissue or subject.
  • multiple doses are provided to produce or maintain a level of the composition, or to produce or maintain a level of the 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 second dose is administered at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer) after the first dose of the pharmaceutical composition.
  • at least one hour e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days,
  • the second dose is administered from 1 hour to 1 year (e.g., from 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day to one week, e.g., two days, three days, four days, five days, six days, or one week, e.g., from one week to one month, e.g., two weeks, three weeks, or one month, e.g., from one month to one year, e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year) after the first dose of the pharmaceutical composition.
  • 1 hour to 1 day e.g., one hour, two hours
  • the second dose is administered from 1 days to 180 days (e.g., from 1 day to 90 days, from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to 7 days, from 2 days to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days, from 3 days to 90 days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3 days to 7 days, from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days to 14 days, from 4 days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days, from 5 days to 14 days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6 days to 30 days, from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to 45 days, from 7 days to 30 days, from 7 days to 14 days, from 14 days, from 6
  • the third dose is administered at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer) after the second dose of the pharmaceutical composition.
  • at least one hour e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days,
  • the third dose is administered from 1 hour to 1 year (e.g., from 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day to one week, e.g., two days, three days, four days, five days, six days, or one week, e.g., from one week to one month, e.g., two weeks, three weeks, or one month, e.g., from one month to one year, e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year) after the second dose of the pharmaceutical composition.
  • 1 hour to 1 day e.g., one hour, two hours
  • the third dose is administered from 1 days to 180 days (e.g., from 1 day to 90 days, from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to 7 days, from 2 days to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days, from 3 days to 90 days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3 days to 7 days, from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days to 14 days, from 4 days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days, from 5 days to 14 days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6 days to 30 days, from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to 45 days, from 7 days to 30 days, from 7 days to 14 days, from 14 days, from 6
  • the second dose is administered before a serum concentration of an polypeptide is less than about 500 ng/mL in serum of the subject.
  • the method maintains a serum concentration of at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL,
  • sequentially administering means that each dose of composition of a nucleic acid molecule described herein is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • a predetermined interval e.g., hours, days, weeks or months.
  • the present invention provides methods which include sequentially administering to the subject a single initial dose of a composition of a nucleic acid molecule described herein, followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition.
  • initial dose refers to the temporal sequence of administration of a composition of a nucleic acid molecule described herein.
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen;
  • the “secondary doses” are the doses which are administered after the initial dose;
  • 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 nucleic acid molecule described herein, and in certain embodiments, may differ from one another in terms of frequency of administration.
  • the amount of a composition of a nucleic acid molecule described herein 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 immediately preceding dose means, in a sequence of multiple administrations, the dose of the composition of a nucleic acid molecule described herein 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 include administering to a subject any number of secondary and/or tertiary doses of a composition of a nucleic acid molecule described herein.
  • a subject any number of secondary and/or tertiary doses of a composition of a nucleic acid molecule described herein.
  • 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 administered to the subject.
  • 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.
  • the method includes 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 includes 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.
  • 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 includes a first amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein.
  • a second composition in a multiple dosing regimen includes a second amount of the nucleic acid molecule (e.g., circular 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 includes a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein.
  • the second amount of the nucleic acid molecule e.g., circular polyribonucleotide
  • the second amount of the nucleic acid molecule is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the third amount of the nucleic acid molecule is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the second amount of the nucleic acid molecule is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the third amount of the nucleic acid molecule is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the second amount of the nucleic acid molecule is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the third amount of the nucleic acid molecule is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition varies by no more than 1%, 5%, 10%, 15%, 20%, or 25% of an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition is no more than 1%, 5%, 10%, 15%, 20%, or 25% less than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1-fold to 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular 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 nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular 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 nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1-fold to 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular 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 nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular 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 nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition is from 0.1-fold to 1000-fold higher or lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular 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 nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • a first composition includes 1-fold nucleic acid molecule (e.g., circular polyribonucleotide), a second composition includes 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition, and a third composition includes 0.2-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition.
  • the second composition includes at least 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to an amount of nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • the first composition includes a higher amount of the nucleic acid molecule (e.g., circular polyribonucleotide) than the second composition. In some embodiments, the first composition includes a higher amount of the nucleic acid molecules (e.g., circular polyribonucleotides) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition. In some embodiments, the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding a polypeptide, which are administered in a multiple dosing regimen as described herein, are the same compositions.
  • the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding a polypeptide, which are administered in a multiple dosing regimen as described herein, are different compositions.
  • the same compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding the same polypeptide.
  • the different compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding different polypeptides, or a combination thereof.
  • the method of administering the nucleic acid molecule includes administering to a subject in need thereof the nucleic acid molecule for multiple times (multiple doses), e.g., at least 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 nucleic acid molecule for at least 3 times, with an interval of about 7 days.
  • a level of the polypeptide e.g., a plasma polypeptide
  • a level of the 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 polypeptide e.g., a plasma polypeptide level
  • a level of the 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 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 polypeptide e.g., a plasma polypeptide level
  • a level of the 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 polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).
  • Example 1 Complexation of one or more carbohydrates to a circular polyribonucleotide This example demonstrates the complexation of a carbohydrate targeting moiety to a circular polyribonucleotide. A non-naturally occurring circular RNA complexed with a carbohydrate targeting moiety was produced. As shown in the following example, a trivalent GalNAc moiety was complexed to a circular RNA.
  • the oligomer conjugated to the triethylene glycol (TEG) linker conjugated to the trivalent GalNAc moiety was then annealed to the circular polyribonucleotide in a thermocycler by heating at 75°C for 2 minutes, cooling to 25°C for 2 minutes, and repeating one time.
  • the resulting circular polyribonucleotide complexed to a GalNAc targeting moiety was stored at room temperature. These conditions resulted in the circular polyribonucleotide as described in the schematic in FIG.3.
  • Example 2 Example 2.
  • the oligomer conjugated to each targeting moiety by way of a linker was annealed to the region of the circular polyribonucleotide containing the portion of the 5’ exon fragment and 3’ exon fragment remaining after circularization of the polyribonucleotide (E1/E2) or just the 5’ exon fragment remaining after circularization (E1).
  • a circular polyribonucleotide complexed to one or more of: (a) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to E1 by way of a TEG linker (A); (b) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to E1/E2 by way of a TEG linker (B); (c) an oligomer conjugated to a GalNAc targeting moiety at the 5’ end of the oligomer bound to E1/E2 by way of a TEG linker (C); (d) an oligomer conjugated to a cholesterol targeting moiety at the 3’ end of the oligomer bound to E1/E2 by way of a TEG linker (I); (e) an oligomer conjugated to a cholesterol targeting moiety at the 3’ end of the oligomer bound to E1 by way of a TEG linker (A); (b
  • the oligomer conjugated to the circular polyribonucleotide had to be annealed to the circular polyribonucleotide in order for there to be some enhancement of expression. Furthermore, it was observed that certain combinations of targeting moieties led to enhanced expression in comparison to complexes having only one targeting moiety. Various linker lengths were also tested for oligomers that were annealed to the ORF to identify if a longer linker length was required for enhanced expression of the polypeptide encoded by the circular polyribonucleotide.
  • a circularized polyribonucleotide encoding a luciferase polypeptide was complexed to an oligomer conjugated to a TEG linker or PEG24 linker which was conjugated to a targeting moiety of a GalNAc moiety as described in Example 1.
  • the oligomer conjugated to each targeting moiety by way of a linker was annealed to the ORF, which encoded a luciferase polypeptide.
  • a circular polyribonucleotide complexed to one or more of: (a) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to ORF by way of a TEG linker (FA and FB); (b) an oligomer conjugated to a GalNAc targeting moiety at the 5’ end of the oligomer bound to ORF by way of a TEG linker (FC and FD); or (c) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to ORF by way of a PEG24 linker (FE24).
  • FE24 PEG24 linker
  • a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with one or more of: (a) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to E1 by way of a TEG linker (B); (b) an oligomer conjugated to a GalNAc targeting moiety at the 5’ end of the oligomer bound to E1/E2 by way of a TEG linker (C); (c) an oligomer conjugated to a cholesterol targeting moiety at the 3’ end of the oligomer bound to E1/E2 by way of a TEG linker (I); (d) an oligomer conjugated to a by way of a linker (FA, FB, FC, and FD), or (d) an oligomer conjugated to a GalNAc moiety by way of specifically a PEG24 linker (FE24) using the methods described in Example 1.
  • a ribonuclease inhibitor and/or CaCl2 were then added to the solution including the complex including the circular polyribonucleotide and the targeting moiety.
  • the resulting mixture was subsequently transfected into mouse hepatocyte cells, incubated, and luminescence was measured as described in Example 2 to understand the effect of the delivery agents on the polypeptide expression (FIG.6 and FIG.7).
  • Albumin This experiment demonstrates the effect of albumin on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with multiple targeting agents.
  • a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer conjugated to a GalNAc moiety by way of a linker and an oligomer conjugated to tocopherol by way of a linker (BQ), as described in Example 1.
  • Bovine serum albumin (BSA) was added in a 1:1 ratio to the circular RNA-targeting moiety complex, and the resulting mixture was subsequently transfected into mouse hepatocyte cells, incubated, and subsequently the luminescence was measured as described in Example 2.
  • BSA enhanced expression of a polypeptide encoded on the circular polyribonucleotide complexed with the two targeting moieties in comparison to the circular polyribonucleotide complexed with the two targeting moieties that were not mixed with BSA.
  • Endosomal Escape Agent in Combination with Calcium Ions and/or Ribonuclease Inhibitors demonstrate the effect of the endosomal escape agent chloroquine alone and in combination with CaCl2 on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with multiple targeting agents.
  • a circular polyribonucleotide encoding luciferase was complexed with an oligomer conjugated to a GalNAc moiety by way of a linker and an oligomer conjugated to tocopherol by way of a linker (BQ), as described in Example 1.
  • Chloroquine at a concentration of 60 ⁇ M, CaCl2 at a concentration of 20 mM, and/or a ribonuclease inhibitor at a concentration of 0.29 U/mL were added to the solution including the complex including the circular polyribonucleotide and the targeting moiety.
  • This enhanced expression was even further enhanced by addition of CaCl2 and/or ribonuclease inhibitor.
  • Delivery Agents In Vivo This experiment demonstrates the effect of various delivery agents have on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with a cholesterol targeting moiety in a mouse. In a first experiment, the effect of the presence of an endosomal escape agent and/or calcium ion on polypeptide expression was studied in vivo.
  • a circular polyribonucleotide encoding a luciferase polypeptide was complexed with an oligomer conjugated to cholesterol by way of a linker (J), as described in Example 1, and was subsequently mixed with CaCl2 at a concentration of 1.8 mM or 20 mM and/or 60 ⁇ M chloroquine.
  • the resulting mixtures were injected into CD1 mice intradermally, and the mice were imaged to detect polypeptide expression after 5 hours. Results of this experiments are provided in FIG 12B. This experiment demonstrated that in vivo the combination of 20 mM CaCl2 and chloroquine resulted in the most enhanced expression.
  • RNA nanoparticles 75 ⁇ g of the complex was mixed with 1 U/ ⁇ L of a ribonuclease inhibitor (e.g., RNasin® ribonuclease inhibitor), CaCl2 at a final concentration of 1.8 mM, chloroquine at a final concentration of 600 ⁇ M, mouse serum albumin (MSerumAlbumin) at a ratio of 1:3 complex to BSA, 25 ⁇ g of lipid nanoparticles (LNP), or a combination thereof as indicated in FIG.15.
  • a ribonuclease inhibitor e.g., RNasin® ribonuclease inhibitor
  • CaCl2 a final concentration of 1.8 mM
  • chloroquine at a final concentration of 600 ⁇ M
  • MSerumAlbumin mouse serum albumin
  • LNP lipid nanoparticles
  • mice were injected intraperitoneally with d-luciferin over a period of 29 days, and the whole body of the mouse was imaged for luminescence at the indicated time points to assess expression of the polypeptide encoded by the circular polyribonucleotide (FIG.15).
  • ribonuclease inhibitor, CaCl2, and chloroquine singly and in combination with BSA, affected polypeptide expression.
  • Example 4 ribonuclease inhibitor, CaCl2, and chloroquine singly and in combination with BSA, affected polypeptide expression.
  • a circular polyribonucleotide encoding a luciferase polypeptide was complexed with: (a) an oligomer conjugated to cholesterol by way of a linker (J and I), (b) an oligomer conjugated to a GalNAc moiety by way of a linker (A), (c) an oligomer conjugated to tocopherol by way of a linker (Q), (d) an oligomer conjugated to cholesterol by way of a linker and an oligomer conjugated to tocopherol by way of a linker (IQ and JQ), (d) two oligomers that each were conjugated to cholesterol by way of a linker (IJ), or (e) two oligomers that each were conjugated to tocopherol by way of a linker (QR), as described in Example 1.
  • a circular polyribonucleotide encoding a luciferase polypeptide was complexed with: (a) an oligomer conjugated to cholesterol by way of a linker and an oligomer conjugated to tocopherol (Chol+Toco), or (b) an oligomer conjugated by way of linker to an aptamer (Aptamer 7), as described in Example 1, in order to compare the resulting polypeptide expression to a circular polyribonucleotide that is not complexed with a targeting moiety but is administered with lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer conjugated to cholesterol by way of a linker as described in Example 1.
  • CaCl2 was added after the annealing and cooling step to the solution including the complex at a final concentration of 0.3 mM, 0.9 mM, 2.7 mM, 8.0 mM, 10 mM, or 20 mM.
  • the resulting mixture was subsequently transfected into mouse hepatocyte cells, incubated, and expression of the polypeptide was measured as described in Example 2. Results of this experiment are shown in FIG.11A.
  • Example 6 Use of various targeting moieties for the delivery of a circular polyribonucleotide encoding a polypeptide in vivo over time This example demonstrates the effect of the use of various targeting ligands on the in vivo delivery of a circular polyribonucleotide encoding a polypeptide as monitored by expression of the polypeptide in vivo over time.
  • Biotinylated mannose bound to avidin, cholesterol, and tocopherol A circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed to an oligomer conjugated to tocopherol by way of a linker and an oligomer conjugated to cholesterol by way of a linker, or was complexed with an oligomer conjugated to a linker which was conjugated to an avidin molecule which was complexed to one or two biotinylated mannose residues bound to an avidin molecule.
  • the complexes including the circular polyribonucleotide and tocopherol and cholesterol targeting moieties were generated as described in Example 1.
  • the complexes including the circular polyribonucleotide and the avidin bound to the biotinylated mannose were generated by mixing 1 pmol of circular polyribonucleotide and 1 pmol of a biotinylated oligomer in HEPES buffered saline. The oligomer was then annealed to the circular polyribonucleotide in a thermocycler by heating at 75 °C for 2 minutes, cooling to 25 °C for 2 minutes, and repeating one time . To this mixture, biotinylated mannose was added and mixed and then avidin was added in a 1:1 ratio with the circular polyribonucleotide.
  • the solution was then left to equilibrate for 15 minutes at 20 o C.
  • the circular RNA-targeting moiety complexes were subsequently injected intramuscularly into C57BL/J6 mice and imaged to assess polypeptide expression after 4-5 hours, 24 hours, and 72 hours from the dorsal view or the ventral view. Results of these experiments are shown in FIG.14A (dorsal view) and FIG.14B (ventral view). These experiments demonstrated that over time, the circular polyribonucleotide complexed to the targeting moiety had greater expression over time of the polyribonucleotide cargo in comparison to LNP’s which showed reduced expression of the polyribonucleotide cargo over time.
  • Targeting Agents used aptamer, cholesterol, and tocopherol
  • eRNA luciferase polypeptide
  • SARS-CoV-2 polypeptide in vivo encoded by circular polyribonucleotides complexed to a targeting moiety
  • a circular polyribonucleotide encoding a SARS-CoV-2 RBD polypeptide was complexed with: (a) an oligomer bound to cholesterol by way of a TEG linker; or (b) an oligomer bound to an avidin, by way of a biotinylated TEG linker, bound to three biotinylated mannose molecules; using the methods described in Examples 1 and 6.
  • Chloroquine was added after the annealing and cooling step to the solution including the complex.
  • the resulting mixture was injected intradermally into CD1 mice.
  • serum was harvested from the mice and subsequently expression of the RBD polypeptide in the serum was measured. Results of this experiment are shown in FIG.13.
  • the amount of RBD measured in circulating serum was greater when a targeting moiety was complexed with the circular polyribonucleotide in comparison to when the circular polyribonucleotide was not complexed with a targeting moiety.
  • the amount of RBD measured in the serum was greatest when the avidin bound to the biotinylated mannose was used as the targeting moiety.
  • the biotinylated DEC205 antibody was mixed with the circular polyribonucleotide in a ratio of either 1:3 (DEC205 Ab 1:3) or 1:6 (DEC205 Ab 1:6).
  • Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to biotinylated DEC205 aptamer using the methods described in Example 1.
  • the biotinylated DEC205 aptamer was mixed with the circular polyribonucleotide in a ratio of either 1:3 (DEC205 APT 1:3) or 1:6 (DEC205 APT 1:6).
  • Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated Transferrin R (Trf) aptamer using the methods described in Example 1.
  • the biotinylated DEC205 antibody and biotinylated Trf aptamer were mixed in a 1:1 ratio with each other and in a ratio of one circular polyribonucleotide to three targeting moieties (DEC205 Ab+Trf APT 3:3).
  • Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 aptamer and biotinylated Transferrin R (Trf) aptamer using the methods described in Example 1.
  • the biotinylated DEC205 aptamer and biotinylated Trf aptamer were mixed in a 1:1 ratio with each other and in a ratio of one circular polyribonucleotide (eRNA) to three targeting moieties (DEC205 APT+Trf APT 3:3).
  • Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated phosphatidylserine (PS) using the methods described in Example 1.
  • the biotinylated DEC205 antibody and biotinylated PS were mixed in a 1:1 ratio with each other and in a ratio of one circular polyribonucleotide (eRNA) to three targeting moieties (DEC205 Ab+PS 3:3).
  • Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to biotinylated PS using the methods described in Example 1.
  • biotinylated PS and the circular polyribonucleotide were mixed in a ratio of 1:6 circular polyribonucleotide to PS (PS 1:6) or 1:3 circular polyribonucleotide to PS (PS 1:3).
  • an avidin-conjugated FITC was added to the mixture.
  • the circular RNA complexes were added to plated RAW264.7 macrophages, internalized for 4 hours, and then cells were removed and analyzed via flow cytometry in order to measure the percent of cells that internalized the circular polyribonucleotide (FIG.17A), the mean amount of cells that internalized the circular polyribonucleotide (FIG.17B), and specifically the mean amount of cells that internalized the complexes having both the DEC205 antibody and PS targeting moieties, in comparison to a lipofectamine control (MM+Av).
  • Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated PS as described in Example 8. Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated mannose using the methods described in Example 1. Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated DEC205 antibody using the methods described in Example 1.
  • Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated mannose using the methods described in Example 1.
  • Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated Trf aptamer using the methods described in Example 1.
  • Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated PS as described in Example 8.
  • Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated Trf antibody using the methods described in Example 1.
  • the biotinylated Trf antibody was mixed in a ratio of 1 circular polyribonucleotide (eRNA) to 6 targeting moieties (Trf Ab 1:6).
  • the circular RNA complexes were added to RAW264.7 macrophages plated onto #1.5 thickness glass coverslips and incubated for 4 hours before washing cells with Hank’s Buffered Saline solution containing Hoechst 33342 nuclear stain. Live cells were analyzed by confocal fluorescence microscopy which showed that the circular polyribonucleotide annealed to the targeting moiety was successfully internalized by the cells into the endosomes of the cells.
  • Example 10 The effect of using avidin to bind a biotinylated targeting moiety to conjugate the targeting moiety to the moiety that binds the circular polyribonucleotide on polypeptide expression This example demonstrates using an avidin to bind a biotinylated targeting ligand and conjugate it to the moiety that binds the circular polyribonucleotide encoding a polypeptide modestly affects the expression of the polypeptide.
  • a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated Transferrin R (Trf) aptamer as described in Example 8.
  • Another circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated PS as described in Example 8.
  • eRNA luciferase polypeptide
  • eRNA luciferase polypeptide
  • eRNA luciferase polypeptide
  • Another circular polyribonucleotide encoding a luciferase polypeptide was complexed with an oligomer bound to an avidin bound to biotinylated mannose using the methods described in Example 1, wherein the mannose and circular polyribonucleotide where mixed in a ratio of 1 circular polyribonucleotide to 3 mannose molecules.
  • Another circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer bound to cholesterol by way of a linker and an oligomer bound to tocopherol by way of a linker using the methods described in Example 1.
  • the circular polyribonucleotide complexes were added to RAW264.7 macrophage cells and incubated for 48 hours.
  • the complex including the DEC205 antibody as the targeting moiety (DEC205 Ab) and the complex including mannose were also administered in the presence of chloroquine (CQ).
  • Luminescence assays were performed using a PierceTM Firefly Luciferase Flash Assay Kit (ThermoFisher) to assess the amount of protein expression. Results of this experiment are shown in FIG. 19. Addition of the chloroquine to the complex mixtures resulted in increased polypeptide expression.
  • a circular polyribonucleotide encoding a luciferase polypeptide was complexed to: (a) two biotinylated oligomers, (b) two biotinylated oligomers that were each independently bound to and avidin bound to biotinylated Trf antibodies; or (c) two biotinylated oligomers that were each independently bound to and avidin bound to biotinylated DEC205 antibodies.
  • the circular polyribonucleotide complexes were added to HeLa cells and incubated for 24 hours.
  • Luminescence assays were performed using a PierceTM Firefly Luciferase Flash Assay Kit (ThermoFisher) to assess the amount of protein expression. Results of this experiment are provided in FIG.20.
  • the use of an avidin in the complex modestly affected the expression of the polypeptide.
  • the ability for a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) complexed to a biotinylated oligomer bound to avidin bound to one or more biotinylated Trf antibodies to be internalized by the cell and result in polypeptide expression was studied.
  • Circular polyribonucleotide complexes were prepared with complexes to: (a) a biotinylated oligomer bound to avidin, (b) a biotinylated oligomer bound to avidin bound to a singular biotinylated Trf antibody, and (c) a biotinylated oligomer bound to avidin bound to three biotinylated Trf antibodies.
  • Each of these complexes were transfected into HeLa cells and the resulting fluorescence was assessed using confocal imaging. The results showed that the presence of the Trf antibody as a targeting moiety resulted in enhanced internalization of the circular polyribonucleotide in comparison the complex lacking the Trf antibody.
  • a circular polyribonucleotide encoding a luciferase polypeptide was complexed with a biotinylated oligomer which was bound to an avidin.
  • the complex was then mixed in with a biotinylated Trf antibody in a ratio of 1:1, 1:3, or 1:6 circular polyribonucleotide to Trf antibody to generate the circular polyribonucleotide targeting moiety complex.
  • the resulting complex was transfected into HeLa cells in the presence of calcium and in either the presence or absence of 100x molar excess of transferrin. The resulting luminescence in the cells was measured to assess the polypeptide expression Results of this experiment are provided in FIG.21.
  • Example 11 Covalent attachment of an oligomer binding moiety to complementary, linear RNA by way of a photoreactive crosslinking agent This example demonstrates the covalent attachment of complementary, linear RNA to an oligomer binding moiety by way of a photoreactive crosslinking agent included within the binding moiety.
  • the binding moiety had 96% complementarity to the complement, linear oligomer termed oligomimic.
  • the sequence name, ID number, sequence, linker, position, and target moiety for each binding moiety used in this and the following Examples are listed in Table 3. Table 3. Binding Moiety Name, Sequence, Linker, and Target Moiety Used in Examples
  • E1_cnvK-biotin 10 ⁇ M was annealed to 10 ⁇ M of the oligomimic in a thermocycler by heating at 75°C for 2 minutes, cooling to 25°C for 2 minutes, and repeated one time.
  • the annealing buffer was HEPES buffered saline 1x (HBS) containing 5 mM MgCl2 and 280 mM NaCl.
  • HBS HEPES buffered saline 1x
  • E1_cnvK-biotin was annealed to the oligomimic or annealed then transferred to a cuvette and irradiated with light having a wavelength of 366 nm for 20 minutes to covalently attach E1_cnvK-biotin to the oligomimic.
  • the irradiated complex shows a distinct retention time of ⁇ 10 minutes that is between those of the oligomer binding moiety and the oligomimic, indicating that the covalent attachment by way of irradiation stabilized the complex at conditions that denatured the annealed complex.
  • Example 12 The irradiated complex shows a distinct retention time of ⁇ 10 minutes that is between those of the oligomer binding moiety and the oligomimic, indicating that the covalent attachment by way of irradiation stabilized the complex at conditions that denatured the annealed complex.
  • dfTAT dimeric fluorescent TAT
  • L triethylene glycol linker
  • CHOL-E1_CK and CHOL-E2/E1_CK each include one 3-cyanovinylcarbazole nucleoside ( CNV K) photoreactive crosslinking agent.
  • CHOL-E1_CK includes one Cy5 fluorescent label at the 5’ end
  • CHOL-E2/E1_CK includes one Cy5 fluorescent label at the 3’ end.
  • CHOL-E1_CK is same sequence as Chol J, however, with one CNV K photoreactive crosslinking agent replacing a cytosine nucleotide at an internal position.
  • CHOL-E1_CK had 96% complementarity to just a portion of the 5’ exon fragment remaining after circularization of the polyribonucleotide (E1).
  • CHOL-E2/E1_CK had 96% complementarity to a portion of the 5’ exon fragment and 3’ exon fragment remaining after circularization (E1/E2). Chol J had 100% complementarity to just a portion of the 5’ exon fragment remaining after circularization (E1).
  • the oligomer conjugated to the triethylene glycol (TEG) linker conjugated to the cholesterol moiety was annealed to the circular polyribonucleotide complexed to a dfTAT peptide in a thermocycler by heating at 75°C for 2 minutes, cooling to 25°C for 2 minutes, and repeated one time.
  • the annealing buffer was HBS containing 5 mM MgCl2 and 280 mM NaCl.
  • the resulting circular polyribonucleotide and dfTAT peptide complex further complexed to a cholesterol targeting moiety was stored at room temperature.
  • the resulting circular polyribonucleotide and dfTAT peptide complex further complexed to the cholesterol targeting moiety was then covalently attached to the oligomer by irradiating the complex with light of a wavelength of 366 nm for various lengths of time.0.01 pmol of the complex including the circular polyribonucleotide and the cholesterol targeting moiety was transfected into human embryonic kidney cells and incubated for 24 hours at 37 °C under 5% CO2.
  • a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) and complexed with a dfTAT peptide further complexed CHOL-E1_CK, described earlier in Example 12 as including one CNV K photoreactive crosslinking agent, conjugated to a TEG linker conjugated to a cholesterol targeting moiety by way of: (a) annealing; (b) annealing then irradiating for 1 minute; (c) annealing then irradiating for 5 minutes; or (d) annealing then irradiating for 30 minutes.
  • eRNA luciferase polypeptide
  • CHOL-E1_CK described earlier in Example 12 as including one CNV K photoreactive crosslinking agent, conjugated to a TEG linker conjugated to a cholesterol targeting moiety by way of: (a) annealing; (b) annealing then irradiating for 1 minute; (c) annealing then
  • a luciferase assay was performed to assess polypeptide expression in the cells in comparison to cells transfected with no eRNA complexed with a dfTAT peptide, the eRNA complexed with a dfTAT peptide, the eRNA complexed with a dfTAT peptide further complexed with Chol J conjugated to a TEG linker conjugated to a cholesterol targeting moiety, and the eRNA complexed with a dfTAT peptide delivered with lipofectamine (lipo). All controls were a result of the previously mentioned protocol in Example 12 but were not irradiated. Results of this experiment are shown in FIG.25B.
  • This experiment demonstrates the principle that expression of the polypeptide encoded by the circular polyribonucleotide could be achieved after irradiation to covalently conjugate the oligomer binding moiety to the circular polyribonucleotide via a photoreactive crosslinking agent.
  • This experiment also resulted in enhanced expression of the polypeptide when the complex was annealed then irradiated in comparison to when the complex was only annealed.
  • This experiment also demonstrated enhanced expression of the polypeptide when the complex was annealed then irradiated in comparison to when the complex containing Chol J, described earlier in Example 12 to not contain a photoreactive crosslinking agent, was only annealed.
  • Example 12 the previously mentioned protocol in Example 12 resulted in a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) and complexed with a dfTAT peptide further complexed with CHOL-E2/E1_CK, described earlier in Example 12 as including one CNV K photoreactive crosslinking agent, conjugated to a TEG linker conjugated to a cholesterol target by way of: (a) annealing only; (b) annealing then irradiating for 1 minute; (c) annealing then irradiating for 5 minutes; or (d) annealing then irradiating for 30 minutes.
  • eRNA luciferase polypeptide
  • CHOL-E2/E1_CK CHOL-E2/E1_CK
  • Example 12 After incubation with the cells for 24 hours, a luciferase assay was performed to assess polypeptide expression in the cells and compared to the results of the prior experiment in Example 12 (FIG.25B). Results of this experiment are shown in FIG.25A. This experiment demonstrated enhanced expression occurs after irradiation of the complex when the binding region of the circular polyribonucleotide only contains a portion of E1 rather than a portion of E1/E2. Particularly, this result was achieved when the complexes were irradiated for 30 minutes. Example 13.
  • PS1D and PS2E each have a cholesterol targeting moiety
  • PS1A and PS2B each have a tocopherol targeting moiety.
  • the sequence name, ID number, sequence, position, linker, and target moiety for each binding moiety are listed in Table 3. All oligomers previously listed contain at least one 3- cyanovinylcarbazole nucleoside ( CNV K) photoreactive crosslinking agent except for Chol J.
  • CNV K 3- cyanovinylcarbazole nucleoside
  • PS2E is the same sequence as Chol J, however, with two CNV K photoreactive crosslinking agents each replacing an adenosine nucleotide at an internal position.
  • PS1A is the same sequence as CHOL-E2/E1_CK but without the Cy5 fluorescent label.
  • PS2B is the same sequence as PS1A, however, with a second CNV K photoreactive crosslinking agent replacing an adenosine nucleotide at an internal position.
  • the binding moiety is also Chol J linked via a TEG linker (L) to a tocopherol targeting moiety (Q) or to both a cholesterol and tocopherol targeting moiety.
  • PS1D had 96% complementarity to just a portion of the E1.
  • PS2E had 91% complementarity to just a portion of E1.
  • PS1A had 96% complementarity to a portion of E1/E2.
  • PS2B had 91% complementarity to a portion of E1/E2.
  • the oligomer conjugated to the TEG linker conjugated to the one or more targeting moieties was then annealed to the circular polyribonucleotide in a thermocycler by heating at 75°C for 30 seconds, heating at 75°C for 1 minute and 30 seconds, cooling to 25°C for 2 minutes, and repeating one time.
  • the annealing buffer was HBS containing 5 mM MgCl2 and 280 mM NaCl.
  • the resulting circular polyribonucleotide complexed to the one or more targeting moieties was stored at room temperature.
  • the resulting circular polyribonucleotide complexed to the one or more targeting moieties was then covalently attached to the oligomer by irradiating the complex with light of a wavelength of 366 nm for 30 minutes.1 pmol of the complex including the circular polyribonucleotide and the one or more targeting moieties was transfected into human embryonic kidney cells and incubated for 24 hours at 37 °C under 6% CO2.
  • a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) complexed to a TEG linker (L) and a cholesterol targeting moiety (J) by way of an oligomer binding moiety of one of the following: (a) CHOL-E1_CK, as described in Example 12; (b) PS1D; (c) PS2E; (d) PS1A; or (e) PS2B.
  • a luciferase assay was performed to assess polypeptide expression in the cells in comparison to cells transfected with the eRNA complexed with Chol J complexed to a cholesterol targeting moiety (J) by a TEG linker (L), the eRNA complexed with Chol J complexed to a tocopherol targeting moiety (Q) by a TEG linker (L), and the eRNA complexed with Chol J complexed to both a cholesterol targeting moiety (J) and tocopherol targeting moiety (Q) by a TEG linker (L). All controls were also a result of the previously mentioned protocol in Example 13 but were not irradiated.
  • FIGS.26A and 26B Results of this experiment are shown in FIGS.26A and 26B.
  • This experiment demonstrated the enhanced expression of the polypeptide for a complex containing one or more photoreactive crosslinking agents that was annealed then irradiated is dependent on the binding region of the circular polyribonucleotide, as enhanced expression after irradiation was only achieved for the complex including either CHOL-E1_CK, PS1D, or PS2E that each bind to just a portion of E1.
  • This experiment also resulted in enhanced expression of the polypeptide after irradiation when the complex included PS1D as compared to when the complex included PS2E and when the complex included CHOL- E1_CK.
  • a complex comprising A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5.
  • A is a circular polyribonucleotide
  • each X is independently a moiety that binds specifically to a region of A
  • each L is independently a linker
  • each B is independently a targeting moiety
  • n is an integer from 1 to 20.
  • Xn(L-B)z comprises Xn(L-B1)z and Xn(L- B2)z, where B1 is a first targeting moiety and B2 is a second targeting moiety.
  • Xn(L-B)z comprises Xn(L1-B)z and Xn(L2- B)z, where L1 is a first linker and L2 is a second linker.
  • Xn(L-B)z comprises X1-(L-B)z and X2-(L- B)z, where X1 is a first moiety that binds specifically to a first region of A and X2 is a second moiety that binds specifically to a second region of A.
  • Xn(L-B)z comprises B1-L- X-L-B2, where B1 is a first targeting moiety and B2 is a second targeting moiety.
  • Xn(L-B)z comprises B-L1-X-L2-B, where L1 is a first linker and L2 is a second linker.
  • the targeting moiety comprises a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid, or a combination thereof.
  • the complex of embodiment 12, wherein the targeting moiety comprises a small molecule.
  • the small molecule is selected from folic acid, urea, ⁇ -mannose, high mannose, ursodeoxycholic acid, an endosomal escape agent, or lithocholic acid. 15.
  • the complex of embodiment 12, wherein the targeting moiety comprises a polypeptide.
  • the polypeptide is a cell-penetrating peptide.
  • the polypeptide is selected from ASSLNIA, M12, RGD, melittin, LPS-binding protein (LBP) peptide, an adipose-homing peptide, or an endolytic peptide.
  • LBP LPS-binding protein
  • the complex of embodiment 15, wherein the polypeptide is an antibody or a target-binding fragment thereof. 19.
  • the antibody or target-binding fragment thereof is selected from a monoclonal antibody or target-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, or a tandem scFv (taFv).
  • scFv single-chain Fv molecule
  • the complex of embodiment 18 or embodiment 19, wherein the antibody or target-binding fragment thereof is selected from an anti-FcRn antibody, an anti-MR antibody, an anti-CD205 antibody, an anti-CD169 antibody, an anti-CD14 antibody, an anti-CD36 antibody, an anti-CD5 antibody, an anti - CD71 antibody, an anti-CD38 antibody, or an anti-prohibin antibody.
  • 22. The complex of embodiment 21, wherein the nanobody is selected from an anti-transferrin nanobody, an anti-HER2 nanobody, or an anti-EGFR nanobody.
  • the targeting moiety comprises a carbohydrate. 24.
  • the complex of embodiment 27, wherein the carbohydrate is tri-GalNAc. 29.
  • the complex of embodiment 12, wherein the targeting moiety comprises a lipid.
  • any one of embodiments 30-32, wherein the fatty acid comprises squalene, stearic acid, oleic acid, palmitic acid, linoleic acid, stearic acid, lauric acid, docosahexanoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, ⁇ - tocopherol, ⁇ -tocopherol succinate, or a retinoic acid.
  • DHA docosahexanoic acid
  • DCA docosanoic acid
  • EPA eicosapentaenoic acid
  • octadecanoic acid myristic acid, anadamide, ⁇ - tocopherol, ⁇ -tocopherol succinate, or a retinoic acid.
  • the fatty acid comprises DCA.
  • the fatty acid comprises DHA
  • the complex of embodiment 33, wherein the fatty acid comprises myristic acid. 37. The complex of embodiment 29, wherein the lipid comprises a steroid or sterol selected from cholesterol, tocopherol, ursodeoxycholic acid, or lithocholic acid. 38. The complex of embodiment 37, wherein the steroid or sterol is cholesterol. 39. The complex of embodiment 37, wherein the steroid or sterol is tocopherol. 40. The complex of embodiment 29, wherein the lipid comprises a fat-soluble vitamin selected from vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof. 41. The complex of embodiment 29, wherein the lipid comprises a phospholipid. 42.
  • the complex of embodiment 41 wherein the phospholipid is selected from phosphocholine (PC), PC-docosahexaenoic acid (PC-DHA), PC-docosanoic acid (PC-DCA), PC-eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC-retinoic acid (PC-RA), or PC- ⁇ -tocopherol succinate (PC-TS).
  • PC phosphocholine
  • PC-DHA PC-docosahexaenoic acid
  • PC-DCA PC-docosanoic acid
  • PC-EPA PC-eicosapentaenoic acid
  • PC-LA PC-lithocholic acid
  • PC-RA PC-retinoic acid
  • PC-TS PC- ⁇ -tocopherol succinate
  • 48. The complex of any one of embodiments 1-44, wherein the linker comprises a polypeptide.
  • 49. The complex of any one of embodiments 1-44, wherein the linker comprises at least one PEG unit. 50.
  • the complex of embodiment 49, wherein the PEG is a PEG2-PEG10,000. 51.
  • 58. The complex of any one of embodiments 1-57, wherein X is an oligonucleotide.
  • 59. The complex of embodiment 58, wherein the oligonucleotide comprises a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. 60.
  • RNA recognition motif selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521-B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi-Argonaute-Zwille domain, or intrinsically disordered region. 67.
  • IVS internal ribosomal entry site
  • any one of embodiments 74-76 wherein: (i) the 3’ end of the binding region is at least 5 ribonucleotides from 5’ end of the IRES; (ii) the 3’ end of the binding region is from 5 to 200 ribonucleotides from 5’ end of the IRES; (iii) the 5’ end of the binding region is at least 5 ribonucleotides from 3’ end of the IRES; or (iv) the 5’ end of the binding region is from 5 to 200 ribonucleotides from 3’ end of the IRES. 78.
  • the circular polyribonucleotide comprises the following elements, arranged in the following order: (i) a first spacer region; (ii) at least one coding region comprising an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide; (iii) optionally a second spacer region; and (iv) a binding region comprising from 5 to 200 ribonucleotides, wherein the binding region binds to X. 79.
  • IRS internal ribosomal entry site
  • the complex of any one of embodiments 1-77, wherein the circular polyribonucleotide comprises the following elements, arranged in the following order: (i) a first spacer region; (ii) a target binding region comprising at least one aptamer or at least ribozyme sequence; (iii) optionally a second spacer region; and (iv) a binding region comprising from 5 to 200 ribonucleotides, wherein the binding region binds to X.
  • the second spacer region comprises 10-500 ribonucleotides.
  • a complex comprising A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A and comprises a photoreactive crosslinking agent, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5.
  • A is a circular polyribonucleotide
  • each X is independently a moiety that binds specifically to a region of A and comprises a photoreactive crosslinking agent
  • each L is independently a linker
  • each B is independently a targeting moiety
  • n is an integer from 1 to 20
  • z is an integer from 1 to 5.
  • the complex of embodiment 94 wherein the oligonucleotide comprises a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.
  • 96. The complex of embodiment 94 or 95, wherein the photoreactive crosslinking agent is attached to the 5’ end of the oligonucleotide.
  • 97 The complex of any one of embodiments 94-96, wherein the photoreactive crosslinking agent is attached to the 3’ end of the oligonucleotide. 98.
  • the photoreactive crosslinking agent comprises 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a phenylselenide, a furan, or an abasic site.
  • the carbazole is 3-cyanovinylcarbazole, 4- methylpyranocarbazole, or pyranocarbazole.
  • the coumarin is 7-hydroxycoumarin. 101.
  • 104 The complex of embodiment 103, wherein the 3’ end of the oligonucleotide has from 1 to 10 nucleotides from the photoreactive nucleotide analog.
  • 107. The complex of any one of embodiments 101-106, wherein the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. 108.
  • each of the plurality of photoreactive crosslinking agents comprises 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a pheylselenide, a furan, or an abasic site.
  • the carbazole is 3-cyanovinylcarbazole, 4- methylpyranocarbazole, or pyranocarbazole.

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Abstract

The disclosure provides compositions, pharmaceutical preparations, and uses of circular polyribonucleotides complexed with one or more targeting moieties. The disclosure further discloses methods of using targeting moieties complexed with a circular polyribonucleotide to achieve functional delivery of a polypeptide to a cell.

Description

COMPOSITIONS COMPRISING CIRCULAR POLYRIBONUCLEOTIDES AND USES THEREOF
Cross-Reference To Related Applications
This application claims priority to Greek Provisional Application No. 20230100684, filed August 18, 2023, U.S. Provisional Application No. 63/533,529, filed August 18, 2023, and U.S. Provisional Application No. 63/551 ,834, filed February 9, 2024, each of which is incorporated by reference in entirety.
Sequence Listing
This application contains a Sequence Listing which has been filed electronically in Extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 16, 2024, is named 51509-052WO3_Sequence_Listing_8_16_24.XML and is 100,545 bytes in size.
Background
Thousands of circular polyribonucleotides are expressed from human genomes. Circular polyribonucleotides are produced endogenously by backsplicing, a process in which the spliceosome fuses a splice donor site in a downstream exon to a splice acceptor site in an upstream exon. Circular polyribonucleotides are produced in cells with high cell-type specificity and can exert biologically meaningful and specific functions. Circular polyribonucleotides are being developed for use in modulating biological systems, including in therapeutic applications. There remains a need to develop improved compositions and methods for the delivery of circular polyribonucleotides in vivo.
Summary
This disclosure provides compositions, pharmaceutical preparations, and uses of circular polyribonucleotides complexed with one or more targeting moieties.
In an aspect, the disclosure provides a complex including A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20), and wherein z is an integer from 1 to 5 (1 , 2, 3, 4, or 5). In some embodiments, n is an integer from 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, n is an integer from 1 to 5 (e.g., 1 , 2, 3, 4, or 5). In some embodiments, z is an integer from 1 to 3 (e.g., 1 , 2, or 3). In some embodiments, z is 1 . In some embodiments, z is 2. In some embodiments, Xn(L-B)z includes Xn(L-B1)z and Xn(L-B2)z, where B1 is a first targeting moiety and B2 is a second targeting moiety. In some embodiments, Xn(L-B)z includes Xn(L1-B)z and Xn(L2-B)z, where L1 is a first linker and L2 is a second linker. In some embodiments, Xn(L-B)z includes Xi-(L-B)z and X2-(L-B)Z, where Xi is a first moiety that binds specifically to a first region of A and X2 is a second moiety that binds specifically to a second region of A. In some embodiments, Xn(L-B)z includes B1-L- X-L- B2, where B1 is a first targeting moiety and B2 is a second targeting moiety. In some embodiments, Xn(L-B)z includes B-L1-X-L2-B, where L1 is a first linker and L2 is a second linker. This disclosure also provides compositions, pharmaceutical preparations, and uses of circular polyribonucleotides complexed with one or more targeting moieties with one or more photoreactive crosslinking agents. In some embodiments, each X includes a photoreactive crosslinking agent.
In an aspect, the disclosure provides a complex including A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A and includes a photoreactive crosslinking agent, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20), and wherein z is an integer from 1 to 5 (1 , 2, 3, 4, or 5). In some embodiments, n is an integer from 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, n is an integer from 1 to 5 (e.g., 1 , 2, 3, 4, or 5). In some embodiments, z is an integer from 1 to 3 (e.g., 1 , 2, or 3). In some embodiments, z is 1 . In some embodiments, z is 2. In some embodiments, Xn(L-B)z includes Xn(L-B1)z and Xn(L-B2)z, where B1 is a first targeting moiety and B2 is a second targeting moiety. In some embodiments, Xn(L-B)z includes Xn(L1-B)z and Xn(l_2-B)z, where L1 is a first linker and L2 is a second linker. In some embodiments, Xn(L-B)z includes Xi-(L-B)z and X2-(L-B)Z, where Xi is a first moiety that binds specifically to a first region of A and X2 is a second moiety that binds specifically to a second region of A. In some embodiments, Xn(L-B)z includes B1-L- X-L-B2, where B1 is a first targeting moiety and B2 is a second targeting moiety. In some embodiments, Xn(L-B)z includes B-L1- X-L2-B, where L1 is a first linker and L2 is a second linker.
In some embodiments, X is an oligonucleotide. In some embodiments, the oligonucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. In some embodiments, the oligonucleotide includes a photoreactive crosslinking agent. In some embodiments, the photoreactive crosslinking agent is attached to the 5’ end of the oligonucleotide. In some embodiments, the photoreactive crosslinking reagent is attached to the 3’ end of the oligonucleotide. In some embodiments, the photoreactive crosslinking agent is a photoreactive nucleotide analog. In some embodiments, the photoreactive crosslinking agent is a photoreactive nucleotide analog, optionally wherein the photoreactive nucleotide analog replaces a single nucleotide within the oligonucleotide. In some embodiments, the photoreactive nucleotide analog is located at an internal position within the oligonucleotide. In some embodiments, the 3’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog. In some embodiments, the 3’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the photoreactive nucleotide analog. In some embodiments, the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation.
In some embodiments, the photoreactive crosslinking agent includes 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a phenylselenide, a furan, or an abasic site. In some embodiments, the carbazole is a vinylcarbazole, e.g., 3-cyanovinylcarbazole, 4- methylpyranocarbazole, or pyranocarbazole.
In some embodiments, the coumarin is 7-hydroxycoumarin.
In some embodiments, the oligonucleotide includes a plurality of photoreactive crosslinking agents. In some embodiments, each of the plurality of photoreactive crosslinking agents is a photoreactive nucleotide analog. In some embodiments, each photoreactive nucleotide analog replaces a single nucleotide within the oligonucleotide. In some embodiments, each photoreactive nucleotide analog is located at an internal position within the oligonucleotide. In some embodiments, the 3’ end of the oligonucleotide has at least 1 , at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 3’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the nearest photoreactive nucleotide analog.
In some embodiments, at least one of the photoreactive nucleotide analogs is attached to the 3’ end of the oligonucleotide. In some embodiments, at least one of the photoreactive nucleotide analogs is attached to the 5’ end of the oligonucleotide.
In some embodiments, the oligonucleotide includes 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides between each of the photoreactive nucleotide analogs. In some embodiments, each of the photoreactive nucleotide analogs crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. In some embodiments, the oligonucleotide includes an aptamer.
In some embodiments, the circular polyribonucleotide is bound to from 1 to 100, 1 and 50, 1 and 20, or 1 and 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety. In some embodiments, the oligonucleotide is from 5 to 100 (e.g., 5 to 80, 5 to 60, 5 to 40, 5 to 20, 20 to 100, 40 to 100, 60 to 100, 80 to 100, 20 to 60, or 10 to 50) nucleotides in length.
In some embodiments, the circular polynucleotide includes a binding region that anneals to the oligonucleotide. In some embodiments, the circular polynucleotide includes one or more binding regions each including from 5 to 200, e.g., 6 to 200, e.g., 7 to 200, e.g., 8 to 200 (e.g., from 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 75, 8 to 50, 8 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each binding region binds to an oligonucleotide. In some embodiments, each binding region includes at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 100% complementarity to the oligonucleotide. In some embodiments, the binding region has zero or one mismatch with the oligonucleotide. In some embodiments, X is a polypeptide. In some embodiments, the polypeptide includes a photoreactive crosslinking agent. In some embodiments, the photoreactive crosslinking agent is a photoreactive amino acid analog. In some embodiments, the photoreactive crosslinking agent is a photoreactive amino acid analog, optionally wherein the photoreactive amino acid analog replaces a single amino acid within the polypeptide. In some embodiments, the photoreactive amino acid analog is located at an internal position within the polypeptide. In some embodiments, the N-terminus of the polypeptide has at least 1 , e.g., at least 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids from the photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids from the photoreactive amino acid analog. In some embodiments, the photoreactive amino acid analog is attached to the N-terminus of the polypeptide. In some embodiments, the photoreactive nucleotide analog is attached to the C-terminus of the polypeptide. In some embodiments, the photoreactive amino acid analog crosslinks to a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. In some embodiments, the photoreactive amino acid analog is an alkyl diazirene-based, arylazide-based, benzophenone-based unnatural amino acid, or A/-e-[2-(furan-2-yl)ethoxy]carbonyl-lysine.
In some embodiments, the polypeptide includes a plurality of photoreactive crosslinking agents. In some embodiments, each of the plurality of photoreactive crosslinking agents is a photoreactive amino acid analog. In some embodiments, each of the plurality of photoreactive crosslinking agents replaces a single amino acid within the polypeptide. In some embodiments, at least one photoreactive amino acid analog is located at an internal position within the polypeptide. In some embodiments, the N-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids from the nearest photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids from the nearest photoreactive amino acid analog. In some embodiments, at least one of the photoreactive amino acid analogs is attached to the N-terminus of the polypeptide. In some embodiments, at least one of the photoreactive amino acid analogs is attached to the C-terminus of the polypeptide. In some embodiments, the polypeptide includes 1 to 50, e.g., 2 to 50, e.g., 8 to 50 (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids between each of the photoreactive amino acid analogs. In some embodiments, each of the photoreactive amino acids crosslinks to a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. In some embodiments, each photoreactive amino acid analog is an alkyl diazirene-based, arylazide-based, benzophenone-based unnatural amino acid, or A/-e-[2-(furan-2-yl)ethoxy]carbonyl-lysine.
In some embodiments, the polypeptide includes an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521 -B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.
In some embodiments, the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide includes one or more protein binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each protein binding region binds to a polypeptide.
In another aspect, the disclosure provides a complex that includes A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically and is covalently attached to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20), and wherein z is an integer from 1 to 5 (1 , 2, 3, 4, or 5). In some embodiments, n is an integer from 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, n is an integer from 1 to 5 (e.g., 1 , 2, 3, 4, or 5). In some embodiments, z is an integer from 1 to 3 (e.g., 1 , 2, or 3). In some embodiments, z is 1 . In some embodiments, z is 2. In some embodiments, Xn(L-B)z includes Xn(L-B1)z and Xn(L-B2)z, where B1 is a first targeting moiety and B2 is a second targeting moiety. In some embodiments, Xn(L-B)z includes Xn(L1-B)z and Xn(L2-B)z, where L1 is a first linker and L2 is a second linker. In some embodiments, Xn(L-B)z includes Xi-(L-B)z and X2-(L-B)Z, where Xi is a first moiety that binds specifically and covalently attaches to a first region of A and X2 is a second moiety that binds specifically and covalently attaches to a second region of A. In some embodiments, Xn(L-B)z includes B1- L- X-L-B2, where B1 is a first targeting moiety and B2 is a second targeting moiety. In some embodiments, Xn(L-B)z includes B-L1-X-L2-B, where L1 is a first linker and L2 is a second linker.
In some embodiments, X is an oligonucleotide. In some embodiments, the oligonucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. In some embodiments, the oligonucleotide includes an aptamer.
In some embodiments, the circular polyribonucleotide is bound to from 1 to 100, 1 and 50, 1 and 20, or 1 and 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety. In some embodiments, the oligonucleotide is from 5 to 100 (e.g., 5 to 80, 5 to 60, 5 to 40, 5 to 20, 20 to 100, 40 to 100, 60 to 100, 80 to 100, 20 to 60, or 10 to 50) nucleotides in length. In some embodiments, the circular polynucleotide includes a binding region that anneals to the oligonucleotide. In some embodiments, the circular polynucleotide includes one or more binding regions each including from 5 to 200, e.g., 6 to 200, e.g., 7 to 200, e.g., 8 to 200 (e.g., from 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 75, 8 to 50, 8 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each binding region binds to an oligonucleotide. In some embodiments, each binding region includes at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 100% complementarity to the oligonucleotide. In some embodiments, the binding region has zero or one mismatch with the oligonucleotide.
In some embodiments, X is a polypeptide. In some embodiments, the polypeptide includes an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521 -B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.
In some embodiments, the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide includes one or more protein binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each protein binding region binds to a polypeptide.
In some embodiments, the targeting moiety includes a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid, or a combination thereof. In some embodiments, the targeting moiety includes a small molecule.
In some embodiments, the small molecule is selected from folic acid, urea, a-mannose, high mannose, ursodeoxycholic acid, an endosomal escape agent, or lithocholic acid.
In some embodiments, the targeting moiety includes a polypeptide. In some embodiments, the polypeptide is a cell-penetrating peptide. In some embodiments, the polypeptide is selected from ASSLNIA (SEQ ID NO: 19), M12, RGD, melittin, LPS-binding protein (LBP) peptide, an adipose-homing peptide, or an endolytic peptide. In some embodiments, the polypeptide is an antibody or a target-binding fragment thereof. In some embodiments, the antibody or target-binding fragment thereof is selected from a monoclonal antibody or target-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, or a tandem scFv (taFv). In some embodiments, the antibody or target- binding fragment thereof is selected from an anti-FcRn antibody, an anti-MR antibody, an anti-CD205 antibody, an anti-CD169 antibody, an anti-CD14 antibody, an anti-CD36 antibody, an anti-CD5 antibody, an anti -CD71 antibody, an anti-CD38 antibody, or an anti-prohibin antibody.
In some embodiments, the polypeptide is a nanobody. In some embodiments, the nanobody is selected from an anti-transferrin nanobody, an anti-HER2 nanobody, or an anti-EGFR nanobody. In some embodiments, the targeting moiety includes a carbohydrate. In some embodiments, the carbohydrate includes a saccharide, disaccharide, or polysaccharide. In some embodiments, the carbohydrate includes mannose, galactose, or glucose. In some embodiments, the carbohydrate includes GalNAc or mannose 6-phosphate. In some embodiments, the carbohydrate includes a mono-, di-, tri-, or tetra-GalNAc. In some embodiments, the carbohydrate is tri-GalNAc.
In some embodiments, the targeting moiety includes a lipid. In some embodiments, the lipid includes a fatty acid. In some embodiments, the fatty acid is a saturated, monounsaturated, or polyunsaturated fatty acid. In some embodiments, the fatty acid is a branched or unbranched chain including from 4 to 40 (e.g., 4, 5, 6, 7, 8, 9, 10, 1 1 , 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, or 40) main-chain carbon atoms. In some embodiments, the fatty acid includes squalene, stearic acid, oleic acid, palmitic acid, linoleic acid, stearic acid, lauric acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, a-tocopherol, a-tocopherol succinate, or a retinoic acid. In some embodiments, the fatty acid includes docosanoic acid. In some embodiments, the fatty acid includes docosahexanoic acid. In some embodiments, the fatty acid includes myristic acid. In some embodiments, the lipid includes a steroid or sterol selected from cholesterol, tocopherol, ursodeoxycholic acid, or lithocholic acid. In some embodiments, the steroid or sterol is cholesterol. In some embodiments, the steroid or sterol is tocopherol. In some embodiments, the lipid includes a fat-soluble vitamin selected from vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof. In some embodiments, the lipid includes a phospholipid. In some embodiments, the phospholipid is selected from phosphocholine (PC), PC-docosahexaenoic acid (PC-DHA), PC-docosanoic acid (PC-DCA), PC- eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC-retinoic acid (PC-RA), and PC-a- tocopherol succinate (PC-TS).
In some embodiments, the targeting moiety includes an oligonucleotide. In some embodiments, the targeting moiety includes an aptamer.
In some embodiments, the linker is a bond. In some embodiments, the linker includes 1 to 250 (e.g., 1 to 225, 1 to 200, 1 to 175, 1 to 150, 1 to 125, 1 to 100, 1 to 75, 1 to 50, 1 to 25, 25 to 250, 50 to 250, 75 to 250, 100 to 250, 125 to 250, 150 to 250, 175 to 250, 200 to 250, 225 to 250, 5 to 50, or 5 to 30) backbone atoms, wherein the backbone atoms are selected from C, N, O, and S. In some embodiments, the linker includes an oligonucleotide. In some embodiments, the linker includes a polypeptide. In some embodiments, the linker includes at least one PEG unit. In some embodiments, the
Figure imgf000008_0001
Figure imgf000008_0005
. In some embodiments, the linker includes at least one TEG unit. In some embodiments, the TEG is a
Figure imgf000008_0004
Figure imgf000008_0002
In some embodiments, the linker is an enzymatically cleavable
Figure imgf000008_0003
linker. In some embodiments, the linker has a length of at least 0.1 nm. In some embodiments, the linker has a length of from 0.1 nm to 20 nm (e.g., 0.1 nm to 18 nm, 0.1 nm to 16 nm, 0.1 nm to 14 nm, 0.1 nm to 12 nm, 0.1 nm to 10 nm, 0.1 nm to 8 nm, 0.1 nm to 6 nm, 0.1 nm to 4 nm, 0.1 nm to 2 nm, 0.1 nm to 0.5 nm, 0.5 nm to 20 nm, 2 nm to 20 nm, 4 nm to 20 nm, 6 nm to 20 nm, 8 nm to 20 nm, 10 nm to 20 nm, 12 nm to 20 nm, 14 nm to 20 nm, 16 nm to 20 nm, 18 nm to 20 nm, 0.2 nm to 15 nm, or 0.5 nm to 18 nm). In some embodiments, the linker is further conjugated to avidin. In some embodiments, the avidin binds from 1 to 4 (e.g., 1 , 2, 3, or 4) targeting moieties. In some embodiments, the avidin binds from 1 to 4 (e.g., 1 , 2, 3, or 4) targeting moieties, wherein the targeting moieties are conjugated to at least one biotin compound.
In some embodiments, X is an oligonucleotide. In some embodiments, the oligonucleotide includes a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. In some embodiments, the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety. In some embodiments, the oligonucleotide is from 5 to 100 (e.g., 5 to 80, 5 to 60, 5 to 40, 5 to 20, 20 to 100, 40 to 100, 60 to 100, 80 to 100, 20 to 60, or 10 to 50) nucleotides in length.
In some embodiments, the circular polynucleotide includes one or more binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each binding region binds to an oligonucleotide. In some embodiments, each binding region includes at least 50%, 60%, 70%, 80%, 90%, or 100% complementarity to the oligonucleotide.
In some embodiments, X is a polypeptide. In some embodiments, the polypeptide includes an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521 -B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.
In some embodiments, the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide includes one or more protein binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each protein binding region binds to a polypeptide. In some embodiments, the circular polyribonucleotide further includes at least one coding region. In some embodiments, the at least one coding region includes an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide. In some embodiments, the polypeptide is expressed in the cell. In some embodiments, the polypeptide is expressed in the cell, optionally wherein the polypeptide expressed in the cell is functional.
In some embodiments, the circular polyribonucleotide includes one or more binding regions each including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein each binding region binds to X. In some embodiments, the binding region is contained within or overlaps in-part with an expression sequence or with a spacer region. In some embodiments, the binding region is not contained within nor does it overlap in-part with an IRES. In some embodiments, the 3’ end of the binding region is at least 5 ribonucleotides from 5’ end of the IRES; the 3’ end of the binding region is from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides from 5’ end of the IRES; the 5’ end of the binding region is at least 5 ribonucleotides from 3’ end of the IRES; or the 5’ end of the binding region is from 5 to 200 ribonucleotides from 3’ end of the IRES. In some embodiments, the circular polyribonucleotide includes the following elements, arranged in the following order: (i) a first spacer region; (ii) at least one coding region including an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide; (iii) optionally a second spacer region; and (iv) a binding region including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein the binding region binds to X. In some embodiments, the circular polyribonucleotide includes the following elements, arranged in the following order: (i) a first spacer region; (ii) a target binding region including at least one aptamer or at least ribozyme sequence; (iii) optionally a second spacer region; and (iv) a binding region including from 5 to 200 (e.g., from 5 to 175, 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 125 to 200, 150 to 200, 175 to 200, 25 to 100, or 50 to 150) ribonucleotides, wherein the binding region binds to X.
In some embodiments, the first spacer region includes from 10 to 500 (e.g., from 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to 500, 100 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 150, or 100 to 300) ribonucleotides. In some embodiments, the second spacer region includes 10 to 500 ribonucleotides (e.g., 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to 500, 100 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 150, or 100 to 300).
In some embodiments, the binding region is contained within or overlaps in-part with the expression sequence. In some embodiments, the binding region is contained within or overlaps the first spacer region. In some embodiments, the binding region is contained within or overlaps the second spacer region. In some embodiments, the first spacer and second spacer are adjacent to one another. In some embodiments, the binding region overlaps the first and second spacer regions. In some embodiments, the binding region is not contained within nor does it overlap in-part with the expression sequence or with the first or second spacer region.
In some embodiments, the circular polyribonucleotide includes at least 1 ,000 ribonucleotides. In some embodiments, the circular polyribonucleotide includes at least 3,000 ribonucleotides. In some embodiments, the circular polyribonucleotide includes from 1 ,000 to 20,000 (e.g., from 1 ,000 to 18,000; 1 ,000 to 16,000; 1 ,000 to 14,000; 1 ,000 to 12,000; 1 ,000 to 10,000; 1 ,000 to 8,000; 1 ,000 to 6,000; 1 ,000 to 4,000; 1 ,000 to 2,000; 2,000 to 20,000; 4,000 to 20,000; 6,000 to 20,000; 8,000 to 20,000; 10,000 to 20,000; 12,000 to 20,000; 14,000 to 20,000; 16,000 to 20,000; 18,000 to 20,000; 5,000 to 10,000; or 3,000 to 12,000) ribonucleotides.
In some embodiments, the circular polyribonucleotide encodes a polypeptide. In some embodiments, the polypeptide is a eukaryotic polypeptide. In some embodiments, the polypeptide is a mammalian polypeptide. In some embodiments, the polypeptide is a mammalian polypeptide, optionally wherein the polypeptide is a human polypeptide. In some embodiments, the polypeptide is a viral, bacterial, or fungal polypeptide. In some embodiments, the polypeptide is for therapeutic use. In some embodiments, the polypeptide is an immunogen.
In some embodiments, the circular polyribonucleotide includes an IRES. In some embodiments, the IRES is selected from 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, fuman poliovirus 1 , Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1 , Human Immunodeficiency Virus type 1 , Homalodisca coagulata virus- 1 , Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 , Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1 , Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1 , Human AML1 /RUNX1 , Drosophila antennapedia, Human AQP4, Human AT1 R, Human BAG-I, Human BCL2 , Human BiP, Human c-IAPI , Human c- myc, Human elF4G, Mouse NDST4L, Human LEF1 , Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-I, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1 , Human c-src, Human FGF-I, Simian picomavirus, Turnip crinkle virus, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 /2).
In an aspect, the disclosure provides a pharmaceutical composition including any one of the circular polyribonucleotides described herein and one or more delivery agents. In some embodiments, the delivery agent is selected from calcium, magnesium, manganese, or strontium. In some embodiments, the delivery agent is an endosomal escape agent. In some embodiments, the endosomal escape agent includes chloroquine, amantadine, ammonium chloride, 4-bromobenzaldehyde N-(2,6- dimethylphenyl)semicarbazone (EGA), UNC-108, or any combination thereof. In some embodiments, the delivery agent is a globular protein. In some embodiments, the globular protein is albumin. In some embodiments, the delivery agent is ribonuclease inhibitor.
In another aspect, the disclosure provides a method of delivering a circular polyribonucleotide to a cell, the method including contacting the cell with any one of the complexes or pharmaceutical compositions described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the circular polyribonucleotide is delivered to the cell ex-vivo. In some embodiments, the cell is administered to a subject after the delivery of the circular polyribonucleotide to the cell. In some embodiments, administration of the cell to the subject treats a disease, disorder, or condition in the subject.
In an aspect, the disclosure provides a method of delivering a circular polyribonucleotide to a subject, the method including administering to the subject any one of the complexes or pharmaceutical compositions described herein.
In another aspect, the disclosure provides a method of treating a disease, disorder, or condition in a subject, the method including administering to the subject any one of the complexes or pharmaceutical compositions described herein. In another aspect, the disclosure provides a method of inducing an immune response in a subject, the method including administering to the subject any one of the complexes or pharmaceutical compositions described herein. In some embodiments, the complex or pharmaceutical composition is administered intramuscularly, subcutaneously, intravenously, intraperitoneally, topically, or orally.
In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the non-human mammal is a cow, a sheep, a goat, a pig, a dog, a horse, or a cat. In some embodiments, the subject is a bird. In some embodiments, the bird is a hen, a rooster, a turkey, or a parrot.
In an aspect, the disclosure provides a method of covalently attaching a targeting moiety to a circular polyribonucleotide. The method includes forming a complex as described herein (e.g., of any of the above embodiments) and irradiating the complex with light. In some embodiments, the wavelength of the irradiated light is from 350-370 (e.g., 350, 351 , 352, 353. 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, or 370) nm. In some embodiments, the complex is irradiated for 1 to 120 (e.g., 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes.
In another aspect, the disclosure provides a method of removing the covalent attachment between the circular polyribonucleotide and the moiety that binds specifically to a region of the circular polyribonucleotide and includes one or more photoreactive crosslinking agent. The method includes irradiating any one of the complexes described herein with light at a second wavelength. In some embodiments, the second wavelength of the irradiated light is from 300-320 (e.g., 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311 , 312, 313, 314, 315, 316, 317, 318, 319, or 320) nm. In some embodiments, the complex is irradiated at the second wavelength for 1 to 120 (e.g., 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes.
In another aspect, the disclosure provides a method of delivering a circular polyribonucleotide to a cell. The method includes contacting the cell with any one of the complexes or pharmaceutical compositions described herein. In some embodiments, the complex is irradiated prior to contacting the cell. In some embodiments, the complex is irradiated after contacting the cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the circular polyribonucleotide is delivered to the cell ex-vivo. In some embodiments, the complex is delivered to the cell in-vivo. In some embodiments, the cell is administered to a subject after the delivery of the circular polyribonucleotide to the cell. In some embodiments, the complex is irradiated prior to administration to the subject. In some embodiments, the complex is irradiated after administration to the subject. In some embodiments, administration of the cell to the subject treats a disease, disorder, or condition in the subject.
In an aspect, the disclosure provides a method of delivering a circular polyribonucleotide to a subject. The method includes administering to the subject any one of the complexes or pharmaceutical compositions described herein.
In another aspect, the disclosure provides a method of treating a disease, disorder, or condition in a subject. The method includes administering to the subject any one of the complexes or pharmaceutical compositions described herein.
In another aspect, the disclosure provides a covalent complex produced by any of the methods described herein. Definitions
To facilitate the understanding of this disclosure, several terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the disclosure. Terms such as "a," "an," and "the" are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The term "or" is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or”. The terminology herein is used to describe specific embodiments, but their usage is not to be taken as limiting, except as outlined in the claims.
As used herein, 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.
As used herein, the term “about” refers to a value that is within ± 10% of a recited value.
The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129- 134 (2003). For a review of scFv fragments, see e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571 ,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments including salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161 ; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments including all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single- domain antibody. Antibody fragments can be made by various techniques.
As used herein, the term “aptamer” is a non-naturally occurring or synthetic oligonucleotide that specifically binds to a target molecule. Typically, an aptamer is from 20 to 500 nucleotides. Typically, an aptamer binds to its target through secondary structure rather than sequence homology.
As used herein, the term “binding moiety” refers to a moiety that specifically binds to (e.g., hybridized or hybridized and covalently bound to) a portion of the circular polyribonucleotide. The binding moiety may include one or more photoreactive crosslinking agents.
As used herein, the term “binding region” refers to a portion of the circular polyribonucleotide to which one or more moieties specifically bind. The one or more moieties specifically bind to the portion of the circular polyribonucleotides by non-covalent interactions. For example, an oligonucleotide may anneal to the binding region by way of hydrogen bonds, or a polypeptide may bind to a binding region by way of hydrogen bonds, hydrophobic interactions, or Van der Waals interactions. As used herein, the term “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. 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).
As used herein, the term “cell-penetrating peptide” refers to a peptide having from about 5 to about 30 amino acids residues which has a net positive charge, which facilitate penetration into cells across the cell membrane.
As used herein, the terms “circular polyribonucleotide” and “circular RNA” are used interchangeably and mean a polyribonucleotide molecule that has a structure having no free ends (i.e. , no free 3’ or 5’ ends), for example a polyribonucleotide molecule that forms a circular or end-less structure through covalent or non-covalent bonds. The circular polyribonucleotide may be, e.g., a covalently closed polyribonucleotide.
As used herein, the term “circularization efficiency” is a measurement of resultant circular polyribonucleotide versus its non-circular starting material.
As used herein, the term “coding region” refers to a region of the polynucleotide including an expression sequence encoding a polypeptide and an IRES and/or another regulatory element.
As used herein, the term “crosslink” refers to forming a covalent bond between two molecules. For example, a photoactivatable crosslinking agent may form a crosslink between the agent and a ribonucleotide upon irradiation with light.
As used herein, the term “delivery agent” refers to an agent that, when added to the pharmaceutical composition including the circular polyribonucleotides described herein, increases cellular delivery, functional delivery, or endosomal escape of the circular polyribonucleotide relative to the same pharmaceutical composition that lacks the agent. The increase in cellular delivery, functional delivery, or endosomal escape of the circular polyribonucleotide may be measured, e.g., by assessing the amount of expression of a polypeptide encoded by the circular polyribonucleotide in comparison to a pharmaceutical composition that does not include the delivery agent.
The term “derived from” refers to in the present specification in the context of a nucleic acid, i.e., for a nucleic acid “derived from” (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares e.g. at least 60%, 70%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived. The skilled person is aware that sequence identity is typically calculated for the same types of nucleic acids, i.e., for DNA sequences or for RNA sequences. Thus, it is understood, if a DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, in a first step the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by U throughout the sequence). Thereafter, the sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined. Preferably, a nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g., in order to increase RNA stability even further and/or to prolong and/or increase protein production. In the context of amino acid sequences, the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares e.g. at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence from which it is derived.
The term “diluent” means a vehicle including an inactive solvent in which a composition described herein (e.g., a composition including a circular polyribonucleotide) may be diluted or dissolved. A diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof. A diluent can be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3- butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetra hydrofurfury I alcohol, polyethylene glycols and fatty acid esters of sorbitan, and 1 ,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, or powdered sugar.
As used herein, 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.
As used herein, the term “expression sequence” is a nucleic acid sequence that encodes a product, e.g., a peptide or polypeptide, beginning with a start codon and ending with a stop codon. An exemplary expression sequence that codes for a peptide or polypeptide can include a plurality of nucleotide triads, each of which can code for an amino acid and is termed as a “codon.”
As used herein, the term “fragment” with respect to a polypeptide or a nucleic acid sequence refers to a continuous, less than a whole portion of a sequence of the polypeptide or the nucleic acid. A fragment of a polypeptide or a nucleic acid sequence encoding a polypeptide, 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.
As used herein, the term “functional delivery” refers to delivery of a polyribonucleotide encoding a polypeptide into a cell, wherein the polypeptide is expressed in the cell.
By “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. For example, 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. The term "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. As used herein, the term “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 identity) as a circular polyribonucleotide and having two free ends (i.e., the uncircularized version (and its fragments) of the circularized polyribonucleotide). In some embodiments, the linear counterpart (e.g., a pre-circularized version) 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 identity) 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). In some embodiments, 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 identity) 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). In some embodiments, 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. In some embodiments, the linear counterpart further includes a 5’ cap. In some embodiments, the linear counterpart further includes a poly adenosine tail. In some embodiments, the linear counterpart further includes a 3’ UTR. In some embodiments, the linear counterpart further includes a 5’ UTR.
As used herein, the terms “linear RNA,” “linear polyribonucleotide,” and “linear polyribonucleotide molecule” are used interchangeably and mean 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. Linear RNA includes RNA that has not undergone circularization (e.g., is pre-circularized) and can be used as a starting material for circularization.
As used herein, the term “linker” refers to a covalent linkage or connection between two or more components in a conjugate (e.g., between a polyribonucleotide or oligonucleotide and a lipid moiety and a PEG, as described herein). Molecules that may be used as linkers include at least two functional groups, which may be the same or different, e.g., two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid group, an amine group and a maleimide group, an amine group and an alkyne group, or an amine group and a sulfonic acid group. The first functional group may form a covalent linkage with a first component in the conjugate and the second functional group may form a covalent linkage with the second component in the conjugate. A linker typically provides space, rigidity, and/or flexibility between the two or more components.
As used herein, the terms “lipid” or “lipid moiety” refer to organic compounds, most preferably organic biomolecules, that are selectively soluble in nonpolar solvents over water. Lipid moieties specifically contemplated by the present invention include, for example, fatty acids, steroids or sterols, glycerides (e.g., monoglycerides, diglycerides, and triglycerides), phospholipids, and fat-soluble vitamins (e.g., vitamins A, D, E, and K).
As used herein, the term “modified ribonucleotide” means a nucleotide with at least one modification to the sugar, the nucleobase, or the internucleoside linkage. As used herein, the term “naked delivery” is a formulation for delivery to a cell without the aid of a carrier and without covalent modification to a moiety that aids in delivery to a cell. A naked delivery formulation is free from any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, naked delivery formulation of a circular polyribonucleotide is a formulation that includes a circular polyribonucleotide without covalent modification and is free from a carrier.
As used herein, the term “nanobody” refers to a polypeptide having a single monomeric variable domain and has a molecular weight of between about 12 kDa and about 15 kDa. A nanobody is able to selectively bind to a specific antigen.
The term “photoreactive crosslinking agent”, as used herein, refers to any compound that is configured to form a covalent adduct with another molecule upon irradiation with light. For example, a photoreactive crosslinking agent may covalently attach to a ribonucleotide within the circular polyribonucleotide upon irradiation with light. In embodiments, a photoreactive crosslinking agent may be able to form a covalent attachment to a ribonucleotide within the circular polyribonucleotide that is reversible. In embodiments, the photoreactive crosslinking agent can be a photoreactive nucleotide analog or photoreactive amino acid analog.
The term “photoreactive nucleotide analog” refers to any nucleic acid analog that is configured to form a covalent adduct with another molecule upon irradiation with light. For example, the photoreactive nucleotide analog may be present within an oligonucleotide and be configured to covalently attach to a circular polyribonucleotide upon irradiation with light. In some embodiments, the photoreactive nucleotide analog can covalently attach to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. Examples of photoreactive nucleotide analogs include, but are not limited to, 4- thiouridene (4sU), 5-bromo-2’-deoxyuridine (BrdU), coumarin derivatives, 3-cyanovinylcarbazole D- threoninol (cnvD) derivatives, 3-cyanovinylcarbazole nucleoside (CNVK) derivatives, diazirene derivatives, phenylselenide derivatives, psoralen derivatives, or pyranocarbazole (PCX) derivatives. For a review of certain photoreactive nucleotide analogs, see Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 and Tavakoli et al., RSC Adv. 12, 6484-6507, 2022. In embodiments, the covalent attachment of the photoreactive nucleotide analog to the ribonucleotide is reversible, e.g., it may be removed when irradiated with light, e.g., of a second different wavelength.
The term “photoreactive amino acid analog” refers to any amino acid analog that is configured to form a covalent adduct with another molecule upon irradiation with light. For example, the photoreactive amino acid analog may be present within a polypeptide that covalently attaches to a circular polyribonucleotide upon irradiation with light. In some embodiments, the photoreactive amino acid analog can covalently attach to a nearby ribonucleotide within the circular polyribonucleotide upon photoirradiation. Examples of photoreactive amino acid analogs include but are not limited to diazirene- based, aryl azide-based, benzophenone-based unnatural amino acid, or A/-e-[2-(furan-2- yl)ethoxy]carbonyl-lysine. In embodiments, the covalent attachment of the photoreactive amino acid analog to the ribonucleotide is reversible, e.g., it may be removed when irradiated with light, e.g., a second different wavelength.
The term “pharmaceutical composition” is intended to also disclose that the circular or linear polyribonucleotide included within a pharmaceutical composition can be used for the treatment of the human or animal body by therapy. It is thus meant to be equivalent to “a polyribonucleotide for use in therapy.”
The term “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.
As used herein, the term “polyribonucleotide cargo” herein includes any sequence including at least one polyribonucleotide. In embodiments, the polyribonucleotide cargo includes one or multiple expression sequences, wherein each expression sequence encodes a polypeptide. In embodiments, the polyribonucleotide cargo includes one or multiple noncoding sequences, such as a polyribonucleotide having regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo includes a combination of expression and noncoding sequences. In embodiments, 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 sequences.
As used interchangeably herein, the terms “polyA,” “polyA sequence,” and “polyA tail” refer to an untranslated, contiguous region of a nucleic acid molecule of at least 5 nucleotides in length and consisting of adenosine residues. In some embodiments, a polyA tail is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. In some embodiments, a polyA tail is located 3’ to (e.g., downstream of) an open reading frame (e.g., an open reading frame encoding a polypeptide), and the polyA tail is 3’ to a termination element (e.g., a stop codon) such that the polyA is not translated. In some embodiments, a polyA tail is located 3’ to a termination element and a 3’ untranslated region.
As used herein, the elements of a nucleic acid are “operably connected” if they are positioned on the vector such that they can be transcribed to form a linear RNA that can then be circularized into a circular RNA using the methods provided herein.
“Polydeoxyribonucleotides,” “deoxyribonucleic acids,” and “DNA” 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, that include detectable tags, such as luminescent tags or markers (e.g., fluorophores). A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. 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). In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. In some cases, 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. In some cases, 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.
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. Examples of modified nucleotides include, but are not limited to diaminopurine, 5-fluorouracil, 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, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6 -isopentenyladenine, uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid(v), 5-methyl-2- thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, 3-(3-amino-3-carboxypropyl)uridine 2,6-diaminopurine and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Non-limiting examples of such modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates). 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-hydroxysuccinimide 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 photo-programmed 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.
As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. 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 include 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. The term 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.
As used herein, 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. A therapeutic agent can be administered to a subject who is at increased risk of developing a viral infection relative to a member of the general population in order to prevent the development of, or lessen the severity of, the disease or condition. A therapeutic agent can be administered as a prophylactic, e.g., before development of any symptom or manifestation of a viral infection.
As used herein, the term “regulatory element” is a moiety, such as a nucleic acid sequence, that modifies expression of an expression sequence within the circular or linear polyribonucleotide.
As used herein, the term “replication element” is a sequence and/or motif useful for replication or that initiate transcription of the circular polyribonucleotide.
As used herein, the term “small molecule” refers to a low molecular weight compound (e.g., a compound (e.g., an organic compound) having less than 1000 Da, that may regulate a biological process, with a size on the order of 1 nm. In some instances, a therapeutic agent is a small molecule therapeutic agent. In some instances, the small molecule agent is from about 300 to about 700 Da (e.g., about 325 Da, about 350 Da, about 375 Da, about 400 Da, about 425 Da, about 450 Da, about 475 Da, about 500 Da, about 525 Da, about 550 Da, about 575 Da, about 600 Da, about 625 Da, about 650 Da, or about 675 Da).
As used herein, a “spacer” refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.
A “signal sequence” refers to a polypeptide sequence, e.g., from 10 to 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.
As used herein, the term “sequence identity” is determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm. Sequences are referred to as "substantially identical” or “essentially similar” when they share at least a certain minimal percentage of sequence identity when optimally aligned (e.g., when aligned by programs such as GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna, and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity are determined, e.g., 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”). Alternatively, or additionally, percent identity is determined by searching against databases, e.g., using algorithms such as FASTA, BLAST, etc. Sequence identity refers to the sequence identity over the entire length of the sequence.
As used herein, the term "subject" refers to an organism, such as an animal, plant, or microbe. In embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In embodiments, the subject is a human. In embodiments, the subject is a non-human mammal. In embodiments, 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). In embodiments, 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). In embodiments, the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusk. In embodiments, the subject is an invertebrate agricultural pest or an invertebrate that is parasitic on an invertebrate or vertebrate host. In embodiments, 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. In embodiments, the subject is a eukaryotic alga (unicellular or multicellular). In embodiments, 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.
As used here, the term “targeting moiety” refers to a moiety that binds specifically to a cell or a portion thereof (e.g., an extracellular, intracellular, or membrane portion thereof) thereby promoting intracellular delivery of the moiety and any cargo bound or complexed thereto (e.g., a polyribonucleotide). A targeting moiety may include a lipid, a small molecule, a carbohydrate, a polypeptide, a nucleic acid (e.g., an aptamer), or a combination thereof. In some embodiments, a targeting moiety interacts with the cellular membrane thereby promoting intracellular delivery. In some embodiments, a targeting moiety promotes endosomal delivery and/or endosomal escape. In some embodiments a targeting moiety promotes cellular delivery that is not specific to any cell type. In some embodiments, the targeting moiety binds preferentially to a particular cell type and therefore promotes cell-type specific delivery.
As used herein, the term “termination element” is a moiety, such as a nucleic acid sequence, that terminates translation of the expression sequence in the circular or linear polyribonucleotide.
As used herein, the term “translation efficiency” is a rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, 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., a cell-free translation system like rabbit reticulocyte lysate.
As used herein, the term “translation initiation sequence” is a nucleic acid sequence that initiates translation of an expression sequence in the circular or linear polyribonucleotide.
As used herein, the terms “treat” and “treating” refer to a therapeutic 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 therapeutic treatment.
As used herein, a ’’vector" means a polynucleotide (e.g., DNA) that is synthesized (e.g., using PCR), or that is taken from a virus, plasmid, or cell of a higher organism into which a foreign DNA fragment can be or has been inserted for cloning or expression purposes. In some embodiments, a vector can be stably maintained in an organism. A vector can include, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, or a multiple cloning site (MCS) . The term includes linear DNA fragments (e.g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like. In one embodiment, the vectors provided herein include a multiple cloning site (MCS). In another embodiment, the vectors provided herein do not include an MCS.
Brief Description of the Drawings
FIG. 1 is a schematic of an exemplary circular polyribonucleotide complexed with Xn(L-B)z, where X is a moiety that binds specifically to a region of the circular polyribonucleotide, L is a linker, and B is a targeting moiety, wherein n is 2, and wherein z is 1 in this schematic.
FIG. 2 is a schematic of a circular polyribonucleotide having an annealing or binding region in the spacer region and/or an annealing or binding region in the coding region.
FIG. 3 is schematic of a circular polyribonucleotide bound to an oligonucleotide covalently linked to a trivalent GalNAc moiety. The oligonucleotide-linker-trivalent GalNAc is bound to either the spacer region or the coding region of the circular polyribonucleotide.
FIG. 4 shows the amount of luminescence measured from circular polyribonucleotides encoding a luciferase polypeptide bound to one or two targeting moieties in comparison to a circular polyribonucleotide without a targeting moiety, wherein the targeting moieties are GalNAc (A or B), cholesterol (I), tocopherol (Q), or a combination thereof.
FIG. 5 shows the amount of luminescence measured from circular polyribonucleotides encoding a luciferase polypeptide bound to one or two targeting moieties in comparison to a circular polyribonucleotide without a targeting moiety, wherein the targeting moieties are GalNAc (B or C), cholesterol (I or J), tocopherol (Q or R), or a combination thereof.
FIG. 6 shows the amount of luminescence measured for circular polyribonucleotides from circular polyribonucleotides encoding a luciferase polypeptide bound to a GalNAc and/or tocopherol targeting moiety in the presence of a ribonuclease inhibitor and/or various concentration of calcium chloride in comparison to a circular polyribonucleotide without a targeting moiety.
FIG. 7 shows the amount of luminescence measured from circular polyribonucleotides encoding a luciferase polypeptide bound to one or two targeting moieties in comparison to a circular polyribonucleotide without a targeting moiety, wherein the targeting moieties are GalNAc, cholesterol, or tocopherol in the absence of calcium chloride.
FIG. 8A and FIG. 8B shows the amount of luminescence measured for circular polyribonucleotides encoding a luciferase polypeptide bound to a GalNAc and tocopherol targeting moiety in the presence of or absence of BSA in a 1 :1 ratio with the circular polyribonucleotide (FIG. 8A) or 60 pM of chloroquine and/or 20 mM of calcium chloride (FIG. 8B) in comparison to in combination with lipofectamine.
FIG. 9A shows the amount of luminescence measured in primary adipocytes for circular polyribonucleotides encoding a luciferase polypeptide complexed with an oligomer conjugated to a cholesterol by way of a linker and/or an oligomer conjugated to a tocopherol by way of a linker in the presence or absence of calcium in comparison to a circular polyribonucleotide in the presence of lipofectamine.
FIG. 9B shows the amount of flux measured for circular polyribonucleotides encoding a luciferase polypeptide complexed with: (a) an oligomer conjugated to a cholesterol by way of a linker and an oligomer conjugated to a tocopherol, or (b) an oligomer conjugated to an aptamer by way of a linker 6 hours, 24 hours, and 48 hours after subcutaneous administration to mice in comparison to a circular polyribonucleotide administered with lipid nanoparticles (LNP).
FIG. 10 shows a schematic of circular polyribonucleotide complexed with a biotinylated oligomer bound to avidin having three additional biotin binding sites which may bind one or more biotinylated targeting moieties such as biotinylated lipids, biotinylated sugars, biotinylated peptides, or biotinylated antibodies.
FIG. 11 A shows the amount of luminescence measured in HEK293T cells transfected with circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to cholesterol by way of a linker in various concentrations of calcium.
FIG. 11 B shows the amount of flux measured in mice 72 hours after they were administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to cholesterol by way of a linker in various concentrations of calcium in comparison to a circular polyribonucleotide administered with an LNP.
FIG. 12A shows the amount of luminescence measured in primary mouse hepatocytes transfected with circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to tocopherol by way of a linker and an oligomer conjugated to GalNAc by way of a linker in various concentrations of calcium, chloroquine, and/or ribonuclease inhibitor in comparison to a circular polyribonucleotide in lipofectamine.
FIG. 12B shows the amount of flux measured in mice 5 hours after they were administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to cholesterol by way of a linker in various concentrations of calcium and/or chloroquine.
FIG. 13 show the polypeptide expression resulting from administering a circular polyribonucleotide encoding a SARS-CoV-2 RNA binding protein (RBD) that is complexed either to (a) an oligomer conjugated to cholesterol by way of a linker, or (b) a biotinylated oligomer bound to an avidin bound to biotinylated mannose in the presence or absence of chloroquine.
FIG. 14A and FIG. 14B shows the amount of flux measured in mice 4-5 hours, 24 hours, and 72 hours after they were intramuscularly administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with: (a) an oligomer conjugated to a biotinylated avidin conjugated to biotinylated mannose by way of a linker, or (b) an oligomer conjugated to a tocopherol by way of a linker and an oligomer conjugated to a cholesterol by way of a linker in comparison to a circular polyribonucleotide administered with an LNP and visualized by the dorsal (FIG. 14A) or ventral view (FIG. 14B). FIG. 15 shows the amount of flux measured in mice over a period of 30 days after they were intradermally administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with an oligomer conjugated to cholesterol by way of a linker or an oligomer, wherein the complex is mixed with chloroquine, calcium, albumin, and ribonuclease inhibitor, in comparison to a circular polyribonucleotide administered with an LNP and visualized by the dorsal view.
FIG. 16 shows the amount of flux measured in mice 6 hours, 24 hours, or 48 hours after they were subcutaneously administered circular polyribonucleotides encoding a luciferase polypeptide either alone or complexed with: (a) an oligomer conjugated to cholesterol by way of a linker and an oligomer and an oligomer conjugated to tocopherol by way of a linker, (b) a oligomer conjugated to an aptamer by way of a linker, and (c) within an LNP and visualized by the dorsal view.
FIG. 17A and FIG. 17B show the percentage of cells that internalized (FIG. 17A) and the mean uptake of cells (FIG. 17B) of the circular polyribonucleotides complexed to: (a) a biotinylated oligomer bound to an avidin bound to an antibody at various concentrations; (b) two biotinylated oligomers each bound to an avidin bound to an antibody and a biotinylated oligomer bound to an avidin conjugated to a mixture of two different antibodies; (c) two biotinylated oligomers each bound to an avidin bound to an antibody and a biotinylated oligomer bound to an avidin conjugated to a mixture of two different aptamers; (d) two biotinylated oligomers each bound to an avidin bound to an antibody and a biotinylated oligomer bound to an avidin conjugated to a mixture of an antibody and phosphatidylserine; or (e) a biotinylated oligomer bound to an avidin conjugated to a phosphatidylserine.
FIG. 18 shows the number copies of the circular polyribonucleotide measured to be internalized by Raw264.7 macrophages, wherein the circular polyribonucleotide is complexed with: (a) two biotinylated oligomers each of which are bound to an avidin which is bound to a mixture of antibodies, aptamers, phosphatidylserine, and/or mannose; or (b) an oligomer conjugated to tocopherol by way of a linker and an oligomer conjugated to cholesterol by way of a linker in the presence or absence of calcium. FIG. 18 demonstrates that the various complexed circular polyribonucleotides were localized inside the cells.
FIG. 19 shows the amount of luminescence measured in Raw264.7 macrophages having been transfected with a circular polyribonucleotide complexed two biotinylated oligomers each of which are bound to an avidin which is bound to one or more targeting moieties including: an aptamer, antibody, mannose, phosphatidylserine or a mixture thereof in the presence or absence of chloroquine.
FIG. 20 shows the amount of luminescence measured for circular polyribonucleotides complexed with either two biotinylated oligomers in comparison to a circular polyribonucleotide complexed with to oligomers each of which independently were bound to an avidin which was bound to biotinylated antibodies.
FIG. 21 shows the amount of luminescence measured in HeLa cells after having been transfected with a circular polyribonucleotide encoding a luciferase polypeptide complexed with two biotinylated oligomers each bound to avidin which was bound to Trf antibodies, wherein the complex was administered in the presence or absence of calcium and/or excess transferrin.
FIG. 22 shows the amount of luminescence measured from circular polyribonucleotides encoding a luciferase polypeptide bound to a GalNAc targeting moiety in comparison to a circular polyribonucleotide without a targeting moiety. The GalNAc targeting moiety was conjugated by way of a linker at the 5’ end (FC and FC) or 3’ end (FA and FB) an oligomer annealed to the ORF of the circular polyribonucleotide. The linker was either a TEG linker (FA, FB, FC, and FD) or PEG24 linker (FE24).
FIG. 23 is a schematic of an exemplary circular polyribonucleotide complexed with X(L-B), where X is a moiety that binds specifically to a region of the circular polyribonucleotide and includes a photoreactive crosslinking agent, L is a linker, and B is a targeting moiety.
FIG. 24 is an HPLC chromatogram showing a linear, complementary oligomer (oligomimic) and a binding moiety that includes a 23-mer ribonucleotide, a 3-cyanovinylcarbazole nucleoside (CNVK) photoreactive crosslinking agent, a linker, and a biotin targeting moiety, wherein the linear complementary oligomer and the binding moiety were bound by annealing or by annealing and irradiation.
FIG. 25A and 25B are graphs showing the amount of luminescence measured in HEK293 cells transfected with circular polyribonucleotides encoding a luciferase polypeptide complexed with a dimeric fluorescent TAT (dfTAT) peptide (eRNA) complexed by way of annealing then irradiation for 0, 1 , 5, or 30 minutes. The circular polyribonucleotide was complexed with an oligomer bound to an E1 binding region (Chol-E1-cnvK) of the circular polyribonucleotide or an oligomer bound to an E1/E2 binding region (Chol- E2/E1-cnvK) of the circular polyribonucleotide. The results are compared to no circular polyribonucleotides, the circular polyribonucleotides without a binding moiety, the circular polyribonucleotides without a binding moiety, and transfected with lipofectamine MessengerMax (lipo). J=Chol J including a cholesterol targeting moiety. FIG. 25B shows the same data as FIG. 25A but is rearranged to emphasize the significant expression of the complex including Chol-E1-cnvK as a binding moiety after irradiation for 30 minutes.
FIG. 26A and 26B are graphs showing the amount of luminescence measured in HEK cells transfected with circular polyribonucleotides encoding a luciferase polypeptide (eRNA) complexed by way of annealing and irradiation for 0 or 30 minutes. The circular polyribonucleotide was complexed with an oligomer including one internally located photoreactive crosslinking agent (PS1 D and CHOL-E1_CK) or an oligomer including two internally located photoreactive crosslinking agents (PS2E). J=Chol J including a cholesterol targeting moiety; Q=Chol J including a tocopherol binding moiety. The results are compared to circular polyribonucleotides without a binding moiety. FIG. 26Bshows the same data from FIG. 26A but is rearranged to emphasize complexes including oligomers with significant expression after irradiation for 30 minutes.
Detailed Description
This disclosure provides compositions, pharmaceutical preparations, and uses of circular polyribonucleotides complexed with one or more targeting moieties. The circular polyribonucleotide complexes described herein improve the efficiency of delivery to a cell or a subject, as compared to the same circular polyribonucleotide alone.
This disclosure provides compositions, pharmaceutical preparations, and uses of circular polyribonucleotides complexed by one or more photoreactive crosslinking agents with one or more targeting moieties. Also featured are covalent complexes produced with photoreactive crosslinking agents. The circular polyribonucleotide complexes described herein improve the efficiency of delivery to a cell or a subject, as compared to the same circular polyribonucleotide alone. In particular, the photoreactive crosslinking agent is able to form a covalent adduct upon irradiation between the targeting moiety and the circular polyribonucleotide, providing increase stability of the targeting complex to more efficiently deliver the complex to a desired intracellular or extracellular location. The targeting moiety may be, for example, a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid (e.g., an aptamer), or a combination thereof.
In some embodiments, the circular polyribonucleotide encodes a therapeutic agent (e.g., a therapeutic polypeptide), the complex improves therapeutic efficacy. In some embodiments, the circular polyribonucleotide encodes a polypeptide, the complex improves translation efficiency.
Complexes and compositions described herein may be complexed with or encapsulated by a carrier (e.g., a cell, a vesicle, a membrane-based carrier, a lipid nanoparticle (LNP), a polymer nanoparticle, a viral particle, or a microbubble). Complexes and compositions described herein may have no carrier. Complexes and compositions described herein may be formulated as a pharmaceutical composition, e.g., for therapeutic use. Complexes and compositions described herein may be used to treat a disease, disorder, or condition, as described herein.
The disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide and contains one or more photoactivatable crosslinking agents, a linker, and targeting moiety. For example, the circular polyribonucleotide may be complexed with a molecule of Xn(L-B)z, where each X is independently a moiety that binds specifically to a region of the circular polyribonucleotide and contains one or more photoactivatable crosslinking agents, each L is independently a linker, each B is independently a targeting moiety, n is an integer, e.g., from 1 to 20, and z is an integer, e.g., from 1 to 5 (see, e.g., FIG. 23). The moiety that binds the circular polyribonucleotide may be, for example, an oligonucleotide, a polypeptide, or a small molecule.
This disclosure also provides compositions of and methods for forming a covalent complex including A and Xn(L-B)z, where is a circular polyribonucleotide, each X is independently a moiety that binds specifically and is covalently attached to a region of A, each L is independently a linker, each B is independently a targeting moiety, n is an integer, e.g., from 1 to 20, and z is an integer, e.g., from 1 to 5. The moiety that binds the circular polyribonucleotide may be, for example, an oligonucleotide, a polypeptide, or a small molecule.
Targeting Moieties
The disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide, a linker, and targeting moiety. In some embodiments, the disclosure provides a circular polyribonucleotide including from 1 to 20 moieties (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, and 20) that binds specifically to a region of the circular polyribonucleotide conjugated to a linker and a targeting moiety. For example, the circular polyribonucleotide may be complexed with Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5; for example, as shown in FIG. 1 . In some embodiments, n is an integer from 1 to 5 (e.g., 1 , 2, 3, 4, and 5).
In some embodiments, the disclosure provides a complex including a circular polyribonucleotide, one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) moieties that each binds specifically to a region of the circular polyribonucleotide and includes one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) photoreactive crosslinking agents or covalent attachments, a linker, and one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeting moieties.
In some embodiments, the disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide, a linker, and two or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeting moieties. In some embodiments, the target moieties are the same targeting moiety. In some embodiments, the targeting moieties are different from one another. Examples of combinations of targeting moieties are described in Table 1. The targeting moiety may be a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid (e.g., an aptamer), or a combination thereof. In some embodiments, the targeting moiety is not an aptamer. In some embodiments, the targeting moiety is biotinylated.
Table 1. Combinations of Targeting Moieties
Figure imgf000027_0001
The circular polyribonucleotide may be bound to two targeting moieties (e.g., Xn(L-B)z includes B1-L-X-L-B2 wherein each of B1 and B2 is an independently selected targeting moiety selected from any targeting moiety described herein, and wherein z is 2). For example, that circular polyribonucleotide may be bound to a GalNAc targeting moiety and a tocopherol targeting moiety. In some embodiments, the two targeting moieties are a GalNAc targeting moiety and a cholesterol targeting moiety. In some embodiments, both targeting moieties are lipid targeting moieties. For example, the two lipid targeting moieties may be a tocopherol and a cholesterol targeting moiety.
The circular polyribonucleotide may be bound to 1 , 2, 3, 4, or 5 targeting moieties (e.g., Xn(L-B)z, wherein z is 1 , 2, 3, 4, or 5, and Bz is up to 5 independently selected targeting moieties selected from any targeting moiety described herein). In some embodiments, all targeting moieties are different. For example, the circular polyribonucleotide may be bound to a GalNAc targeting moiety, a tocopherol targeting moiety, a urea targeting moiety, a melittin targeting moiety, and an aptamer targeting moiety. In some embodiments, all targeting moieties are the same class of moiety, but differ within that class. For example, the circular polyribonucleotide may be bound to four lipid targeting moieties, wherein the lipid targeting moieties are cholesterol, tocopherol, vitamin A, and phosphocholine. In some embodiments, all targeting moieties are the same. For example, the circular polyribonucleotide may be bound to 1 , 2, 3, 4, or 5 GalNAc targeting moieties. In some embodiments, 2, 3, or 4 targeting moieties are the same and 1 targeting moiety is different. In some embodiments, 2 or 3 targeting moieties are the same and 1 or 2 are different. In some embodiments, 2 targeting moieties are the same and 1 , 2, or 3 are different. For example, the circular polyribonucleotide may be bound to two tocopherol targeting moieties, one cholesterol targeting moiety, and one aptamer targeting moiety. In another example, the circular polyribonucleotide may be bound to two tocopherol targeting moieties and two cholesterol moieties. In some embodiments, 2 sets of 2 targeting moieties are the same and one is different. For example, a circular polyribonucleotide may be bound to two GalNAc targeting moieties, two cellulose targeting moieties, and one Transportan targeting moiety.
In some embodiments, the targeting moiety targets (e.g., binds to or delivers the circular polynucleotide preferentially to) a specific cell type; for example, the polypeptide targeting moiety may preferentially target hepatic cells, muscle cells (e.g., skeletal muscle cell), blood cells, bone cells, fat cells, skin cells, nerve cells, endothelial cells, stem cells, sex cells, pancreatic cells, or cancer cells. In particular embodiments, the polypeptides target cancer cells (e.g., tumor cells). In some embodiments, the polypeptide targeting moiety targets cells of a specific organ; for example, the polypeptide targeting moiety may preferentially target cells in the heart, lungs, liver, or kidneys.
Lipid Targeting Moieties
In some embodiments, the targeting moiety complexed to the circular polyribonucleotide is a lipid. Lipid moieties include organic compounds, most preferably organic biomolecules, that are selectively soluble in nonpolar solvents over water. Lipid moieties specifically contemplated by the present invention include, for example, fatty acids, steroids or sterols, glycerides (e.g., monoglycerides, diglycerides, and triglycerides), phospholipids (e.g., phosphatidylcholines), fat-soluble vitamins (e.g., vitamins A, D, E, and K), cationic lipids, ionizable lipids, or zwitterionic lipids.
In some embodiments, the lipid moiety is a fatty acid. A fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. The fatty acid may be naturally occurring, a modified variant of a naturally occurring fatty acid, or a synthetic (e.g., non-naturally occurring fatty acid). Fatty acids are known to those of skill in the art. In some embodiments the fatty acid is a saturated fatty acid. In some embodiments, the fatty acid is an unsaturated fatty acid. In some embodiments, the fatty acid includes a branched or unbranched chain including from 4 to 40 main-chain carbon atoms (e.g., from 4 to 28 main-chain carbon atoms, from 4 to 15 main-chain carbon atoms, from 10 to 30 main-chain carbon atoms, or from 15 to 40 main-chain carbon atoms). In some embodiments, the fatty acid includes squalene, stearic acid, oleic acid, palmitic acid, linoleic acid, stearic acid, lauric acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, a-tocopherol, a-tocopherol succinate, or a retinoic acid (e.g., all-trans-retinoic acid), or an analog or metabolite thereof. In some embodiments, the fatty acid includes one, two, three, four, or five lipid chains (e.g., aliphatic lipids selected from above).
In some embodiments, the lipid moiety is a steroid or a sterol. Steroid and sterols are known to those of skill in the art. In general, a steroid is an organic compound with four rings in a fused ring molecular configuration, in particular, a having a core cyclopentanoperhydrophenanthrene ring system. A sterol is any steroid-based alcohol. Steroids and sterols may be naturally occurring or synthetic. In some embodiments, the steroid or sterol is selected from cholesterol, ursodeoxycholic acid, lithocholic acid, or an analog or metabolite thereof.
In some embodiments, the lipid moiety is a fat-soluble vitamin. In some embodiments, the fat- soluble vitamin is selected from vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof.
In some embodiments, the lipid moiety is a phospholipid. Phospholipids are a class of lipids whose molecule has a hydrophilic "head" containing a phosphate group, and one or more (e.g., two) hydrophobic "tails" derived from fatty acids, often joined by an alcohol residue. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine. In some embodiments, the phospholipid is a phosphatidylcholine (e.g., phospholipids that incorporate choline as a headgroup). In some embodiments, the phosphatidylcholine is selected from phosphocholine (PC), PC- docosahexaenoic acid (PC-DHA), PC-docosanoic acid (PC-DCA), PC-eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC-retinoic acid (PC-RA), or PC-a-tocopherol succinate (PC-TS).
Any of the above-described lipid moieties may be chemically-modified (e.g., derivatized or conjugate to a linker) to allow for conjugation to a nucleic acid (e.g., an oligonucleotide or a circular polyribonucleotide). Any of the above-described lipid moieties may also be biotinylated to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
Small Molecule Targeting Moieties
The disclosure provides small molecule targeting moieties which may form a complex with a circular polyribonucleotide. Small molecule targeting moiety have an average molecular weight less than 1 kDa, corresponding to a Stokes-Einstein radius of 1 nm. For example, the small molecule may have a molecular weight of from about 300 to about 700 Da (e.g., about 325 Da, about 350 Da, about 375 Da, about 400 Da, about 425 Da, about 450 Da, about 475 Da, about 500 Da, about 525 Da, about 550 Da, about 575 Da, about 600 Da, about 625 Da, about 650 Da, or about 675 Da). In some embodiments, the small molecule may have a molecular weight of from 500 Da to 1000 Da (e.g., from about 500 Da to 900 Da, about 500 Da to 800 Da, 500 Da to 700 Da, 500 Da to 600 Da, 600 Da to 1000 Da, 700 Da to 1000 Da, 800 Da to 1000 Da, or 900 Da to 1000 Da).
Small molecule targeting moieties, upon reaching the target tissue, must cross the plasma membrane to reach the cytosol or nucleus of the cell. The plasma membrane is relatively permeable to hydrophobic small molecules. However, the transport of macromolecules requires an active uptake mechanism, which requires the small molecule have efficient interactions with the plasma membrane and subsequent endocytosis processes, such as phagocytic-, clathrin-, and caveolae-mediated endocytosis. The small molecule binding targeting moieties specifically bind to a protein of a cell and the protein mediates internalization of the circular polyribonucleotide bound to the targeting small molecule moiety into the cell upon binding to the targeting small molecule moiety.
In some embodiments, the small molecule targeting moiety is folic acid. Folic acid may enable efficient intracellular delivery of the circular polyribonucleotide, following folic acid receptor mediated endocytosis. In some embodiments, the small molecule targeting molecule is urea. Urea may be internalized into the cell using clathrin-mediated endocytosis to deliver the attached circular polyribonucleotide. In some embodiments, the small molecule may be glutamate urea or 2-[3-(1 ,3- dicarboxypropyl)-ureido] pentanedioic acid. Urea may be used as a small molecule targeting moiety to target the prostate specific membrane antigen (PSMAIn some embodiments, the small molecule is ursodexoycholic acid. In some embodiments, the small molecule is lithocholic acid. In some embodiments, glycyrrhetinic acid derivatives may be the small molecule targeting moiety. Glycyrrhetinic acid derivatives may be used for hepatocellular applications due to presence of glycyrrhetinic acid receptors. In some embodiments, a sulfonamide may be used as the small molecule targeting moiety. Sulfonamide derivatives may be used to target tumors expressing carbonic anhydrase IX. In some embodiments, a benzamide may be used as the small molecule targeting moiety. Benzamides, such as anisamide, may be used to specifically target sigma-1 receptors. In some embodiments, phenyl boronic acid may be used as the small molecule targeting moiety. Phenyl boronic acid may be used to target components of sialic acid. In some embodiments, hyaluronic acid may be used as the small molecule targeting moiety. Hyaluronic acid has been shown to have affinity toward CD44 receptors. In some embodiments, bisphosphonate may be used as the small molecule targeting moiety. Bisphosphonates may be used to effectively target bone. In some embodiments, biotin may be used as the small molecule targeting moiety. Biotin-mediated drug delivery may be used for tumor targeting applications by way of the biotin receptor. In some embodiments, biotin may be conjugated to any of the above-described small molecules to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
Carbohydrate Targeting Moieties
The disclosure provides carbohydrate targeting moieties which may form a complex with a circular polyribonucleotide. The carbohydrate targeting moiety may, for example, include a saccharide, disaccharide, or polysaccharide. In some embodiments, the carbohydrate includes mannose, galactose, or glucose. In some embodiments, a carbohydrate moiety described herein includes one or more monosaccharide moieties. In some embodiments, the one or more monosaccharide moieties includes at least 3 carbon atoms (e.g., arranged in a linear, branched, or cyclic structure) and an oxygen, nitrogen, or sulfur atom, or a fragment or variant of a monosaccharide moiety including at least 3 carbon atoms (e.g., arranged in a linear, branched, or cyclic structure) and an oxygen, nitrogen, or sulfur atom. Each monosaccharide moiety or fragment or variant thereof may be a tetrose, pentose, hexose, or heptose . Each monosaccharide moiety or fragment or variant thereof may exist as an aldose, ketose, sugar alcohol, and, where appropriate, in the L or D form. Exemplary monosaccharide moieties may be amino sugars, N-acetylamino sugars, imino sugars, deoxysugars, or sugar acids. Carbohydrates may include individual monosaccharide moieties, or may further include a disaccharide, oligosaccharide (e.g., a trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide), a polysaccharide, or combinations thereof. Exemplary carbohydrates include ribose, arabinose, lyxose, xylose, deoxyribose, ribulose, xylulose, glucose, galactose, mannose, gulose, idose, talose, allose, altrose, psicose, fructose, sorbose, tagatose, rhamnose, pneumose, quinovose, fucose, mannuheptulose, sedoheptulose, galactosamine, mannosamine, glucosamine, N-acetylglucosamine, N- acetylgalactosamine, N-acetylmannosamine, glucuronic acid, galacturonic acid, mannuronic acid, guluronic acid, iduronic acid, tagaturonic acid, frucuronic acid, galactosaminuronic acid, mannosaminuronic acid, glucosaminuronic acid, N-acetylglucosaminuronic acid, N- acetylgalactosaminuronic acid, N-acetylmannosaminuronic acid, maltose, lactose, sucrose, trehalose, gentiobiose, cellobiose, chitobiose, kojibiose, nigerose, sophorose, trehalulose, isomaltose, xylobiose, starch, cellulose, chitin, and dextran. The carbohydrate moiety may include one or more monosaccharide moieties linked by a glycosidic bond. In some embodiments, the glycosidic bond includes a 1 ^2 glycosidic bond, a 1^3 glycosidic bond, a 1^4 glycosidic bond, or a 1^6 glycosidic bond. In some embodiments, each glycosidic bonds may be present in the alpha or beta configuration. In an embodiment, the one or more monosaccharide moieties are linked directly by a glycosidic bond or are separated by a linker.
The term “carbohydrate” as used herein refers to compound including one or more monosaccharide moieties including at least 3 carbon atoms (e.g., arranged in a linear, branched, or cyclic structure) and an oxygen, nitrogen, or sulfur atom, or a fragment or variant of a monosaccharide moiety including at least 3 carbon atoms (e.g., arranged in a linear, branched, or cyclic structure) and an oxygen, nitrogen, or sulfur atom. Each monosaccharide moiety or fragment or variant thereof may be a tetrose, pentose, hexose, or heptose. Each monosaccharide moiety or fragment or variant thereof may exist as an aldose, ketose, sugar alcohol, and, where appropriate, in the L or D form. Exemplary monosaccharide moieties may be amino sugars, N-acetylamino sugars, imino sugars, deoxysugars, or sugar acids. Carbohydrates may include individual monosaccharide moieties, or may further include a disaccharide, oligosaccharide (e.g., a trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide), a polysaccharide, or combinations thereof. Exemplary carbohydrates include ribose, arabinose, lyxose, xylose, deoxyribose, ribulose, xylulose, glucose, galactose, mannose, gulose, idose, talose, allose, altrose, psicose, fructose, sorbose, tagatose, rhamnose, pneumose, quinovose, fucose, mannuheptulose, sedoheptulose, galactosamine, mannosamine, glucosamine, N- acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, glucuronic acid, galacturonic acid, mannuronic acid, guluronic acid, iduronic acid, tagaturonic acid, frucuronic acid, galactosaminuronic acid, mannosaminuronic acid, glucosaminuronic acid, N-acetylglucosaminuronic acid, N- acetylgalactosaminuronic acid, N-acetylmannosaminuronic acid, maltose, lactose, sucrose, trehalose, gentiobiose, cellobiose, chitobiose, kojibiose, nigerose, sophorose, trehalulose, isomaltose, xylobiose, starch, cellulose, chitin, and dextran.
The carbohydrate may include one or more monosaccharide moieties linked by a glycosidic bond. In some embodiments, the glycosidic bond includes a 1->2 glycosidic bond, a 1->3 glycosidic bond, a 1->4 glycosidic bond, or a 1->6 glycosidic bond. In some embodiments, each glycosidic bonds may be present in the alpha or beta configuration. In an embodiment, the one or more monosaccharide moieties are linked directly by a glycosidic bond or are separated by a linker.
In some embodiments, the present disclosure features a circular polyribonucleotide complexed with a carbohydrate targeting moiety, wherein the carbohydrate targeting moiety includes an asialoglycoprotein receptor (ASGPR) binding moiety. The ASGPR is a C-type lectin primarily expressed on the sinusoidal surface of hepatocytes, and includes a major (48 kDa, ASGPR-1) and a minor (40 kDa, ASGPR-2) subunit. The ASGPR is involved in the binding, internalization, and subsequent clearance of glycoproteins containing an N-terminal galactose (Gal) or N-terminal N-acetylgalactosamine (GalNAc) residues from circulation, such as antibodies. ASGPRs have also been shown to be involved in the clearance of low-density lipoprotein, fibronection, and certain immune cells, and may be utilized by certain viruses for hepatocyte entry (see, e.g., Yang J., et al (2006) J Viral Hepat 13:158-165 and Guy, CS et al (2011) Nat Rev Immunol 8:874-887).
In some embodiments, the carbohydrate targeting moiety is mannose. For example, carbohydrate targeting moiety may be a-mannose or high-mannose. In some embodiments, the carbohydrate targeting moiety includes a mannose 6-phosphate (M6P) or analog thereof. In an embodiment, the carbohydrate targeting moiety include a plurality of M6P moieties (e.g., M6Ps), e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more M6P moieties. In an embodiment, the carbohydrate targeting moiety includes from 2 to 20 M6P moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 M6P moieties). In an embodiment, the carbohydrate targeting moiety includes from 2 to 10 M6P moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 M6P moieties). In an embodiment, the carbohydrate targeting moiety includes from 2 to 5 M6P moieties (e.g., 2, 3, 4, or 5 M6P moieties).
In some embodiments, the carbohydrate targeting moiety includes a galactose (Gal), galactosamine (GalNH2), or an N-acetylgalactosamine (GalNAc) moiety, for example, a Gal, GalNH2, or GalNAc, or an analog thereof. In an embodiment, the carbohydrate targeting moiety includes a GalNAc moiety (e.g., GalNAc). In an embodiment, the carbohydrate targeting moiety includes a plurality of GalNAc moieties (e.g., GalNAcs), e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more GalNAc moieties (e.g., GalNAcs). In an embodiment, the carbohydrate targeting moiety includes from 2 to 20 GalNAcs moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 GalNAc moieties). In an embodiment, the carbohydrate targeting moiety includes from 2 to 10 GalNAc moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 GalNAc moieties). In an embodiment, the carbohydrate targeting moiety includes from 2 to 5 GalNAc moieties (e.g., 2, 3, 4, or 5 GalNAc moieties). In an embodiment, the carbohydrate targeting moiety includes 2 GalNAc moieties. In an embodiment, the carbohydrate targeting moiety includes 3 GalNAc moieties. In an embodiment, the carbohydrate targeting moiety includes 4 GalNAc moieties. In an embodiment, the carbohydrate targeting moieties includes 5 GalNAc moieties. In some embodiments, the carbohydrate targeting moiety includes a mono-, di-, tri-, or tetra-GalNAc
In some embodiments, the GalNAc moiety includes a structure of Formula (I):
Figure imgf000032_0001
salt thereof, wherein X is O, N(R7), or S; each of R1, R3, R4, and R5 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, C(O)-alkyl, C(O)-alkenyl, C(O)-alkynyl, C(O)-heteroalkyl, C(O)-haloalkyl, C(O)-aryl, C(O)- heteroaryl, C(O)-cycloalkyl, or C(O)-heterocyclyl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl is optionally substituted with one or more R8; R2a is hydrogen or alkyl; R2b is -C(O)alkyl (e.g., C(O)CH3); each of R6a and R6b is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, halo, cyano, nitro, -ORA, aryl, heteroaryl, cycloalkyl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl is optionally substituted with one or more R9; R7 is hydrogen, alkyl, or C(O)-alkyl; each of R8 and R9 is independently hydrogen, halo, cyano, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, cycloalkyl, or heterocyclyl; and RA is hydrogen, or alkyl, alkenyl, alkynyl, wherein the structure of Formula (I) may be connected to a linker at any position.
In some embodiments, X is O. In some embodiments, each of R1, R3, R4, and R5 are independently hydrogen or alkyl (e.g., CH3). In some embodiments, R2a is hydrogen. In some embodiments, R2b is C(O)CH3. In some embodiments, each of R6a and R6b is hydrogen. In some embodiments, the GalNAc moiety is connected to a linker at R2a. In some embodiments, the GalNAc moiety is connected to a linker at R2b. In some embodiments, the GalNAc moiety is connected to a linker or at R3. In some embodiments, the GalNAc moiety is connected to a linker at R4. In some embodiments, the GalNAc moiety is connected to a linker at R5. In some embodiments, the GalNAc moiety is connected to a linker at R6a or R6b. In some embodiments, the GalNAc moiety is connected to a linker at a plurality of positions, e.g., at least two of R1, R2a, R2b, R3, R4, R5, R6a, and R6b.
In some embodiments, the GalNAc moiety is includes a structure of Formula (l-a)
Figure imgf000033_0001
(|_a) or a sa|| thereof, wherein R2a is hydrogen or alkyl; R2b is -C(O)alkyl (e.g., C(O)CH3); each of R3, R4, and R5 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, C(O)-alkyl, C(O)-alkenyl, C(O)-alkynyl, C(O)- heteroalkyl, C(O)-haloalkyl, C(O)-aryl, C(O)-heteroaryl, C(O)-cycloalkyl, or C(O)-heterocyclyl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl is optionally substituted with one or more R8; and R8 is hydrogen, halo, cyano, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, cycloalkyl, or heterocyclyl, wherein the represents a bond in any configuration, and represents an attachment point to a linker.
In some embodiments, each of R3, R4, and R5 are independently hydrogen or alkyl (e.g., CH3). In some embodiments, R2a is hydrogen. In some embodiments, R2b is C(O)CH3.
In some embodiments, the carbohydrate targeting moiety includes a structure of Formula (II):
Figure imgf000033_0002
(II), or a salt thereof, wherein each of R1, R2a, R2b, R3,
R4, R5, R6a, and R6b and subvariables thereof are as defined for Formula (I), L is a linker, and n is an integer from 1 to 100, wherein represents an attachment point to a branching point, additional linker.
In some embodiments, X is O. In some embodiments, each of R1, R3, R4, and R5 are independently hydrogen or alkyl (e.g., CH3). In some embodiments, R2a is hydrogen. In some embodiments, R2b is C(O)CH3. In some embodiments, each of R6a and R6b is hydrogen. In some embodiments, n is an integer from 1 to 50. In some embodiments, n is an integer from 1 to 25. In some embodiments, n is an integer from 1 to 10. In some embodiments, n is an integer from 1 to 5. In some embodiments, n is 1 , 2, 3, 4, or 5. In some embodiments, n is 1.
In some embodiments, the carbohydrate targeting moiety includes a structure of Formula (ll-a):
Figure imgf000034_0001
(I l-a) , or a salt thereof, wherein each of
R1, R2a, R2b, R3, R4, R5, R6a, and R6b and subvariables thereof are as defined for Formula (I), each of L1 and L2 is independently a linker, each of m and n is independently an integer from 1 to 100, and M is a linker, wherein represents an attachment point to a branching point, additional linker.
In some embodiments, X is O (e.g., X in each of A and B is O). In some embodiments, each of R1, R3, R4, and R5 are independently hydrogen or alkyl (e.g., CH3) (e.g., R1, R3, R4, and R5 in each of A and B is independently hydrogen or alkyl). In some embodiments, R2a is hydrogen (e.g., R2a in each of A and B is hydrogen). In some embodiments, R2b is C(O) CH3 (e.g., R2b in each of A and B is C(O)CH3). In some embodiments, each of R6a and R6b is hydrogen (e.g., R6a and R6b in each of A and B is hydrogen). In some embodiments, each of m and n is independently an integer from 1 to 50. In some embodiments, each of m and n is independently an integer from 1 to 25. In some embodiments, each of m and n is independently an integer from 1 to 10. In some embodiments, each of m and n is independently an integer from 1 to 5. In some embodiments, each of m and n is independently 1 , 2, 3, 4, or 5. In some embodiments, each of m and n is independently 1.
In an embodiment, each of L1 and L2 independently includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, each of L1 and L2 independently includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, each of L1 and L2 independently is cleavable or non-cleavable.
In some embodiments, M includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, M includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, M is cleavable or non-cleavable. In some embodiments, the ASGPR moiety includes a structure of Formula (I l-b):
Figure imgf000035_0001
(I l-b), or a salt thereof, wherein each of R1,
R2a, R2b, R3, R4, R5, R6a, and R6b and subvariables thereof are as defined for Formula (I), each of L1 , L2, and L3 is independently a linker, each of m, n, and o is independently an integer from 1 to 100, and M is a linker, wherein represents an attachment point to a branching point, additional linker.
In some embodiments, X is O (e.g., X in each of A, B, and C is O). In some embodiments, each of R1, R3, R4, and R5 are independently hydrogen or alkyl (e.g., CH3) (e.g., R1, R3, R4, and R5 in each of A, B, and C is independently hydrogen or alkyl). In some embodiments, R2a is hydrogen (e.g., R2a in each of A, B, and C is hydrogen). In some embodiments, R2b is C(O)CH3 (e.g., R2b in each of A, B, and C is C(O)CH3). In some embodiments, each of R6a and R6b is hydrogen (e.g., R6a and R6b in each of A, B, and
C is hydrogen). In some embodiments, each of m, n, and 0 is independently an integer from 1 to 50. In some embodiments, each of m, n, and 0 is independently an integer from 1 to 25. In some embodiments, each of m, n, and 0 is independently an integer from 1 to 10. In some embodiments, each of m, n, and 0 is independently an integer from 1 to 5. In some embodiments, each of m, n, and 0 is independently 1 , 2, 3, 4, or 5. In some embodiments, each of m, n, and 0 is independently 1 .
In an embodiment, each of L1, L2, and L3 independently includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, each of L1 , L2, and L3 independently includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, each of L1, L2, and L3 independently is cleavable or non-cleavable.
In some embodiments, M includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, M includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, M is cleavable or non-cleavable. In some embodiments, the carbohydrate targeting moiety includes a structure of Formula (I l-c):
Figure imgf000036_0002
(I l-c) , or a salt thereof, wherein each of R2a, R2b, R3, R4,
R5, and subvariables thereof are as defined for Formula (I), each of L1, L2, and L3 is independently a linker, and M is a linker, wherein
Figure imgf000036_0001
represents an attachment point to a branching point, additional linker.
In some embodiments, each of R3, R4, and R5 are independently hydrogen or alkyl (e.g., CH3). In some embodiments, R2a is hydrogen. In some embodiments, R2b is C(O)CH3.
In an embodiment, each of L1, L2, and L3 independently includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, each of L1 , L2, and L3 independently includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, each of L1, L2, and L3 independently is cleavable or non-cleavable.
In some embodiments, M includes an alkylene, alkenylene, alkynylene, heteroalkylene, or haloalkylene group. In an embodiment, M includes an ester, amide, disulfide, ether, carbonate, aryl, heteroaryl, cycloalkyl, or heterocyclyl group. In an embodiment, M is cleavable or non-cleavable.
In some embodiments, the carbohydrate targeting moiety includes a compound selected from:
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
In some embodiments, the carbohydrate targeting moiety includes a linker including a cyclic moiety, such as a pyrroline ring. In an embodiment, the carbohydrate targeting moiety includes a structure of Formula (CH):
Formula (CII)
Figure imgf000040_0002
, or a salt thereof, wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO2, or SO2NH; R11, R12, R13, R14, R15, R16, R17, and R18 are each independently for each occurrence H, — CH2ORa, or ORb; Ra and Rb are each independently for each occurrence hydrogen, a hydroxyl protecting group, optionally substituted alkyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted alkenyl, optionally substituted heteroaryl, polyethyleneglycol (PEG), a phosphate, a diphosphate, a triphosphate, a phosphonate, a phosphonothioate, a phosphonodithioate, a phosphorothioate, a phosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a phosphotriester, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, — P(Z1)(Z2) — O- nucleoside, — P(Z1)(Z2) — O-oligonucleotide, — P(Z1)(O-linker-RL) — O-nucleoside, or — P(Z1)(O-linker-
RL) — O-oligonucleotide; R30 is independently for each occurrence -linker-RL or R31; RL is hydrogen or a ligand; R31 is — C(O)CH(N(R32)2)(CH2)hN(R32)2; R32 is independently for each occurrence H, — RL, -linker- RL or R31; Z1 is independently for each occurrence O or S; Z2 is independently for each occurrence O, S, N(alkyl) or optionally substituted alkyl; and h is independently for each occurrence 1 -20. In some embodiments, the compound of Formula (Cll) is selected from:
Figure imgf000041_0001
Figure imgf000042_0001
In some embodiments, the carbohydrate targeting moiety is a compound or substructure disclosed in U.S. Patent No. 8,106,022, which is incorporated herein by reference in its entirety. In other embodiments, the carbohydrate targeting moiety is selected from:
Figure imgf000042_0002
Figure imgf000043_0001
wherein one ofX or Y is a branching point or a linker, and the other of X and Y is hydrogen.
In an embodiment, the ASGPR moiety includes a structure of Formula (Xll-a):
Figure imgf000044_0001
. In an embodiment, the carbohydrate targeting moiety is a compound or substructure disclosed in Nucleic Acids (2016) 5:e317 or WO2015/042447, each of which is incorporated herein by reference in its entirety.
In some embodiments, the carbohydrate targeting moiety includes a structure of Formula (V-a):
Figure imgf000044_0002
wherein n is an integer from 1 to 20.
In some embodiments, the compound of Formula (V-a) is selected from:
Figure imgf000045_0001
(V-a-iii), wherein Z is an oligomeric compound, e.g., a linker.
In another embodiment, the carbohydrate binding moiety includes a structure of Formula (V-b):
Figure imgf000046_0001
wherein A is O or S, A’ is O, S, or
NH, and Z is an oligomeric compound, e.g., a linker.
In some embodiments, the carbohydrate targeting moiety includes
Figure imgf000046_0002
In some embodiments, the carbohydrate targeting moiety is selected from:
Figure imgf000046_0003
(V-c-i),
Figure imgf000047_0001
(V-e-i).
In an embodiment, the carbohydrate targeting moiety is a compound or substructure disclosed in WO 2017/156012, which is incorporated herein by reference in its entirety. In some embodiments, a hydroxyl group within a carbohydrate targeting moiety is protected, for example, with an acetyl or acetonide moiety. In some embodiments, a hydroxyl group within a carbohydrate targeting moiety is protected with an acetyl group. In some embodiments, a hydroxyl group within an ASGPR moiety is protected with acetonide group. For example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or more hydroxyl groups within a carbohydrate targeting moiety may be protected, e.g., with an acetyl group or an acetonide group. In some embodiments, all of the hydroxyl groups within a carbohydrate targeting moiety are protected.
In some embodiments, the carbohydrate targeting moiety includes an additional active agent, such as a ligand (e.g., a steroid). The ligand may be covalently or non-covalently associated with the carbohydrate targeting moiety. For example, the ligand may be covalently bound to a carbohydrate, linker, or a branching point within the carbohydrate targeting moiety. In some embodiments, the carbohydrate targeting moiety includes
Figure imgf000048_0001
wherein one of X or Y is a branching point or linker, and the other of X and Y is hydrogen.
Additional exemplary carbohydrate targeting moieties are described in further detail in U.S.
Patent Nos. 8,828,956; 9,867,882; 10,450,568; and 10,808,246, each of which are incorporated herein by reference in its entirety.
Any of the above-described carbohydrate targeting moieties may also be biotinylated to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
Polypeptide Targeting Moieties
The disclosure provides polypeptide targeting moieties which may form a complex with a circular polyribonucleotide. The polypeptide targeting moieties may also be referred to as protein drug conjugates. The polypeptide targeting moieties specifically bind to a protein of a cell and the protein mediates internalization of the circular polyribonucleotide bound to the targeting polypeptide moiety into the cell upon binding to the targeting polypeptide moiety.
The polypeptide targeting moiety may include all natural amino acids. In some embodiments, the polypeptide targeting moiety includes at least one unnatural amino acid reside; for example, the polypeptide targeting moiety may include 1 , 2, 3, 4, 5, 6, 8, 9, 10, or more unnatural amino acid residues. In some embodiments, the polypeptide targeting moiety may include all unnatural amino acid residues.
In some embodiments, the polypeptide targeting moiety is a cell-penetrating peptide. A cell penetrating peptide may include from 2 to 100 amino acid residues (e.g., from 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 10 to 100, 20 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, and 90 to 100 amino acid residues). In some embodiments, the polypeptide targeting moiety includes from 2 to 50 amino acids (e.g., from 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 and 20, 2 to 15, 2 to 10, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, and 45 to 50 amino acid residues).
Cell-penetrating peptides may be characterized as a synthetic cell-penetrating peptides, protein- derived cell-penetrating peptides, and chimeric cell-penetrating peptides. Furthermore, cell-penetrating peptides may be either linear or cyclic. Cell-penetrating peptides may also be categorized by physical properties and include cationic cell-penetrating peptides, amphipathic cell-penetrating peptides, or hydrophobic cell-penetrating peptides. The cell-penetrating peptide may be a one of the as described in Table 2. In some embodiments, the cell-penetrating peptide is melittin. In some embodiments, the cell-penetrating peptide is a TAT peptide. In some embodiments, the cell-penetrating peptide is Pep-1. In some embodiments, the cell- penetrating peptide is Transportan. In some embodiments, the cell-penetrating peptide is Penetratin. Further examples of cell-penetrating peptides may be found on the CPPsite 2.0 Database of Cell- Penetrating Peptides (webs.iiitd.edu.in/raghava/cppsite/information.php). This database contains >1500 cell-penetrating peptide sequences.
Table 2. Exemplary Cell-Penetrating Peptides
Figure imgf000049_0001
In some embodiments, cell-penetrating peptides may transport hydrophilic macromolecules to a cell through energy-independent pathways. Cell-penetrating peptides may enter the cell in a noninvasive way, as they usually do not disturb the structure of the plasma membranes and are considered safe and highly efficient. Some cell-penetrating peptides are reported to cross the cell membrane by an energy- dependent cellular process using, for example, endocytosis or receptor-mediated uptake, whereas others use energy-independent non-endocytic translocation pathways.
In some embodiments, the polypeptide targeting moiety binds to a receptor on a cell surface. In some embodiments, the polypeptide binds to the receptor with high affinity. In some embodiments, the receptor is overexpressed on the cell surface. In some embodiments, the polypeptide includes an Arg- Gly-Asp (RGD) motif. In some embodiments, the RGD motif binds integrins. In some embodiments, the RGD motif is a cyclic RGD motif. In some embodiments, the cyclic RGD motif includes the amino acid sequence of RGDFK (SEQ ID NO: 16). In some embodiments, the cyclic RGD may be an internalizing RGD conjugate having an amino acid sequence of CRGDKRGPDC (SEQ ID NO: 17).
In some embodiments, the polypeptide targeting moiety targets the circular polyribonucleotide to muscle cells. In some embodiments, this polypeptide is a M12 peptide having the amino acid sequence RRQPPRSISSHP (SEQ ID NO: 18). In some embodiments, the muscle targeting polypeptide is the 7 amino acid residue peptide ASSLNIA (SEQ ID NO: 19). In some embodiments, the polypeptide is an LPS-binding protein (LBP) peptide. In some embodiments, the polypeptide is an adipose-homing peptide. In some embodiments, the peptide is an endolytic peptide. For example, the endolytic peptide may have the structure H2N-CKRKKRRQRRRG(dPEG6)GWWG(K/AzidePEG10)-amide. In some embodiments, the peptide is cyclo-F<t>RRRRQ.
In some embodiments, the polypeptide is melittin. In some embodiments, the melittin targets tumor cells.
In some embodiments, the polypeptide targets a specific cell type; for example, the polypeptide may preferentially target muscle cells (e.g., skeletal muscle cell), blood cells, bone cells, fat cells, skin cells, nerve cells, endothelial cells, stem cells, sex cells, pancreatic cells, or cancer cells. In particular embodiments, the polypeptides target cancer cells (e.g., tumor cells). In some embodiments, the polypeptide targets cells of a specific organ; for example, the polypeptide may preferentially target cells in the heart, lungs, liver, or kidneys.
In some embodiments, the polypeptide targeting moiety is an antibody, which may also then be known an antibody drug conjugate. The antibody may target an overexpressed antigen or receptor on the surface of a cell. In some embodiments, the polypeptide targeting moiety is an antibody fragment. In some embodiments, the polypeptide targeting moiety is a single chain Fv molecule (scFv), a diabody, a triabody, a nanobody, a domain antibody, an antibody-like protein scaffold, a Fab fragment, a Fv fragment, a Fab’, a F(ab’)2, a tandem scFv (taFv), or an scFv-Fc. In some embodiments, the polypeptide targeting moiety is a bispecific antibody. In particular embodiments, the peptide targeting moiety is an scFv. In certain embodiments, the polypeptide targeting moiety is a nanobody.
In some embodiments, the antibody may bind to a tumor cell-specific antigen. For example, the antibody may be an anti-CD33 antibody, an anti-CD30 antibody, an anti-HER2 antibody, an anti-CD22 antibody, an anti-EGFR antibody, an anti-nectin4 antibody, an anti HER2 antibody, an anti-AXL antibody, an anti-CD74 antibody, an anti-ALK antibody, an anti-PTK antibody, anti-MR antibody, an anti-CD205 antibody, an anti-CD169 antibody, an anti-CD14 antibody, an anti-CD36 antibody, an anti-CD5 antibody, an anti -CD71 antibody, an anti-CD38 antibody, or an anti-prohibin antibody. In some embodiments, the antibody may bind to a tumor tissue-associated antigen. For example, the antibody may be an anti-PSMA antibody, an anti-TM4SF1 antibody, or an anti-CD276 antibody. In particular embodiments, the peptide targeting moiety is an anti-FcRn antibody. In some embodiments, the peptide targeting moiety is an anti- MR antibody. In some embodiments, the peptide targeting moiety is an anti-CD205. In some embodiments, the peptide targeting moiety is an anti-CD14 antibody. In some embodiments, the peptide targeting moiety is an anti-CD36 antibody. In some embodiments, the peptide targeting moiety is an anti- CD5 antibody. In some embodiments, the peptide targeting moiety is an anti-CD71 Fab. In some embodiments, the peptide targeting moiety is an anti-prohibin. In some embodiments, the peptide targeting moiety is an anti-DEC205 antibody.
In some embodiments, the nanobody may bind to a tumor cell-specific antigen. For example, the nanobody may be an anti-CD33 nanobody, an anti-CD30 nanobody, an anti-HER2 nanobody, an anti- CD22 nanobody, an anti-EGFR nanobody, an anti-transferrin nanobody, an anti-nectin4 nanobody, an anti HER2 nanobody, an anti-AXL nanobody, and anti-CD74 nanobody, an anti-ALK nanobody, or an anti-PTK nanobody. In some embodiments, the nanobody may bind to a tumor tissue-associated antigen. For example, the antibody may be an anti-PSMA nanobody, an anti-TM4SF1 nanobody, or an anti- CD276 nanobody. In some embodiments, the polypeptide targeting moiety is an anti-transferrin nanobody. In some embodiments, the polypeptide targeting moiety is an anti-EGFR nanobody. In some embodiments, the polypeptide targeting moiety is an anti-HER2 nanobody.
Any of the above-described polypeptide moieties may also be biotinylated to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
Aptamer Targeting Moieties
In some embodiments, a targeting moiety is an aptamer. An aptamer is an isolated nucleic acid molecule that binds with high specificity and affinity to a binding moiety or target molecule, such as a protein. An aptamer is a three-dimensional structure held in certain conformation(s) that provides chemical contacts to specifically bind its given target. Although aptamers are nucleic acid-based molecules, there is a fundamental difference between aptamers and other nucleic acid molecules such as genes and mRNA. In the latter, the nucleic acid structure encodes information through its linear base sequence and thus this sequence is of importance to the function of information storage. In complete contrast, aptamer function, which is based upon the specific binding of a target molecule, is not entirely dependent on a conserved linear base sequence (a non-coding sequence), but rather a particular secondary /tertiary/quaternary structure. Any coding potential that an aptamer may possess is fortuitous and is not thought to play a role in the binding of an aptamer to its cognate target. Aptamers are differentiated from naturally occurring nucleic acid sequences that bind to certain proteins. These latter sequences are naturally occurring sequences embedded within the genome of the organism that bind to a specialized sub-group of proteins that are involved in the transcription, translation, and transportation of naturally occurring nucleic acids (e.g., nucleic acid-binding proteins). Aptamers on the other hand are non-naturally occurring nucleic acid molecules. While aptamers can be identified that bind nucleic acid- binding proteins, in most cases such aptamers have little or no sequence identity to the sequences recognized by the nucleic acid-binding proteins in nature. More importantly, aptamers can bind virtually any protein (not just nucleic acid-binding proteins) as well as almost any partner of interest including small molecules, carbohydrates, peptides, etc. For most partners, even proteins, a naturally occurring nucleic acid sequence to which it binds does not exist. For those partners that do have such a sequence, e.g., nucleic acid-binding proteins, such sequences will differ from aptamers as a result of the relatively low binding affinity used in nature as compared to tightly binding aptamers. Aptamers are capable of specifically binding to selected partners and modulating the partner’s activity or binding interactions, e.g., through binding, aptamers may block their partner’s ability to function. The functional property of specific binding to a partner is an inherent property an aptamer. An aptamer can be 6-35 kDa. An aptamer can be from 20 to 500 nucleotides. An aptamer can bind its partner with micromolar to sub-nanomolar affinity, and may discriminate against closely related targets (e.g., aptamers may selectively bind related proteins from the same gene family). In some cases, an aptamer only binds one molecule. In some cases, an aptamer binds family members of a molecule of interest. An aptamer, in some cases, binds to multiple different molecules. Aptamers are capable of using commonly seen intermolecular interactions such as hydrogen bonding, electrostatic complementarities, hydrophobic contacts, and steric exclusion to bind with a specific partner. Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, low immunogenicity, biological efficacy, and excellent pharmacokinetic properties. An aptamer can include a molecular stem and loop structure formed from the ou hybridization of complementary polynucleotides that are covalently linked (e.g., a hairpin loop structure). The stem includes the hybridized polynucleotides and the loop is the region that covalently links the two complementary polynucleotides. An aptamer can be a linear ribonucleic acid (e.g., linear aptamer) including an aptamer or a circular polyribonucleic acid including an aptamer (e.g., a circular aptamer).
In some embodiments, one or more of the aptamer targeting moieties includes aptamer K19, M2 aptamer, Ly75 aptamer, aptamer CTApt-268, Aptamer14, Aptamer NAFLD01 , TfnR-aptamer, DEC205 aptamer (GGGAGGUGUGUUAGCACACGAUUCAUAAUCAGCUACCCUCCC (SEQ ID NO: 20)),, or MA33 aptamer (GTTACCGCGGTGAAGGGTGGATGTGTCTGGA (SEQ ID NO: 21)), A01 B aptamer (CAGGAGCCGAGAACCGGTTGGTGGGTAATCCTGTTAGCGC (SEQ ID NO: 22)), HG19 aptamer (GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCC (SEQ ID NO: 23)), C2.min aptamer (GGGGGAUCAAUCCAAGGGACCCGGAAACGCUCCCUUACACCCC (SEQ ID NO: 87)), DC aptamer (AGGGTTACTCGGAT (SEQ ID NO: 24)), A2 aptamer (GAAGAGTAGATGAAACGTTTTTTCGCCCGATAAAAGGGACGTGCGTCAGACA (SEQ ID NO: 25)), or WAZ aptamer (GGGUUCUACGAUAAACGGUUAAUGACCAGCUUAUGGCUGGCAGUUCCC (SEQ ID NO: 26)), or A1411 aptamer (GGTGGTGGTGGTTGTGGTGGTGGTGG (SEQ ID NO: 95).
Any of the above-described aptamer targeting moieties may also be biotinylated to facilitate binding to a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin).
Linkers
In some embodiments, a linker provides space, rigidity, and/or flexibility between the moiety which forms a complex with the circular polyribonucleotide and the targeting moiety described herein. In some embodiments, the disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide, two or more linkers (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), and two or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeting moieties (e.g., X-(L-B)Z, wherein z is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the linkers are the same linker. In some embodiments, the linkers are different from one another. In some embodiments, some of the linkers are the same linker and some of the linkers are different from one another.
In some embodiments, a linker may be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a C-0 bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation. In some embodiments, a linker includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1- 180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). In some embodiments, a linker includes no more than 250 non-hydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1- 30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1- 130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 non-hydrogen atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non- hydrogen atom(s)). In some embodiments, the backbone of a linker includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). In some embodiments, the linker includes from 1 to 1000 atoms (e.g., from 1 to 750 atoms, 1 to 500 atoms, 1 to 250 atoms, 1 to 100 atoms, 1 to 75 atoms, 1 to 50 atoms, 1 to 25 atoms, and 1 to 10 atoms). In some embodiments, the linker includes from 1 to 100 atoms. In some embodiments, the linker includes from 1 to 50 atoms. In some embodiments, the linker includes from 1 to 25 atoms.
In some embodiments, the linker is linear and includes from 1 to 1000 atoms (e.g., from 1 to 750 atoms, 1 to 500 atoms, 1 to 250 atoms, 1 to 100 atoms, 1 to 75 atoms, 1 to 50 atoms, 1 to 25 atoms, and 1 to 10 atoms). In some embodiments, the linker is linear and includes from 1 to 100 atoms. In some embodiments, the linker is linear and includes from 1 to 50 atoms. In some embodiments, the linker is linear and includes from 1 to 25 atoms.
In some embodiments, the linker is branched, and each branch includes from 1 to 1000 atoms (e.g., from 1 to 750 atoms, 1 to 500 atoms, 1 to 250 atoms, 1 to 100 atoms, 1 to 75 atoms, 1 to 50 atoms, 1 to 25 atoms, and 1 to 10 atoms). In some embodiments, the linker is branched, and each branch includes from 1 to 100 atoms. In some embodiments, the linker is branched, and each branch includes from 1 to 50 atoms. In some embodiments, the linker is branched, and each branch includes from 1 to 25 atoms.
The “backbone” of a linker refers to the atoms in the linker that together form the shortest path from one part of the conjugate to another part of the conjugate. The atoms in the backbone of the linker are directly involved in linking one part of the conjugate to another part of the conjugate. For examples, hydrogen atoms attached to carbons in the backbone of the linker are not considered as directly involved in linking one part of the conjugate to another part of the conjugate.
In some embodiments, a linker includes a heteroatom, such as a nitrogen, sulfur, oxygen, phosphorus, silicon, or boron atom. In some embodiments, the linker includes a cyclic group (e.g., an aryl, heteroaryl, cycloalkyl, or heterocyclyl group). In some embodiments, a linker includes a functional group such as an amide, ketone, ester, ether, thioester, thioether, thiol, hydroxyl, amine, cyano, nitro, azide, triazole, pyrroline, p-nitrophenyl, alkene, or alkyne group. Any atom within a linker may be substituted or unsubstituted. In some embodiments, a linker includes an arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alky ny larylalky ny I, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alky ny lary I, alkylheteroaryl, alkenylheteroaryl, or alkynylhereroaryl group.
In some embodiments, a linker includes a polyethylene glycol group (e.g., PEG1 , PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG10, PEG12, PEGU, PEG16, PEG18, PEG20, PEG24, PEG28, PEG32, PEG100, PEG200, PEG250, PEG500, PEG600, PEG700, PEG750, PEG800, PEG900, PEG1000, PEG2000, PEG3000, PEG4000, PEG5000, PEG6000, PEG7000, PEG8000, PEG9000, and PEG10000). In some embodiments, the linker includes at least one PEG unit. For example, the linker may include PEG2 to PEG10000 (e.g., PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG10, PEG12, PEGU, PEG16, PEG18, PEG20, PEG24, PEG28, PEG32, PEG100, PEG200, PEG250, PEG500, PEG600, PEG700, PEG750, PEG800, PEG900, PEG1000, PEG2000, PEG3000, PEG4000, PEG5000, PEG6000, PEG7000, PEG8000, PEG9000, and PEG10000).
In some embodiments, a linker includes a triethylene glycol group (e.g., TEG1 , TEG2, TEG3, TEG4, TEG5, TEG6, TEG7, TEG8, TEG10, TEG12, TEG14, TEG16, TEG18, TEG20, TEG24, TEG28, TEG32, TEG100, TEG200, TEG250, TEG500, TEG600, TEG700, TEG750, TEG800, TEG900, TEG1000, TEG2000, TEG3000, TEG4000, TEG5000, TEG6000, TEG7000, TEG8000, TEG9000, and TEG10000). In some embodiments, the linker includes at least one TEG unit. For example, the linker may include TEG2 to TEG10000 (e.g., TEG2, TEG3, TEG4, TEG5, TEG6, TEG7, TEG8, TEG10, TEG12, TEG14, TEG16, TEG18, TEG20, TEG24, TEG28, TEG32, TEG100, TEG200, TEG250, TEG500, TEG600, TEG700, TEG750, TEG800, TEG900, TEG1000, TEG2000, TEG3000, TEG4000, TEG5000, TEG6000, TEG7000, TEG8000, TEG9000, and TEG10000).
The composition includes at least one linker that connects the targeting moiety to the moiety that binds or conjugates to the circular polyribonucleotide. In some embodiments, the moiety that binds or conjugates to the circular polyribonucleotide is connected to one or more targeting moieties, through a linker as described herein. The linker may be monovalent or multivalent, e.g., bivalent, trivalent, tetravalent, or pentavalent. In some embodiments, the linker includes a structure selected from:
Figure imgf000054_0002
wherein q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, are each independently for each occurrence absent, CO, NH,
Figure imgf000054_0001
O, S, OC(O), NHC(O), CH2, CH2NH or CH2O; Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of
Figure imgf000055_0001
( ) ( ) ( ) ( )
Figure imgf000055_0002
occurrence a targeting moiety; and Ra is H or amino acid side chain.
In some embodiments, the linker includes:
Figure imgf000055_0003
wherein L5A, L5B and L5C represent a targeting moiety, e.g., as described herein.
In some embodiments, molecules that may be used to make linkers include at least two functional groups, e.g., two carboxylic acid groups. In some embodiments of a trivalent linker, two arms of a linker may contain two dicarboxylic acids. In some embodiments, dicarboxylic acid molecules may be used as linkers (e.g., a dicarboxylic acid linker).
Examples of dicarboxylic acids molecules that may be used to form linkers include, but are not limited to,
Figure imgf000055_0004
Figure imgf000056_0001
wherein n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20). Other examples of dicarboxylic acids molecules that may be used to form linkers include, but are not limited to,
Figure imgf000057_0001
Figure imgf000058_0001
In some embodiments, dicarboxylic acid molecules, such as the ones described herein, may be further functionalized to contain one or more additional functional groups. Dicarboxylic acids may be further functionalized, for example, to provide an attachment point to one or more targeting moieties and a moiety which forms a complex with the circular polyribonucleotide.
In some embodiments, when the targeting moiety is attached to a moiety which forms a complex with the targeting circular polyribonucleotide, the linking group may include a moiety including a carboxylic acid moiety and an amino moiety that are spaced by from 1 to 25 atoms. Examples of such linking groups include, but are not limited to,
Figure imgf000058_0002
Figure imgf000059_0001
wherein n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, a linking group may include a moiety including a carboxylic acid moiety and an amino moiety, such as the ones described herein, may be further functionalized to contain one or more additional functional groups. Such linking groups may be further functionalized, for example, to provide an attachment point to a targeting moiety.
In some embodiments, when the targeting moiety is attached to the moiety which forms a complex with the circular polyribonucleotide, the linking group may include a moiety including two or amino moieties (e.g., a diamino moiety) that are spaced by from 1 to 25 atoms. Examples of such linking groups include, but are not limited to,
Figure imgf000059_0002
Figure imgf000060_0001
wherein n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20).
In some embodiments, a linking group may include a diamino moiety, such as the ones described herein, may be further functionalized to contain one or more additional functional groups. Such diamino linking groups may be further functionalized, for example, to provide an attachment point to a targeting moiety.
In some embodiments, a molecule containing an azide group may be used to form a linker, in which the azide group may undergo cycloaddition with an alkyne to form a 1 ,2,3-triazole linkage. In some embodiments, a molecule containing an alkyne group may be used to form a linker, in which the alkyne group may undergo cycloaddition with an azide to form a 1 ,2,3-triazole linkage. In some embodiments, a molecule containing a maleimide group may be used to form a linker, in which the maleimide group may react with a cysteine to form a C-S linkage. In some embodiments, a molecule containing one or more haloalkyl groups may be used to form a linker, in which the haloalky I group may form a covalent linkage, e.g., C-N and C-0 linkages, with a targeting moiety.
In some embodiments, the linker may be an oligonucleotide, which includes a string of nucleic acids. In some embodiments, the targeting moiety described herein include a string of nucleic acids, which is in turn linked a moiety which complexes with the circular polyribonucleotide. The linker can have any sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence specifically chosen for its molecular or biochemical properties. In some embodiments, the linker includes 20 one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof. In some embodiments, the linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof. In some embodiments, the string of nucleic acids includes from 1 to 50 nucleic acid residues (e.g., from 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, 5 to 50, 10 to 50, 20 to 50, 30 to 50, or 40 to 50 residues). In some embodiments, the string of nucleic acids includes from 5 to 30 nucleic acid residues (e.g., from 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 30, 15 to 30, 20 to 30, or 25 to 30 residues). In some embodiments, the linker includes one or more guanines, for example from 1-10 guanines (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 guanine residues).
In some embodiments, the linker is a polypeptide. For example, the linker may include at least one amino acid. In some embodiments, the polypeptide linker may include from two to 100 amino acid residues (e.g., from 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 amino acid residues). In certain embodiments, the linker is a single amino acid (e.g., any naturally occurring amino acid such as Cys). In some embodiments, the linker includes a series of glycine residue to form a polyglycine linker. In some embodiments, the linker includes an amino acid sequence of (G ly) n, wherein n may be from 2 to 20 residues (e.g., from 2 to 15, 2 to 10, 2 to 5, 5 to 20, 10 to 20, or 15 to 20 glycine residues). Examples of polyglycine linkers include but are not limited to GGG, GGGA (SEQ ID NO: 27), GGGG (SEQ ID NO: 28), GGGAG (SEQ ID NO: 29), GGGAGG (SEQ ID NO: 30), GGGAGGG (SEQ ID NO: 31), GGAG (SEQ ID NO: 32),GGSG (SEQ ID NO: 33), AGGG (SEQ ID NO: 34), SGGG (SEQ ID NO: 35), GGAGGA (SEQ ID NO: 36), GGSGGS (SEQ ID NO: 37), GGAGGAGGA (SEQ ID NO: 38), GGSGGSGGS (SEQ ID NO: 39), GGAGGAGGAGGA (SEQ ID NO: 40), GGSGGSGGSGGS (SEQ ID NO: 41), GGAGGGAG (SEQ ID NO: 42), GGSGGGSG (SEQ ID NO: 43), GGAGGGAGGGAG (SEQ ID NO: 44), GGSGGGSGGGSG (SEQ ID NO: 45), GGGGAGGGGAGGGGA (SEQ ID NO: 46), GGGGSGGGGSGGGGS (SEQ ID NO: 47), and GGGSGGGS (SEQ ID NO: 48). In other embodiments, the polypeptide linker is a glycine-rich polypeptide such as a polypeptide having the sequence [Gly-Gly- Gly-Gly-Ser]n (SEQ ID NO: 49) where n is 1 , 2, 3, 4, 5 or 6 is used. In some embodiments, the polypeptide linker is a serine-rich polypeptide linker. Serine rich peptide linkers include those of the formula [X-X-X-X-Gly]y (SEQ ID NO: 50), where up to two of the X are Thr, and the remaining X are Ser, and y is 1 to 5 (e.g., Ser-Ser-Ser-Ser-Gly (SEQ ID NO: 51), where y is greater than 1). In some cases, the linker is a single amino acid (e.g., any amino acid, such as Gly or Cys).
Amino acid linkers may be selected for flexibility (e.g., flexible or rigid) or may be selected on the basis of charge (e.g., positive, negative, or neutral). Flexible linkers typically include those with Gly resides (e.g., [Gly-Gly-Gly-Gly-Ser]n where n is 1 , 2, 3, 4, 5 or 6). Other linkers include rigid linkers (e.g., PAPAP (SEQ ID NO: 52) and (PT)nP (SEQ ID NO: 53), where n is 2, 3, 4, 5, 6, or 7) and a-helical linkers (e.g., A(EAAAK)nA (SEQ ID NO: 54), where n is 1 , 2, 3, 4, or 5).
Examples of suitable linkers are succinyl, Lys, Glu, and Asp, or a dipeptide such as Gly-Lys. When the linker is succinyl, one carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the other carboxyl group thereof may, for example, form an ester bond with a hydroxyl group of opioid moiety. When the linker is Lys, Glu, or Asp, the carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the amino group thereof may, for example, form an amide bond with a carboxyl group of the opioid moiety. When Lys is used as the linker, a further linker may be inserted between the ε-amino group of Lys and the opioid moiety. In one particular embodiment, the further linker is succinyl acid, which can form an amide bond with the ε- amino group of Lys and an ester bond with a hydroxyl group present in the opioid moiety. In one embodiment, the further linker is Glu or Asp (e.g., which forms an amide bond with the ε-amino group of Lys and an ester or amide bond with a hydroxyl or amino group present in the opioid moiety). In other embodiments, the peptide linker is a branched polypeptide.
In some embodiments, the linker is an enzymatically cleavable linker. A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 -7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis. Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (-S-S-). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular RNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
In another embodiment, a cleavable linker includes a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)- O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S- P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O- P(S)( Rk)-S-. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S- P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O- P(S)(H)-O-, -S-P(O)(H)-O, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O- P(O)(OH)-O-. These candidates can be evaluated using methods analogous to those described above.
In another embodiment, a cleavable linker includes an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.
In another embodiment, a cleavable linker includes an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O-, or - OC(O)-. These candidates can be evaluated using methods analogous to those described above.
In yet another embodiment, a cleavable linker includes a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula - NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
In some additional embodiments, the linker further includes a biotin-binding protein (e.g., avidin, streptavidin, NeutrAvidin). For example, the biotin-binding protein may be avidin. In some embodiments, the avidin binds a biotinylated targeting moiety, as shown for example in FIG. 10. In some embodiments, the avidin binds 1 , 2, 3, 4, or more biotinylated targeting moieties. In some embodiments, the biotinylated targeting moiety includes a lipid, small molecule, carbohydrate, polypeptide, and/or aptamer. In some embodiments, two or more of the biotinylated targeting moieties are the same targeting moiety. In some embodiments, two or more of the biotinylated targeting moieties that are different targeting moieties from one another.
Photoreactive Crosslinking Agents
The disclosure provides a complex that includes a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide, a photoreactive crosslinking agent, a linker, and targeting moiety. The photoreactive crosslinking agent provides a covalent attachment between the circular polyribonucleotide and the moiety that attaches to a region of the circular polyribonucleotide upon irradiation with light, e.g., at a first wavelength. In some embodiments, the photoreactive crosslinking agent is reversibly attached. For example, the covalent attachment may be removed upon irradiation with light, e.g., at a second wavelength that is different from the first wavelength. In some embodiments, the photoreactive crosslinking agent is attached to the end of the moiety. In some embodiments, the photoreactive crosslinking agent is located at an internal position within the moiety.
In some embodiments, the disclosure provides a complex including a circular polyribonucleotide, a moiety that binds specifically to a region of the circular polyribonucleotide and includes one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) photoreactive crosslinking agents, a linker, and a targeting moiety. In some embodiments, the photoreactive crosslinking agents are the same photoreactive crosslinking agent. In some embodiments, the photoreactive crosslinking agents are different from one another. In some embodiments, each of the photoreactive crosslinking agents is located at an internal position within the moiety. In some embodiments, at least one of the photoreactive crosslinking agents is attached to an end of the moiety. In some embodiments, at least one of the photoreactive crosslinking agents is attached to a second end of the moiety. The photoreactive crosslinking agent may be a photoreactive nucleotide analog or a photoreactive amino acid analog.
Photoreactive Nucleotide Analogs
In some embodiments, the moiety that specifically binds a region of the circular polyribonucleotide and includes one or more photoreactive crosslinking agents is an oligonucleotide. Oligonucleotide binding moieties are described in greater detail in a later section of the Detailed Description. In some embodiments, the moiety that specifically binds a region of the circular polyribonucleotide and includes one or more photoreactive crosslinking agent is an oligonucleotide, wherein each of the one or more photoreactive crosslinking agents is a photoreactive nucleotide analog. In some embodiments, each of the one or more photoreactive nucleotide analogs replaces a single nucleotide within the oligonucleotide. Each of the one or more photoreactive nucleotide analogs may include a nucleotide or nucleoside modified to contain a photoreactive group. In some embodiments, the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the opposing ribonucleotide within the circular polyribonucleotide upon photoirradiation. The disclosure provides photoreactive nucleotide analogs included within the oligonucleotide. Photoreactive nucleotide analogs include, for example, 4-thiouridene (4sU), 5-bromo-2’-deoxyuridine (BrdU), coumarin derivatives, D-threoninol (CNVD) derivatives, 3- cyanovinylcarbazole nucleoside (CNVK) derivatives, diazirene derivatives, phenylselenide derivatives, psoralen derivatives, or pyranovinylcarbazole (PCX) derivatives.
In some embodiments, the photoreactive crosslinking agent includes 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a pheylselenide, a furan, or an abasic site.
In some embodiments, the carbazole is 3-cyanovinylcarbazole, 4-methylpyranocarbazole, or pyranocarbazole.
In some embodiments, the coumarin is 7-hydroxycoumarin.
In some embodiments, the photoreactive nucleotide analog is one disclosed in U.S. Pat. No. 8,481 ,714, the disclosure of which is hereby incorporated by reference in its entirety.
In some embodiments, the photoreactive crosslinking agent includes a compound of Formula (I):
Figure imgf000065_0001
wherein, in the formula (I), Ra represents a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen, wherein, in the formula (I), Ri and R2 each independently represent a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen, and wherein, in the formula (I), Rb represents a sugar, a polysaccharide, a polyether, a polyol, a polypeptide chain, or a water-soluble synthetic polymer.
In some embodiments, the photoreactive crosslinking agent is the compound according to Formula (I), wherein R& is represented by any one of Formulae (II) - (V):
Figure imgf000065_0002
Figure imgf000066_0001
3, 4, or 5). In some embodiments, the photoreactive crosslinking agent is the compound of any one of
Formulae (VI) - (X):
Figure imgf000066_0002
Figure imgf000067_0001
Figure imgf000068_0001
wherein, in Formula (IX), n is represented as any number 2 to 5 (e.g., 2, 3, 4, or 5).
In some embodiments, the photoreactive crosslinking agent includes a compound of Formula
Figure imgf000068_0002
wherein, in Formula (XII), R1 represents a hydrogen or methyl group, wherein, in Formula (XII), R2 represents a hydrogen or methoxy group, wherein in Formula (XII), R3 represents a hydrogen, a methyl group, or a methoxy group, wherein in Formula (XII), R4 represents an aminomethyl group, an azidomethyl group, or a hydrogen group, wherein in Formula (XII), R5 represents a hydrogen or methyl group. In some embodiments, the photoreactive crosslinking agent is the compound of any one of Formulae (XIII) - (XVII):
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
In some embodiments, the photoreactive nucleotide analog may be 4sU. The structure of 4sU mimics the structure of uridine analogs but gains photoreactive properties by replacing the carbonyl oxygen at position 4 of the ring with a sulfur atom. Upon irradiation with light having a wavelength of 356 nm, photoreactive 4sU within an oligonucleotide covalently binds to a nearby cytosine nucleobase through a [2+2] cycloaddition reaction.
In some embodiments, the photoreactive nucleotide analog may be BrdU. Upon irradiation with light having wavelength of 308 nm, BrdU generates 2’-deoxyuridin-5-yl radicals.
In some embodiments, the photoreactive nucleotide analog may be a coumarin, a coumarin analog, or a derivative thereof. Coumarin and derivatives thereof located within the oligonucleotide covalently bind to a nearby thymine nucleobase through a [2+2] cycloaddition reaction upon irradiation with light having a wavelength of 250 nm, resulting in the formation of a syn-cycloaddition photo-adduct. Irradiating the syn-cycloaddition photo-adduct with light having a wavelength of 254 nm reverses the reaction. An example of a coumarin derivative that is a photoreactive nucleotide analog is 7- hydroxycoumarin (see, e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 , which is hereby incorporated by reference in its entirety).
In some embodiments, the photoreactive nucleotide analog may be CNVK or derivatives thereof. CNVK and derivatives thereof within an oligonucleotide have a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby oligonucleotide. Upon irradiation with light having a wavelength of 366 nm, CNVK or a derivative thereof covalently binds with a nearby pyrimidine-containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct. Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents the photo-induced DNA damage at shorter wavelengths. The trans isomer compared to the cis isomer of CNVKis the reactive species for photoreactive crosslinking (see, e.g., Fujimoto et al. J. Am. Chem. Soc.135, 16161-16167, 2013 which is hereby incorporated by reference in its entirety). Another example of a CNVK derivative is n- CNVK which includes a linker of variable lengths (n = 2-5). Upon irradiation, n- CNVK can form a covalent bond with a nearby pyrimidine nucleobase at locations other than the 1 base downstream position of an opposing nucleotide depending on the linker length (see, e.g., Fujimoto et al. Photochem. Photobiol. Sci. 19, 776-782, 2020, which is hereby incorporated by reference in its entirety).
In some embodiments, the photoreactive nucleotide analog may be CNVD. CNVD and derivatives thereof within an oligonucleotide have a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby oligonucleotide. CNVD is a derivative of CNVK that has the same chemical structure of CNVK except for a D- threoninol linker in the place of the deoxyribose sugar which acts to increase the flexibility as compared to CNVK, increasing the photo-induced crosslinking reaction rate due to minimization of entropic losses during hybridization (see, e.g., Sakamoto et al. Org. Lett. 17, 936-939, 2015, which is hereby incorporated by reference in its entirety). Upon irradiation with light having a wavelength of 365 nm, CNVD or a derivative thereof covalently binds with a nearby pyrimidine-containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct. Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents photo-induced DNA damage at shorter wavelengths.
In some embodiments, the photoreactive nucleotide analog may be a diazirene derivative. Upon irradiation with light having a wavelength of 365 nm, a diazirene derivative becomes a reactive carbene intermediate. The reactive carbene intermediates rapidly form a covalent bond with a nearby nucleobase through C-C, C-H, O-H, or X-H (X = heteroatom) insertions. The reactive carbene intermediate can react with all nucleobases, including adenine, cytosine, guanine, thymine, and uracil, forming a covalent attachment with a nearby nucleobase. An example of a diazirene derivative is 3-phenyl-3-trifluoromethyl-
Figure imgf000072_0001
which is attached to the sugar ring through an acetal linkage (see, e.g., Nakamoto et al. J. Org. Chem. 79, 2463-2472, 2014, which is hereby incorporated by reference in its entirety).
In some embodiments, the photoreactive nucleotide analog may be a phenyl selenide analog. Upon irradiation with light having a wavelength of 350 nm, phenyl selenide becomes a radical intermediate which has the capability of alkylating the N1 or N6 position of nearby purine nucleobases, including adenine. An example of a phenyl selenide analog is phenyl selenide-modified 2’-deoxythymidine (see, e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 , which is hereby incorporated by reference in its entirety). In some embodiments, the photoreactive nucleotide analog may be a psoralen analog or a derivative thereof. Psoralen and derivatives thereof are tricyclic compounds that may intercalate in any AT or AU region of hybridized DNA and RNA sequences, respectively. Psoralen and derivatives thereof react through their furan or pyrone photoreactive site to covalently bind the C5-C6 double bond of pyrimidine residues. This forms a covalent attachment between two nearby pyrimidine nucleobases through a [2+2] cycloaddition reaction upon irradiation with light having a wavelength of 365 nm, resulting in the formation of a cyclobutene photo-adduct. The resulting cyclobutene photo-adduct may be in the syn- or cis- configuration. Pyrimidine residues are present among cytosine, thymine, and uracil bases. Irradiating the cyclobutene photo-adduct with light having a wavelength of 254 nm reverses the reaction. An example of a psoralen derivative is 4’-aminomethyltrioxsalen (see, e.g., Velema et al. JACS Au 3, 316-332, 2023, which is hereby incorporated by reference in its entirety).
In some embodiments, the photoreactive nucleotide analog may be pyranocarbazole and derivatives thereof, such as pyranocarbozole nucleoside (PCX). The photoreactive pyranocarbozole and derivatives thereof within an oligonucleotide include a vinyl group that is aligned with the C5-C6 double bond of a pyrimidine nucleobase located 1 base downstream of an opposing nucleotide within a nearby oligonucleotide. Upon irradiation with light having a wavelength of 400 nm, PCX covalently binds with a nearby, pyrimidine-containing nucleobase through a [2+2] cycloaddition, resulting in the formation of the photo-adduct. Pyrimidine-containing nucleobases include cytosine, thymine, and uracil. Irradiating the photo-adduct with light having a wavelength of 312 nm reverses the reaction. Irradiation with longer wavelength light, as compared to the 254 nm wavelength light used for reversing photo-adduct formation, circumvents the photo-induced DNA damage at shorter wavelengths. An example of a pyranocarbozole derivative is PCX with a D-threoninol linker in the place of the deoxyribose sugar (PCXD) which acts to increase the flexibility as compared to PCX, increasing the photo-induced crosslinking reaction rate due to minimization of entropic losses during hybridization (see, e.g., Fujimoto et al. RSC Adv. 9, 30693-30697, 2019, which is hereby incorporated by reference in its entirety). Another example of a pyranocarbozole derivative is pcX that includes a methyl group at the 4-position of PCX. 4-methylpyranocarbozole nucleoside (MEPK) is able to differentiate between thymine and cytosine, enabling selectivity toward thymine (see, e.g., Mihara et al. Org. Biomol. Chem. 19, 9860-9866, 2021 , which is hereby incorporated by reference in its entirety).
In some embodiments, the oligonucleotide may include another photoreactive nucleotide analog which allows for the oligonucleotide binding moiety to covalently attach to the polyribonucleotide described herein. For a review of certain photoreactive nucleotide analogs, see, e.g., Elskens et al., RSC Chem. Biol. 2, 410-422, 2021 ; Tavakoli et al., RSC Adv. 12, 6484-6507, 2022; and Velema et al. JACS Au 3, 316-332, 2023.
Photoreactive Amino Acid Analogs
In some embodiments, the moiety that specifically binds a region of the circular polyribonucleotide is a polypeptide. In some embodiments, the moiety includes one or more photoreactive amino acid analogs. In some embodiments, the photoreactive amino acid analog replaces an amino acid within the polypeptide. The one or more photoreactive amino acid analogs may include an amino acid modified to contain a photoreactive group. Photoreactive nucleotide analogs include, for example, aryl azide-based, benzophenone-based, or diazirene-based unnatural amino acids or
Figure imgf000074_0003
y I) et h oxy] carbonyl-lysine.
In some embodiments, the photoreactive amino acid analog may be an aryl azide-based unnatural amino acid. Upon irradiation with light having a wavelength of 250-350 nm, aryl azide-based unnatural amino acids generate a reactive nitrene intermediate. The reactive nitrene intermediate can covalently bond with a nearby nucleobase through C-H and X-H insertions. An example of an arylazide- based unnatural amino acid is tetrafluorophenylazide (see, e.g., Smith et al Future Med. Chem. 7, 159- 183, 2015, which is hereby incorporated by reference in its entirety).
In some embodiments, the photoreactive amino acid analog may be a benzophenone-based unnatural amino acid. Benzophenone-based unnatural amino acids can be easily prepared and are further beneficial due to their inertness to solvents. Upon irradiation with light having a wavelength of 350- 365 nm, benzophenone-based unnatural amino acids generate a reactive diradical intermediate, containing reactive triplet carbonyl states. The reactive diradical intermediate can covalently bind to a nearby nucleobase through C-H insertion. Benzophenone-based unnatural amino acids are activated with light of a longer wavelength, reducing the risk of damage to biomolecules; however, irradiation times are longer, risking the potential for non-specific interactions.
In some embodiments, the photoreactive amino acid analog may be a diazirene-based unnatural amino acid. Upon irradiation with light having a wavelength of 350-380 nm, diazirine-based unnatural amino acids generate a reactive carbene intermediate. The reactive carbene intermediates rapidly form a covalent bond with a nearby nucleobase through C-C, C-H, O-H, or X-H (X = heteroatom) insertions. The reactive carbene intermediate can react with all nucleobases, including adenine, cytosine, guanine, thymine, and uracil, forming a covalent attachment with a nearby nucleobase. One example of an alkyl diazirene-based amino acid is a pyrrolysine analog that bears an alkyl diazirene reactive group attached by a short, flexible C2 linker (see, e.g., Dziuba et al. ChemBioChem 2020, 21, 88-93, which is hereby incorporated by reference in its entirety).
In some embodiments, the photoreactive amino acid analog may be
Figure imgf000074_0001
yl)ethoxy]carbonyl-lysine )ethoxy]carbonyl-lysine is genetically encodable and can be
Figure imgf000074_0002
irradiated with red light to covalently bind the amino acid to a nearby nucleobase (see, e.g., Moritz et al. Angew. Chem. 2013, 52, 4690-4693, , which is hereby incorporated by reference in its entirety).
Complexation of Circular Polyribonucleotide with a Targeting Moiety
The disclosure also provides circular polyribonucleotide that are complexed with (e.g., hybridized or bound to) one or more moieties that specifically bind a region of the circular polyribonucleotide, wherein each of the one or more moieties that specifically bind a region of the circular polyribonucleotide is conjugated to (e.g., directly, chemically-covalently conjugated) to one or more targeting moieties. In some embodiments, the moieties that specifically bind a region of the circular polyribonucleotide are the same moiety that specifically binds a region of the circular polyribonucleotide. In some embodiments, the moieties that specifically bind a region of the circular polyribonucleotide are different from one another. For example, the moieties that specifically bind to the region of the circular polyribonucleotide may be one or more polypeptide binding moieties and one or more oligonucleotide binding moieties. In some embodiments, the moieties that specifically bind to the region of the circular polyribonucleotide may be two or more polypeptide binding moieties that are different polypeptides from one another. The moieties that specifically bind to the region of the circular polyribonucleotide may be one or more oligonucleotide binding moieties that have different sequences from one another.
The disclosure also provides circular polyribonucleotide complexed with (e.g., hybridized or hybridized and covalently attached to) a moiety that specifically binds a region of the circular polyribonucleotide. The moiety that specifically binds a region of the circular polyribonucleotide includes one or more photoreactive crosslinking agents. The binding moiety may be, for example, a polypeptide or an oligonucleotide. In some embodiments, the complex includes from 1 to 20 moieties (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, to 20) that each bind specifically to a region of the circular polyribonucleotide. In some embodiments, the moieties that specifically bind a region of the circular polyribonucleotide are the same moiety that specifically binds a region of the circular polyribonucleotide. In some embodiments, the moieties that specifically bind a region of the circular polyribonucleotide are different from one another. For example, the moieties that specifically bind to the region of the circular polyribonucleotide may be one or more polypeptide binding moieties and/or one or more oligonucleotide binding moieties. In some embodiments, the moieties that specifically bind to the region of the circular polyribonucleotide may be two or more polypeptide binding moieties that are different polypeptides from one another. The moieties that specifically bind to the region of the circular polyribonucleotide may be one or more oligonucleotide binding moieties that have different sequences from one another.
Each moiety that specifically binds a region of the circular polyribonucleotide may also include a linker covalently connecting the moieties that specifically bind a region of the circular polyribonucleotide and the targeting moiety. The moiety that specifically binds a region of the circular polyribonucleotide may be, for example an oligonucleotide or a polypeptide.
In some embodiments, the moiety binds a region of the circular polyribonucleotide that includes one or more of an IRES, a spacer sequence, or an expression sequence. In some embodiments, the moiety binds to one of the binding regions as shown in FIG. 2. In some embodiments, the moiety binds a region in an IRES. In some embodiments, the moiety does not bind a region in an IRES. In some embodiments, the moiety binds a region in a spacer sequence. In some embodiments, the moiety does not bind a region in a spacer sequence. In some embodiments, the moiety binds at least part of a spacer sequence. In some embodiments, the moiety binds at least part of two spacer sequences. In some embodiments, the moiety binds a region in a coding region. In some embodiments, the moiety does not bind a region in a coding region. In some embodiments, the moiety binds a region in a spacer sequence and/or a coding region but does not bind a region in an IRES. In some embodiments, the binding region is sufficiently distant in sequence and space from the expression sequence so that it does not interfere with or minimizes interference with translation efficiency. For example, the binding region may be from about 10 nucleotides to about 200 nucleotides (e.g., from about 10 nucleotides to 170 nucleotides, 10 nucleotides to 150 nucleotides, 10 nucleotides to 130 nucleotides, 10 nucleotides to 100 nucleotides, 10 nucleotides to 70 nucleotides, 10 nucleotides to 50 nucleotides, 10 nucleotides to 30 nucleotides, 30 nucleotides to 200 nucleotides, 50 nucleotides to 200 nucleotides, 70 nucleotides to 200 nucleotides, 100 nucleotides to 200 nucleotides, 130 nucleotides to 200 nucleotides, 150 nucleotides to 200 nucleotides, 170 nucleotides to 200 nucleotides, or 50 nucleotides to 100 nucleotides) from the 5’ or 3’ end of the expression sequence. Oligonucleotide Binding Moieties
In some embodiments, the moiety that specifically binds a region of the circular polyribonucleotide an oligonucleotide. The oligonucleotide may be, for example, a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.
In some embodiments, each oligonucleotide is from about 10-500 nucleic acids in length (e.g., about 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 20-50, 20-100, 20-200 20-300, 20-400, 30-50, 30-100, 30-200, 30-300, 30-400, 50-100, 50-200, 50-300, or 50- 400 ribonucleotides in length). In some embodiments, each oligonucleotide has at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%) complementarity to a portion of the sequence of the circular polyribonucleotide. In some embodiments, each oligonucleotide has from 20% to 90% (e.g., from 20-90%, 20-90%, 20-90% 20-90%, 30%-90%, 40%-90%, or 50%-90% complementarity to a portion of the sequence of the circular polyribonucleotide. Each oligonucleotide may be DNA, RNA, a synthetic nucleic acid, or a hybrid. Each oligonucleotide may include one or more nucleic acid modifications as described herein.
In some embodiments, the circular polyribonucleotide is hybridized to one, two, three, four, five, six, seven, eight, nine, ten or more oligonucleotides, each oligonucleotide being covalently conjugated to one or more targeting moieties. In some embodiments, the circular polyribonucleotide is hybridized to one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more oligonucleotides. In some embodiments, the circular polyribonucleotide is hybridized to 10, 20, 30, 40, or 50 or more oligonucleotides. In some embodiments, the circular polyribonucleotide is hybridized to 1-10, 2-10, 2-20, 2-50, 5-10, 5-20, 5-50, 10-20, 20-30, 30- 40, 40-50, 1-50, 20-50, 30-60 or 50-100 targeting moieties.
In some embodiments, each oligonucleotide is hybridized to (e.g., shares at least partial complementarity sufficient for binding with) a sequence with a binding region of the circular polyribonucleotide. In some embodiments, wherein the circular polyribonucleotide includes either a coding region (e.g., including an expression sequence), the binding region is sufficiently distant in sequence and space from the coding region or target binding region so that it does not interfere with or minimizes interference with translation efficiency. In some embodiments, the binding region includes from 1 to 100 ribonucleotides (e.g., from 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 ribonucleotides). For example, the binding region may include from 10 to 90 ribonucleotides (e.g., from 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 80, 30 to 80, 40 to 80, 50 to 80, 60 to 80, or 70 to 80 ribonucleotides). In some embodiments, the oligonucleotide has from 50% to 100% (e.g., from 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%) complementarity to a portion of the binding region of the circular polyribonucleotide. For example, the oligonucleotide may have from 80% to 100% (e.g., from 85% to 100%, 90% to 100%, 95% to 100%, 80% to 95%, 80% to 90%, or 80% to 85%) and complementarity to a portion of the binding region of the circular polyribonucleotide.
In some embodiments, the moiety that specifically binds a region of the circular polyribonucleotide and includes a photoreactive crosslinking agent is an oligonucleotide. The oligonucleotide may be, for example, a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. In some embodiments, each oligonucleotide includes a photoreactive crosslinking agent. In some embodiments, the photoreactive crosslinking agent is attached to the 5’ end of the oligonucleotide. In some embodiments, the photoreactive crosslinking agent is attached to the 3’ end of the oligonucleotide. In some embodiments, the photoreactive crosslinking agent is a photoreactive nucleotide analog. In some embodiments, the photoreactive nucleotide analog replaces a single nucleotide within the oligonucleotide. In some embodiments, the photoreactive nucleotide analog is located at an internal position within the oligonucleotide. In some embodiments, the 3’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog. In some embodiments, the 3’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the photoreactive nucleotide analog.
In some embodiments, each oligonucleotide includes one, two, three, four, five, six, seven, eight, nine, ten or more photoreactive crosslinking agents. In some embodiments, each photoreactive crosslinking agent is a photoreactive nucleotide analog. In some embodiments, each of the photoreactive nucleotide analogs replaces a single nucleotide within the oligonucleotide. In some embodiments, each photoreactive nucleotide analog is located at an internal position within the oligonucleotide. In some embodiments, the 3’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 3’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the nearest photoreactive nucleotide analog. In some embodiments, the 5’ end of the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) from the nearest photoreactive nucleotide analog. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the 5’ end of the oligonucleotide. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the 3’ end of the oligonucleotide. In some embodiments, the oligonucleotide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 nucleotides (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 nucleotides) between each of the photoreactive nucleotide analogs. For example, the oligonucleotide may have one photoreactive nucleotide analog attached to the 5’ end and a second photoreactive nucleotide analog 8 nucleotides away from the 5’ end. The oligonucleotide may have one photoreactive nucleotide analog attached to the 5’ end, a second photoreactive nucleotide analog 10 nucleotides away from the 5’ end, and a third photoreactive nucleotide analog 15 nucleotides away from the 3’ end and 8 nucleotides away from the second photoreactive nucleotide analog.
In some embodiments, each oligonucleotide is from about 5-500 nucleotides in length (e.g., about 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 5-200, 5-300, 5-400, 20-50, 20-100, 20-20020-300, 20-400, 30-50, 30-100, 30-200, 30-300, 30-400, 50-100, 50-200, 50-300, or 50-400 nucleotides in length). In some embodiments, each oligonucleotide has at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%) complementarity to a portion of the sequence of the circular polyribonucleotide. In some embodiments, each oligonucleotide has from 20% to 90% (e.g., from 20-90%, 20-90%, 20-90% 20-90%, 30%-90%, 40%-90%, or 50%-90%) complementarity to a portion of the sequence of the circular polyribonucleotide. Each oligonucleotide may be DNA, RNA, a synthetic nucleic acid, or a hybrid. Each oligonucleotide may include one or more nucleic acid modifications as described herein.
In some embodiments, the oligonucleotide includes an aptamer. In some embodiments, the aptamer specifically binds to the linker or targeting moiety.
In some embodiments, the circular polyribonucleotide is annealed and covalently bound to one, two, three, four, five, six, seven, eight, nine, ten or more oligonucleotides, each oligonucleotide being covalently conjugated to one or more targeting moieties. In some embodiments, the circular polyribonucleotide is annealed and covalently bound to one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more oligonucleotides. In some embodiments, the circular polyribonucleotide is annealed and covalently bound to 10, 20, 30, 40, or 50 or more oligonucleotides. In some embodiments, the circular polyribonucleotide is annealed and covalently bound to 1-10, 2-10, 2-20, 2-50, 5-10, 5-20, 5-50, 10-20, 20-30, 30-40, 40-50, 1- 50, 20-50, 30-60 or 50-100 targeting moieties.
In some embodiments, each oligonucleotide is annealed (e.g., shares at least partial complementarity sufficient for binding with) and covalently bound to (e.g., photo-crosslinked) a sequence with a binding region of the circular polyribonucleotide. In some embodiments, wherein the circular polyribonucleotide includes either a coding region (e.g., including an expression sequence), the binding region is sufficiently distant in sequence and space from the coding region or target binding region so that it does not interfere with or minimizes interference with translation efficiency. In some embodiments, the binding region includes from 1 to 100 ribonucleotides (e.g., from 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 ribonucleotides). For example, the binding region may include from 10 to 90 ribonucleotides (e.g., from 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 80, 30 to 80, 40 to 80, 50 to 80, 60 to 80, or 70 to 80 ribonucleotides). In some embodiments, the oligonucleotide has from 50% to 100% (e.g., from 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%) complementarity to a portion of the binding region of the circular polyribonucleotide. For example, the oligonucleotide may have from 80% to 100% (e.g., from 85% to 100%, 90% to 100%, 95% to 100%, 80% to 95%, 80% to 90%, or 80% to 85%) and complementarity to a portion of the binding region of the circular polyribonucleotide. In some embodiments, the binding region has zero or one mismatch with the oligonucleotide.
Polypeptide Binding Moieties
In some embodiments, the moiety that specifically binds a region of the circular polyribonucleotide and a targeting moiety by way of a linker is polypeptide. In some embodiments, the circular polyribonucleotide is bound to at least one polypeptide, wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 1 to 50 polypeptides (e.g., from 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 50, 10 to 50, 20 to 50, 30 to 50, or 40 to 50 polypeptides), wherein each polypeptide is complexed to at least one targeting moiety. The polypeptide may include an RNA binding protein domain. In some embodiments, the RNA binding protein domain (RBP) may a be an RNA recognition motif (RRM), K homology domain, a zinc finger motif, a Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, a THUMP domain, a YT521-B homology domain, a double stranded RNA binding domain, a helicase domain, a cold shock domain, an S1 domain, an Sm domain, a La motif, a Piwi-Argonaute-Zwille domain, or an intrinsically disordered region.
In some embodiments, the circular polyribonucleotide is bound to at least one polypeptide, wherein each polypeptide is complexed to at least one targeting moiety.
In some embodiments, the moiety that specifically binds a region of the circular polyribonucleotide and includes one or more photoreactive crosslinking agents is a polypeptide. In some embodiments, the circular polyribonucleotide is bound to at least one polypeptide, wherein each polypeptide is complexed to at least one targeting moiety. In some embodiments, the circular polyribonucleotide is bound to from 1 to 50 polypeptides (e.g., from 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 50, 10 to 50, 20 to 50, 30 to 50, or 40 to 50 polypeptides), wherein each polypeptide is complexed to at least one targeting moiety.
In some embodiments, each polypeptide includes a photoreactive crosslinking agent. In some embodiments, the photoreactive crosslinking agent is attached to the N-terminus of the polypeptide. In some embodiments, the photoreactive crosslinking agent is attached to the C-terminus of the polypeptide. In some embodiments, the photoreactive crosslinking agent is a photoreactive amino acid analog. In some embodiments, the photoreactive amino acid analog replaces a single amino acid within the polypeptide. In some embodiments, the photoreactive amino acid analog is located at an internal position within the polypeptide. In some embodiments, the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the photoreactive amino acid analog.
In some embodiments, each polypeptide includes one, two, three, four, five, six, seven, eight, nine, ten or more photoreactive crosslinking agents. In some embodiments, each photoreactive crosslinking agent is a photoreactive amino acid analog. In some embodiments, each of the photoreactive amino acid analogs replaces a single amino acid within the polypeptide. In some embodiments, each photoreactive amino acid analog is located at an internal position within the polypeptide. In some embodiments, the C-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog. In some embodiments, the C-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the nearest photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has at least 1 , e.g., at least 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids from the nearest photoreactive amino acid analog. In some embodiments, the N-terminus of the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) from the nearest photoreactive amino acid analog. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the N-terminus of the polypeptide. In some embodiments, at least one of the photoreactive crosslinking agents is attached to the C-terminus of the polypeptide. In some embodiments, the polypeptide has from 1 to 50, e.g., 2 to 50, e.g., 8 to 50 amino acids (e.g., 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 8 to 35, 8 to 40, 8 to 45, 15 to 50, 20 to 40, or 30 to 35 amino acids) between each of the photoreactive amino acid analogs. For example, a polypeptide may have one photoreactive amino acid analog attached to the N-terminus and a second photoreactive amino acid analog 8 amino acids away from the N-terminus. The polypeptide may have one photoreactive amino acid analog attached to the N-terminus, a second photoreactive amino acid analog 10 amino acids away from the N-terminus, and a third photoreactive amino acid analog 15 amino acids away from the C- terminus and 8 amino acids away from the second photoreactive amino acid analog. In some embodiments, the RNA binding protein domain may be an RNA recognition motif. RRMs are the most common and well-studied RNA-binding domain. The Protein Data Bank includes over 500 known structures which have an RRM. RRMs average 90 amino acids in size and adopt a β1α1β2β3α2β4 topology forming two α helices against an antiparallel β sheet, which houses the conserved RNA-binding ribonucleoprotein 1 and ribonucleoprotein 2 motifs in the central β1 and β3 strands. In some embodiments, RRMs interact with from 2 to 8 nucleotides (e.g., 2, 3, 4, 5, 6, 7, and 8 nucleotides) in single-stranded RNA commonly. This interaction may occur through several sequential stacking interactions and hydrogen bonds with ribonucleoprotein motifs, often with nanomolar affinities. Each RRM may have its own sequence preferences, often for degenerate sequences such as GU-rich tracts. In some embodiments, the RBP includes multiple ribonucleoprotein motifs. The combination of consecutive ribonucleoprotein motifs in an RBP dramatically increases binding affinity and specificity. In some embodiments, the polypeptide has a K homology domain. The K homology (KH) domain was first discovered in heterogeneous nuclear ribonucleoprotein K (hnRNPK). The KH domain includes about 70 amino acid residues, and typically recognizes about 4 nucleotides in single stranded RNA. KH domains may adopt either a type I β1α1α2β2β′α′ topology, in eukaryotes, or the reverse type II α′β′β1α1α2β2 topology, in prokaryotes, with a conserved “GXXG” RNA-binding motif (SEQ ID NO: 56) located between the α1 and α2 helices. RNA binding may occur in a hydrophobic pocket of the KH domain and may include several hydrogen bonds coordinated by the “GXXG” motif (SEQ ID NO: 56). Proteins having a KH domain and RNA have few stacking interactions. Furthermore, proteins having a KH domain usually have weak micromolar RNA affinities. Multiple KH domains in an RBP can independently or synergistically increase binding specificity. In some embodiments, the polypeptide includes a zinc finger motif. Zinc finger motifs may be found in a large family of proteins that average 30 amino acids in size and form a simple ββα topology in which residues in the β hairpin turn and α helix are coordinated by a Zn2+ ion. Zinc finger motif polypeptides may bind DNA but have been additionally shown to bind RNA. Zinc finger motifs include subtypes that interact with RNA and include amino acid sequences of CCHC (SEQ ID NO: 57), CCCH (SEQ ID NO: 58), CCCC (SEQ ID NO: 59), and CCHH (SEQ ID NO: 60) subtypes, where C and H refer to the interspersed cysteine and histidine residues that coordinate the zinc atom, respectively. The zinc finger motif subtypes display a range of sequence and structural specificities. The CCHC (SEQ ID NO: 57) subtype recognizes stem-loop elements in RNA through contacts with bases in the loop and the phosphate backbone of the stem. The CCCH (SEQ ID NO: 58) and CCCC (SEQ ID NO: 59) subtypes tend to recognize 3 nucleotide repeats through multiple such zinc finger motifs in one RBP. These contacts may be formed through hydrogen bonds with bases and the insertion of aromatic side chains that stack between bases. The versatile and abundant CCHH (SEQ ID NO: 60) subtype interact swith both single-stranded and double-stranded RNA. Thus, designer zinc finger motifs may be use for directed binding of RNA sequences. In some embodiments, the RNA binding protein includes a Pumilio homology domain. The Pumilio family of proteins occurs in most eukaryotes and is defined by the Pumilio homology domain. The Pumilio homology domain is very large, consisting of eight α-helical repeats of a highly conserved 36- amino acid sequence that forms a concave RNA-binding surface. Each repeat recognizes one unpaired RNA base through hydrogen bonds and a stabilizing stacking interaction, where the full domain recognizes up to 8 nucleotides in single-stranded RNA with low-nanomolar affinity. Wild-type Pumilio homology domain repeats do not specifically recognize cytosine; however, protein engineering has produced repeats that do. This protein engineering combined with the PUF domain’s predictable base recognition code allow modular design of Pumilio proteins that recognize from 8 nucleotides to 10- nucleotides sequences containing all RNA bases. In some embodiments, the polypeptide includes a pentatricopeptide repeat domain. Pentatricopeptide repeats include about 35 amino acid residues in length and form two antiparallel α helices.2–30 repeats form a solenoid-shaped scaffold that binds specific single-stranded RNA sequences with nanomolar affinity. Two residues in each repeat determine base-specific binding through hydrogen bonds, enabling the development of pentatricopeptide repeat domains designed to bind specified single-stranded RNA sequences. In some embodiments, the polypeptide includes a pseudouridine synthase and archaeosine transglycosylase (PUA) domain. PUA domains include a range from 67 to 94 amino acids residues (e.g., 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 94 amino acid residues), with a β1α1β2β3β4β5α2β6 architecture that forms a pseudobarrel encased by two α helices. PUA domains may contact double-stranded RNA, and its adjacent loops or overhangs through extensive hydrogen bonds may contact all parts of the RNA. These contacts are typically formed by a glycine-rich loop between α1 and β2 or α2 and β6. Unlike many other domains, PUA domains are not found as tandem repeats. In some embodiments, the polypeptide includes a THUMP domain. Named for thiouridine synthase, methyltransferase, and pseudouridine synthase, the THUMP domain is found in numerous tRNA-modifying enzymes. THUMP domains are always found in proximity to RNA-modifying domains and often in proximity to an N-terminal ferredoxin-like domain. THUMP domains include about 100 amino acids residues and display a α1α2β1α3β2β2 topology that forms parallel α helices flanking a β sheet. In some embodiments, the polypeptide includes a YT521-B homology (YTH) domain. The YTH domain is found in the YTH family of proteins that identify N6-methyladenosine (m6A) marks in RNA. The YTH domain ranges from 100 to 150 amino acids residues in length and forms a six-stranded β barrel surrounded by four or five α helices. Three residues in the hydrophobic core of the β barrel trap the methyl group of m6A in an aromatic cage consisting of hydrogen bonds with the adenosine and π interactions between tryptophan rings and the methyl group. The YTH domain specifically binds m6A over unmodified adenosines. In some embodiments, the polypeptide includes a double-stranded RNA binding domain or motif. The double-stranded RNA binding domain or motif may include from 65 to 70 amino acids residues. Double-stranded RNA binding domains specifically recognize and bind double-stranded RNA and are found in proteins with roles in viral protection, RNAi, and cellular transport. Double-stranded RNA binding domains often appear as tandem repeats or in combination with other functional RNA-binding domains, such as RNA-editing or helicase domains. The double-stranded RNA binding domain is made up of an α1β1β2β3α2 fold that forms an antiparallel β sheet flanked by α helices on one face. Double-stranded RNA binding domains specifically recognize the structure of an A-form RNA helix, spanning up to 16 base pairs with hydrogen-bond contacts to the phosphodiester backbone and 2′ OH. In some cases, double- stranded RNA binding domains have demonstrated base-specific contacts, such as to bases in adjacent loops. In some embodiments, the polypeptide includes a helicase domain. Helicase domains are found in all forms of life in helicase proteins, which unwind both DNA and double-stranded RNA. Helicases include six superfamilies, of which superfamily 1 and superfamily 2 contain all the eukaryotic RNA helicases. RNA-binding helicases include the Upf1-like family in superfamily 1 and the DEAD-box, DEAH, RIG-I-like, Ski2-like, and NS3 families in superfamily 2. The remaining superfamilies, 3–6, contain bacterial and viral helicases that form multimeric rings. Helicase domains are very large, containing 350– 400 amino acid residues. In superfamily 1 and superfamily 2, the helicase domain is composed of two “recombinase A (recA)-like” subdomains, each of which contains an ATP-catalytic core, a nucleic-acid- binding region, and subdomains that coordinate the two. Within families of helicases these subdomains are quite conserved. Helicase monomers in the ring-forming superfamilies of helicases are similarly quite large and composed of multiple subdomains. Bound RNA is surrounded by recA-like domains or, in the case of multimeric helicases, RNA is pulled through the center of the ring. Contacts with RNA are dominated by hydrogen bonds to phosphate and sugar moieties, but contacts with bases have only occasionally been observed. Affinities to RNA for proteins having a helicase domain are often in the nanomolar range, although they vary greatly by helicase and are modulated by other subdomains of the helicase. ATP binding generally promotes higher affinity to RNA by causing the helicase RNA-binding regions to clamp. In some embodiments, the polypeptide may include a cold shock domain. The cold shock domain is found in a large family of proteins associated with cold adaptation found in all domains of life. Cold shock domains are composed of about 70 amino acids residues or more and five antiparallel β strands that form a common β barrel structure known as an oligosaccharide/oligonucleotide-binding fold. Cold shock domains contain the conserved ribonucleoprotein 1 and ribonucleoprotein 2 motifs common to RNA recognition motifs, which bind single-stranded RNA. Cold shock domains contact 3 or 4 nucleotides through sequential stacking interactions and hydrogen bonds with bases, achieving nanomolar affinities. Cold shock domain containing proteins vary greatly in the types of sequences they recognize. In some embodiments, the polypeptide includes an S1 RNA-binding domain. The S1 domain includes about 70 amino acid residues, which form a 5-stranded antiparallel β barrel in the same oligonucleotide binding fold family as the cold shock domain. S1 domains are additionally found in several exoribonucleases and eukaryotic translation initiation factors and in combination with other RNA-binding domains such as the KH domain or cold shock domains. Despite their abundance, very little structural information is available for S1 domains in complex with RNA. S1 domains interact with both single- stranded RNA and double-stranded RNA in the context of the RNA-binding channel of exoribonucleases.
In some embodiments, the polypeptide includes a Sm-RNA binding motif. The Sm RNA-binding motif is found in Sm and like-Sm proteins in eukaryotes and archaea and in Hfq protein in prokaryotes. The Sm motif consists of about 70 residues with an a1 p1 p2p3p4p5 topology that forms a curved antiparallel p sheet. Sm-containing proteins readily multimerize through interactions between strands p4 and p5 in two Sm motifs. For example, Sm-Sm interactions link the seven human Sm proteins that make up the protein core of small nuclear ribonucleoproteins. The Sm multimers bind RNA with nanomolar affinity. Two Sm motifs form a 6-nucleotide binding surface that binds specific bases, often uridines, through hydrogen bonds and stacking interactions.
In some embodiments, the polypeptide includes a La motif. La motif includes about 90 amino acid residues and is found in eukaryotic La and La-related proteins (LARPs). The La motif consists of five a helices and three p strands that form a small antiparallel p sheet against a modified winged-helix fold. The winged-helix structure itself is common to several other RNA-binding proteins. La motifs are always found adjacent to at least one RNA recognition motif, where the combination of these two domains likely evolved as a unit. In La proteins, the dual La motif and RNA recognition motif region tightly binds the UUU-OH elements at the 3' ends of polymerase-l Il-transcribed small RNAs. Binding occurs in a cleft between the La motif and the RNA recognition motif rather than the traditional RNA-binding surfaces of either the RNA recognition motif or the La motif winged-helix fold. Several uracil bases stack with highly conserved aromatic residues in the La motif, and hydrogen bonds from both the La motif and RNA- recognition motif coordinate bases, phosphates, and the terminating 2' OH. These contacts result in low- nanomolar affinities of the La motif for 3'-terminal UUU-OH elements.
In some embodiments, the polypeptide includes a Piwi-Argonaute-Zwille or PIWI RNA-binding domains. These RNA binding domains define the Argonaute family of proteins found in eukaryotes. These domains are found on opposite sides of the Argonaute protein, both domains facilitate binding of small interfering RNA and microRNA guides to mRNA targets. The Piwi-Argonaute-Zwille domain occurs in Dicer proteins in addition to Argonaute proteins. Crystal structures of the Piwi-Argonaute-Zwille domain display a six-stranded p barrel topped with two a helices and flanked on the opposite side by a special appendage containing a p hairpin and short a helix. A binding pocket formed between this appendage and the p barrel binds the 2 nucleotide 3' overhang in guide RNAs with low-micromolar affinity. Binding is coordinated mostly by conserved tyrosine residues that form hydrogen bonds with the phosphate backbone and sugar hydroxyls of the two terminal nucleotides. The PIWI RNA-binding domain tertiary structure forms an RNase H-like fold consisting of a five-stranded p sheet flanked by a helices on both faces. The PIWI domain has endonucleolytic activity in some cases, but primarily stabilizes the gRNA- mRNA duplex seed region through hydrogen bonds with the gRNA backbone of nucleotides 3-5 and the 5' overhang base.
In some embodiments, the polypeptide includes an intrinsically disordered region. Intrinsically disordered regions are unstructured and often consist of repeats of arginine/serine residues, arginine/glycine, arginine- or lysine-rich patches, or short linear motifs of amino acids. Despite their lack of structure, intrinsically disordered regions have been found to dominate the composition in over 20% of RNA binding proteins. Intrinsically disordered regions may be the sole RNA-binding domain in an RNA binding protein and may actually drive the majority of protein-RNA interactions in the cell. Like globular RNA-binding domains, intrinsically disordered regions are conserved, often occur multiple times in one RNA binding protein, and can coordinate RNA binding in concert with other domains. Intrinsically disordered regions have been shown to drive higher affinity to RNA in RNA binding proteins that contain ordered RNA-binding domains and can themselves transition to an ordered state once bound to RNA. Intrinsically disordered regions show little RNA sequence dependence, however, suggesting that these regions’ high affinity for RNA is predominantly driven by electrostatic attraction to the phosphodiester backbone.
In some embodiments, the polypeptide may include another domain which allows for the protein to complex with the polyribonucleotide described herein.
Circular Polyribonucleotides
The disclosure provides circular polyribonucleotide that are complexed with one or more moieties that specifically bind a region of the circular polyribonucleotide, wherein each of the one or more moieties that specifically bind a region of the circular polyribonucleotide is conjugated to (e.g., directly, chemically- covalently conjugated) to one or more targeting moieties.
Expression Sequences
In some embodiments, the circular polyribonucleotide includes at least one expression sequence (e.g., coding region) that encodes a polypeptide. In some embodiments, the polypeptide, when expressed in the cell is functional. In some embodiments, the moiety that specifically bind a region of the circular polyribonucleotide binds a region of the circular polyribonucleotide includes or more expression sequences.
The encoded polypeptide may have 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. In some embodiments, 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.
Some examples of a polypeptides include a fluorescent tag or marker, an antigen, a peptide therapeutic, a synthetic or analog peptide from a naturally-bioactive peptide, an agonist or antagonist peptide, an anti-microbial peptide, a pore-forming peptide, a bicyclic peptide, a targeting or cytotoxic peptide, a degradation or self-destruction peptide, and degradation or self-destruction peptides. Peptides useful in the invention described herein also include antigen-binding peptides, e.g., antigen binding antibody or antibody-like fragments, such as single chain antibodies, nanobodies (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21 (7): 1076-113). Such antigen binding peptides may bind a cytosolic antigen, a nuclear antigen, an intra- organellar antigen.
The polypeptide may be produced in substantial amounts. As such, the polypeptide may be any proteinaceous molecule that can be produced. A polypeptide can be a polypeptide that can be secreted from a cell, or localized to the cytoplasm, nucleus or membrane compartment of a cell. Some polypeptides include, but are not limited to, at least a portion of a viral envelope protein, metabolic regulatory enzymes (e.g., that regulate lipid or steroid production), an antigen, a toleragen, a cytokine, a toxin, enzymes whose absence is associated with a disease, and polypeptides that are not active in an animal until cleaved (e.g., in the gut of an animal), and a hormone.
In some embodiments, the circular polyribonucleotide includes an expression sequence encoding a protein e.g., a therapeutic protein. In some embodiments, therapeutic proteins that can be expressed from the circular polyribonucleotide disclosed herein have antioxidant activity, binding, cargo receptor activity, catalytic activity, molecular carrier activity, molecular function regulator, molecular transducer activity, nutrient reservoir activity, protein tag, structural molecule activity, toxin activity, transcription regulator activity, translation regulator activity, or transporter activity. Some examples of therapeutic proteins may include, but are not limited to, an enzyme replacement protein, a protein for supplementation, a protein vaccination, antigens (e.g. tumor antigens, viral, bacterial), hormones, cytokines, antibodies, immunotherapy (e.g. cancer), cellular reprogramming/transdifferentiation factor, transcription factors, chimeric antigen receptor, transposase or nuclease, immune effector (e.g., influences susceptibility to an immune response/signal), a regulated death effector protein (e.g., an inducer of apoptosis or necrosis), a non-lytic inhibitor of a tumor (e.g., an inhibitor of an oncoprotein), an epigenetic modifying agent, epigenetic enzyme, a transcription factor, a DNA or protein modification enzyme, a DNA-intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor for an enzyme, a protein synthesis effector or inhibitor, a nuclease, a protein fragment or domain, a ligand or a receptor, and a CRISPR system or component thereof.
In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include human proteins, for instance, receptor binding protein, hormone, growth factor, growth factor receptor modulator, and regenerative protein (e.g., proteins implicated in proliferation and differentiation, e.g., therapeutic protein, for wound healing). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include EGF (epithelial growth factor). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include enzymes, for instance, oxidoreductase enzymes, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzyme, and desaturases. In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include an intracellular protein or cytosolic protein. In some embodiments, the circular polyribonucleotide expresses a NanoLuc® luciferase (nLuc). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include a secretary protein, for instance, a secretary enzyme. In some cases, the circular polyribonucleotide expresses a secretary protein that can have a short half-life therapeutic in the blood, or can be a protein with a subcellular localization signal, or protein with secretory signal peptide. In some embodiments, the circular polyribonucleotide expresses a Gaussia Luciferase (gLuc). In some cases, the circular polyribonucleotide expresses a non-human protein, for instance, a fluorescent protein, an energy- transfer acceptor, or a protein-tag like Flag, Myc, or His. In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide include a GFP. In some embodiments, the circular polyribonucleotide expresses tagged proteins, .e.g., fusion proteins or engineered proteins containing a protein tag, e.g., chitin binding protein (CBP), maltose binding protein (MBP), Fc tag, glutathione-S- transferase (GST), AviTag, Calmodulin-tag, polyglutamate tag; E-tag, FLAG-tag), HA-tag, His-tag, Myc- tag, NE-tag, S-tag, SBP-tag, Softag 1 , Softag 3, Spot-tag, Strep-tag; TC tag, Ty tag, V5 tag ; VSV-tag; or Xpress tag.
In some embodiments, the circular polyribonucleotide encodes the expression of an antibody, e.g., an antibody fragment, or a portion thereof. In some embodiments, the antibody expressed by the circular polyribonucleotide can be of any isotype, such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, 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. In some embodiments, the circular polyribonucleotide expresses one or more portions of an antibody. For instance, 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. In some cases, 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. In some cases, when the circular polyribonucleotide is expressed in a cell or a cell-free environment, the light chain and heavy chain can be subject to appropriate modification, folding, or other post-translation modification to form a functional antibody.
Regulatory Elements
In some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes one or more regulatory elements. In some embodiments, 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 linked operatively 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 exists. In addition, one regulatory element can increase amounts of products expressed for multiple expression sequences attached in tandem. Hence, one regulatory element can enhance the expression of one or more expression sequences. Multiple regulatory elements are well-known to persons of ordinary skill in the art.
In some embodiments, 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. In some embodiments, the polyribonucleotide includes at least one translation modulator adjacent to at least one expression sequence. In some embodiments, the polyribonucleotide includes a translation modulator adjacent each expression sequence. In some embodiments, 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). In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site. Further examples of regulatory elements are described, e.g., in paragraphs [0154] - [0161] of International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
Internal Ribosomal Entry Sites
In some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences). In embodiments, the IRES is located between a heterologous promoter and the 5’ end of a coding sequence. In some embodiments, the moiety that specifically bind a region of the circular polyribonucleotide binds a region of the circular polyribonucleotide includes an IRES.
A suitable IRES element to include in a polyribonucleotide includes an RNA sequence capable of engaging a eukaryotic ribosome. In some embodiments, 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.
In some embodiments, the IRES element is derived from the DNA of an organism including, but not limited to, a virus, a mammal, or a Drosophila. Such viral DNA may be derived from, but is not limited to, picomavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA from which an IRES element is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster.
In some embodiments, if present, 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, fuman poliovirus 1 , Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1 , Human Immunodeficiency Virus type 1 , Homalodisca coagulata virus- 1 , Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 , Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1 , Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1 , Human AML1 /RUNX1 , Drosophila antennapedia, Human AQP4, Human AT1 R, Human BAG-I, Human BCL2 , Human BiP, Human c-IAPI , Human c-myc, Human elF4G, Mouse NDST4L, Human LEF1 , Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-I, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Salivirus, Cosavirus, Parechovirus, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1 , Human c-src, Human FGF-I, Simian picomavirus, Turnip crinkle virus, Aichivirus, Crohivirus, Echovirus 11 , an aptamer to elF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 /2). In yet another embodiment, the IRES is an IRES sequence of Coxsackievirus B3 (CVB3). In a further embodiment, the IRES is an IRES sequence of Encephalomyocarditis virus. In a further embodiment, the IRES is an IRES sequence of Theiler's encephalomyelitis virus. In some embodiments, the IRES sequence has more than 90% sequence identify with one of the foregoing IRES sequences.
The IRES sequence may have the sequence of wild-type CVB3 IRES sequence having the nucleic acid sequence of:
Figure imgf000088_0001
GCAAA (SEQ ID NO: 96).
The IRES sequence may be a CVB3 IRES sequence having the nucleic acid sequence of :
Figure imgf000088_0002
GCAA (SEQ ID NO: 97).
The IRES sequence may have a modified sequence in comparison to the wild-type IRES sequence. In some embodiments, when the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be modified such that it is a cytosine residue. For example, the IRES sequence may be a CVB3 IRES sequence wherein the terminal adenosine residue is modified to cytosine residue. In some embodiments, the modified CVB3 IRES may have the nucleic acid sequence of:
Figure imgf000088_0003
Figure imgf000089_0001
GCAAC (SEQ ID NO: 98).
In some embodiments, the IRES sequence is an encephalomyocarditis virus (EMCV) IRES. In some embodiments, the ECMV IRES may have the nucleic acid sequence of:
Figure imgf000089_0002
( )
In some embodiments, the IRES sequence is an Enterovirus 71 (EV71) IRES. In some embodiments, the IRES sequence is a wild-type EV71 sequence having the nucleic acid sequence of:
Figure imgf000089_0003
AAGCG (SEQ ID NO: 100).
In some embodiments, the terminal guanosine residue of the EV71 IRES sequence is modified to a cytosine residue. In some embodiments, the modified EV71 IRES may have the nucleic acid sequence of:
Figure imgf000089_0004
Figure imgf000090_0001
AAGCC (SEQ ID NO: 101).
In some embodiments, 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. In some embodiments, 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).
In some embodiments, a polyribonucleotide described herein includes an IRES (e.g., an IRES operably linked to a coding region). For example, the polyribonucleotide may include any IRES as described in Fan et al. Nature Communications 13(1):3751 -3765, 2022 doi: 10.1038/s41467-022-31327- y; 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. PNAS 99(8):5301-5306, 2002; Wang et al. Nucleic Acids Research 33(7):2248-2258, 2005; Petz et a. Nucleic Acids Research 35(8):2473-2482, 2007; Chen et al. Science 268:415-417, 1995; and International Publication No. W02020/198403; International Patent Publication No. WO 2021/263124 A2; and International Publication No. WO2022/271965, each of which is hereby incorporated by reference in their entirety.
Signal Sequences
In some embodiments, a polypeptide expressed from a circular polyribonucleotide disclosed herein includes a secreted protein, for example, a protein that naturally includes a signal sequence, or one that does not usually encode a signal sequence but is modified to contain one. In some embodiments, the polypeptide encoded by the circular polyribonucleotide includes a secretion signal. For example, the secretion signal may be the naturally encoded secretion signal for a secreted protein. In another example, the secretion signal may be a modified secretion signal for a secreted protein. In other embodiments, the polypeptide encoded by the circular polyribonucleotide does not include a secretion signal.
In some embodiments, a circular polyribonucleotide encodes multiple copies of the same polypeptide (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more). In some embodiments, at least one copy of the polypeptide 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., a plurality of different polypeptides or a plurality of polypeptides having less than 100% sequence identity), 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.
In some embodiments, the signal sequence is a wild-type signal sequence that is present on the N-terminus of the corresponding wild-type polypeptide, e.g., when expressed endogenously. In some embodiments, the signal sequence is heterologous to the polypeptide, 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. A polypeptide encoded by a polyribonucleotide may include a signal sequence that directs the polypeptide to the secretory pathway. In some embodiments, the signal sequence may direct the polypeptide to reside in certain organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes). In some embodiments, the signal sequence directs the polypeptide to be secreted from the cell. For secreted proteins, the signal sequence may be cleaved after secretion, resulting in a mature protein. In other embodiments, 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. In certain embodiments, the signal sequence of a transmembrane protein is a short sequence at the N-terminal of the polypeptide. In other embodiments, the first transmembrane domain acts as the first signal sequence, which targets the protein to the membrane.
In some embodiments, a polypeptide encoded by a polyribonucleotide includes either a secretion signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence.
Cleavage Domains
A circular polyribonucleotide of the disclosure can include a cleavage domain (e.g., a stagger element or a cleavage sequence).
The term “stagger element” refers to a moiety, such as a nucleotide sequence, that induces ribosomal pausing during translation. In some embodiments, the stagger element is a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence - D(V/l)ExNPGP, where x= any amino acid (SEQ ID NO: 61). In some embodiments, 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.
In some embodiments, 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 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 polyribonucleotide. In some embodiments, 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. In some embodiments, the stagger element is a sequence separate from the one or more expression sequences. In some embodiments, the stagger element includes a portion of an expression sequence of the one or more expression sequences.
In some embodiments, the circular polyribonucleotide includes a stagger element. To avoid production of a continuous expression product, e.g., peptide or polypeptide, while maintaining rolling circle translation, a stagger element may be included to induce ribosomal pausing during translation. In some embodiments, 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 polyribonucleotide. The stagger element may include, but is not limited to a 2A-like, or CHYSEL (SEQ ID NO: 62) (cis-acting hydrolase element) sequence. In some embodiments, the stagger element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP (SEQ ID NO: 63), where X1 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. In some embodiments, this sequence includes a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence -D(V/I)EXNPGP (SEQ ID NO: 64), where x= any amino acid. Some nonlimiting examples of stagger elements includes GDVESNPGP (SEQ ID NO: 65), GDIEENPGP (SEQ ID NO: 66), VEPNPGP (SEQ ID NO: 67), IETNPGP (SEQ ID NO: 68), GDIESNPGP (SEQ ID NO: 69), GDVELNPGP (SEQ ID NO: 70), GDIETNPGP (SEQ ID NO: 71), GDVENPGP (SEQ ID NO: 72), GDVEENPGP (SEQ ID NO: 73), GDVEQNPGP (SEQ ID NO: 74), IESNPGP (SEQ ID NO: 75), GDIELNPGP (SEQ ID NO: 76), HDIETNPGP (SEQ ID NO: 77), HDVETNPGP (SEQ ID NO: 78), HDVEMNPGP (SEQ ID NO: 79), GDMESNPGP (SEQ ID NO: 80), GDVETNPGP (SEQ ID NO: 81), GDIEQNPGP (SEQ ID NO: 82), and DSEFNPGP (SEQ ID NO: 83). In some embodiments, the stagger element described herein cleaves an expression product, such as between G and P of the consensus sequence described herein. As one non-limiting example, the circular polyribonucleotide includes at least one stagger element to cleave the expression product. In some embodiments, the circular polyribonucleotide includes a stagger element adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element after each expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element 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. In some embodiments, 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 / 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. In some embodiments, the stagger element is present in the circular polyribonucleotide in other forms. For example, in some exemplary circular polyribonucleotides, a stagger element includes a termination element of a first expression sequence in the circular 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. In some examples, 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 polyribonucleotide. In some cases, the first expression sequence and the expression sequence succeeding the first expression sequence are two separate expression sequences in the circular 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. In some embodiments, 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. In some cases, the circular polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the succeeding sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular 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. In some cases, there is only one expression sequence in the circular polyribonucleotide, and the first expression sequence and its succeeding expression sequence are the same expression sequence. In some exemplary circular polyribonucleotides, a stagger element includes a first termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a downstream translation initiation sequence. In some such examples, the first stagger element is upstream of (5’ to) a first translation initiation sequence of the first expression sequence in the circular polyribonucleotide. In some cases, 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.
In some embodiments, 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. In some cases, the circular polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the first expression sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide including a stagger element upstream of a second translation initiation sequence of a second expression sequence in the corresponding circular polyribonucleotide is not continuously translated. In some cases, 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 polyribonucleotide than a distance between the first stagger element and the first translation initiation in the circular polyribonucleotide. In some cases, 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. In some embodiments, 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. In some embodiments, the circular polyribonucleotide includes more than one expression sequence.
Examples of stagger elements are described in paragraphs [0172] - [0175] of International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
In some embodiments, a plurality of polypeptides encoded by a circular ribonucleotide may be separated by an IRES between each polypeptide (e.g., each polypeptide is operably linked to a separate IRES). For example, a circular polyribonucleotide may include a first IRES operably 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 polypeptides. The IRES may be different between different polypeptides. In some embodiments, the plurality of polypeptides may be separated by a 2A self-cleaving peptide. For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, a 2A, and a second polypeptide.
In some embodiments, the plurality of polypeptides may be separated by a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, a protease cleavage site (e.g., a furin cleavage site), and a second polypeptide.
In some embodiments, the plurality of polypeptides may be separated by a 2A self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, a 2A, a protease cleavage site (e.g., a furin cleavage site), and a second polypeptide. A circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first polypeptide, a protease cleavage site (e.g., a furin cleavage site), a 2A, and a second polypeptide. 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).
Furthermore, the plurality of polypeptides encoded by the circular ribonucleotide may be separated by both IRES and 2A sequences. For example, an IRES may be between one polypeptide and a second polypeptide while a 2A peptide may be between the second polypeptide and the third polypeptide. The selection of a particular IRES or 2A self-cleaving peptide may be used to control the expression level of a polypeptide 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.
In some embodiments, a circular 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 polyribonucleotide includes from 2 to 10 cleavage sequences. In some embodiments, the circular polyribonucleotide includes from 2 to 5 cleavage sequences. In some embodiments, the multiple cleavage sequences are between multiple expression sequences; for example, a circular polyribonucleotide may include three expression sequences two cleavage sequences such that there is a cleavage sequence in between each expression sequence. In some embodiments, the circular polyribonucleotide includes a cleavage sequence, such as in an immolating circRNA or cleavable circRNA or self-cleaving circRNA. In some embodiments, the circular polyribonucleotide includes two or more cleavage sequences, leading to separation of the circular polyribonucleotide into multiple products, e.g., miRNAs, linear RNAs, smaller circular polyribonucleotide, etc.
In some embodiments, 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. In some embodiments, 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. In some embodiments, aptamers and ribozymes can both be encoded in the same non-coding RNA.
In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, a polyribonucleotide may encode two or more polypeptides, e.g., where the two or more polypeptides are encoded by a single open-reading frame (ORF). For example, two or more polypeptides may be encoded by a single open-reading frame, the expression of which is controlled by an IRES. In some embodiments, the ORF further encodes a polypeptide linker, e.g., such that the expression product of the ORF encodes two or more polypeptides 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). In such embodiments, a single expression product including the amino acid sequence of two or more polypeptides is cleaved upon expression, such that the two or more polypeptides are separated following expression. Exemplary 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 (e.g., furin), urokinase-type plasminogen activator, matriptase, a cysteine protease, an aspartic protease, or a cathepsin protease. In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is matriptase.
In some embodiments, a circular polyribonucleotide described herein is an immolating circular polyribonucleotide, a cleavable circular polyribonucleotide, or a self-cleaving circular polyribonucleotide. A circular polyribonucleotide can deliver cellular components including, for example, RNA, long non-coding RNA (IncRNA), long intergenic non-coding RNA (lincRNA), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), small interfering RNA (siRNA), or small hairpin RNA (shRNA). In some embodiments, a circular polyribonucleotide includes miRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites; (iii) degradable linkers; (iv) chemical linkers; and/or (v) spacer sequences. In some embodiments, 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 sequences. Non-limiting examples of self- cleavable elements include hammerhead, splicing element, hairpin, hepatitis delta virus (HDV), Varkud Satellite (VS), and glmS ribozymes.
Translation Initiation Sequences
In some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes at least one translation initiation sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence operably linked to an expression sequence.
In some embodiments, the polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide includes the translation initiation sequence, e.g., Kozak sequence, adjacent to an expression sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, the translation initiation sequence, e.g., Kozak sequence, is present on one or both sides of each expression sequence, leading to separation of the expression products. In some embodiments, the polyribonucleotide includes at least one translation initiation sequence adjacent to an expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the 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 polyribonucleotide may include more than 1 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.
In some embodiments, the polyribonucleotide may initiate at a codon which is not the first start codon, e.g., AUG. Translation of the 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. In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions. As a non-limiting example, the translation of the polyribonucleotide may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the polyribonucleotide translation may begin at alternative translation initiation sequence, CTG/CUG. As another non-limiting example, the polyribonucleotide translation may begin at alternative translation initiation sequence, GTG/GUG. As another non-limiting example, 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 (SEQ ID NO: 93), CAG, CTG.
Termination Elements
In some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes 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.
In some embodiments, 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.
In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, 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. In some other embodiments, a termination element of an expression sequence can be part of a stagger element. In some embodiments, one or more expression sequences in the circular polyribonucleotide includes a termination element. However, rolling circle translation or expression of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide is performed. In such instances, the expression product may fall off the ribosome when the ribosome encounters the termination element, e.g., a stop codon, and terminates translation. In some embodiments, translation is terminated while the ribosome, e.g., at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.
In some embodiments, the circular polyribonucleotide includes a termination element at the end of one or more expression sequences. In some embodiments, one or more expression sequences includes two or more termination elements in succession. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome completely disengages with the circular polyribonucleotide. In some such embodiments, 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. Generally, termination elements include an in-frame nucleotide triplet that signals termination of translation, e.g., UAA, UGA, UAG. In some embodiments, 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. In some embodiments, the termination element is a stop codon.
Further examples of termination elements are described in paragraphs [0169] - [0170] of International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
Untranslated Regions
In some embodiments, a circular polyribonucleotide includes untranslated regions (UTRs). UTRs of a genomic region including a gene may be transcribed but not translated. In some embodiments, a UTR may be included upstream of the translation initiation sequence of an expression sequence described herein. In some embodiments, a UTR may be included downstream of an expression sequence described herein. In some instances, one UTR for first expression sequence is the same as or continuous with or overlapping with another UTR for a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, e.g., ZKSCAN1.
Exemplary untranslated regions are described in paragraphs [0197] - [201] of International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
In some embodiments, a circular polyribonucleotide includes a polyA tail. Exemplary polyA tails are described in paragraphs [0202] - [0205] of International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety. In some embodiments, a circular polyribonucleotide lacks a polyA tail. In some embodiments, a circular 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.
Introduction, removal, or modification of UTR AU rich elements (AREs) may be useful to modulate the stability, or immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide. When engineering specific circular polyribonucleotides, 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. Likewise, 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.
It should be understood that any UTR from any gene may be incorporated into the respective flanking regions of the circular polyribonucleotide.
In some embodiments, 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 polyribonucleotide lacks a 3’-UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a polyA tail and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a termination element and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular 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 polyribonucleotide lacks a cap and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 5’-UTR, a 3 -UTR, and an IRES, and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular 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 a therapeutic, 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 a therapeutic mRNA or protein.
In some embodiments, a circular polyribonucleotide lacks a 5’-UTR. In some embodiments, the circular polyribonucleotide lacks a 3’-UTR. In some embodiments, the circular polyribonucleotide lacks a polyA tail. In some embodiments, the circular polyribonucleotide lacks a termination element. In some embodiments, the circular polyribonucleotide lacks an internal ribosomal entry site. In some embodiments, the circular polyribonucleotide lacks degradation susceptibility by exonucleases. In some embodiments, 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. In some embodiments, the circular polyribonucleotide is not degraded by exonucleases. In some embodiments, the circular polyribonucleotide has reduced degradation when exposed to exonuclease. In some embodiments, the circular polyribonucleotide lacks binding to a cap-binding protein. In some embodiments, the circular polyribonucleotide lacks a 5’ cap. Spacer Sequences
In some embodiments, the polyribonucleotide described herein includes one or more spacer sequences. In some embodiments, the moiety that specifically bind a region of the circular polyribonucleotide binds a region of the circular polyribonucleotide includes one or more spacers. A spacer refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacers may be present in between any of the nucleic acid elements described herein. Spacer may also be present within a nucleic acid element described herein.
For example, wherein a nucleic acid includes any two or more of the following elements: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment; a spacer region may be present between any one or more of the elements. Any of elements (A), (B), (C), (D), (E), (F), or (G) may be separated by a spacer sequence, as described herein. For example, there may be a spacer between (A) and (B), between (B) and (C), between (C) and (D), between (D) and (E), between (E) and (F), or between (F) and (G).
A spacer sequences may be used to separate an IRES from adjacent structural elements to martini the structure and function of the IRES or the adjacent element. A spacer can be specifically engineered depending on the IRES. In some embodiments, an RNA folding computer software, such as RNAFold, can be utilized to guide designs of the various elements of the vector, including the spacers.
The spacer may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, each spacer region is at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. Each spacer region may be, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a polyA tail (or, e.g., polyA sequence). The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a polyA-C tail (or, e.g., polyA-C sequence). In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a polyA-G tail (or, e.g., polyA-G sequence). In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a polyA-U tail (or, e.g., polyA-U sequence). In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region includes a random sequence.
In some embodiments, the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 30 nucleotides in length. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the spacer sequence is from 20 to 50 nucleotides in length. In certain embodiments, the spacer sequence is 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.
The spacer sequences can be polyA tails, polyA-C tails, polyC tails, or poly-U tails. In some embodiments, the spacer sequences can be polyA-T, polyA-C, polyA-G, or a random sequence.
Exemplary spacer sequences are described in paragraphs [0293] - [0302] of International Patent Publication No. WO2019/1 18919, which is hereby incorporated by reference in its entirety.
Modifications
A polyribonucleotide (e.g., circular polyribonucleotide) as described herein 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.
In some embodiments, a circular polyribonucleotide includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, polyA tail, 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. Nucl Acids Res 27: 196-197). In some embodiments, the first isolated nucleic acid includes messenger RNA (mRNA). In some embodiments, the polyribonucleotide includes at least one nucleoside selected from the group such as those described in [0311] of International Patent Publication No. WO2019/1 18919, 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 / 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). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.
In some embodiments, a polyribonucleotide includes at least one N(6)methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the m6A modification can reduce immunogenicity (e.g., reduce the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide.
In some embodiments, a modification may include a chemical or cellular induced modification. For example, some non-limiting examples of intracellular 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.
In some embodiments, chemical modifications to the ribonucleotides of a circular polyribonucleotide may enhance immune evasion. The circular 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. In some embodiments, base modifications may modulate expression, immune response, stability, subcellular localization, to name a few functional effects, of the circular polyribonucleotide. In some embodiments, the modification includes a bi-orthogonal nucleotide, e.g., an unnatural base. See for example, Kimoto et al, Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039/c7cc06661a, which is hereby incorporated by reference.
In some embodiments, sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar one or more ribonucleotides of the circular polyribonucleotide may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of circular polyribonucleotide include, but are not limited to, circular polyribonucleotide including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Circular polyribonucleotides having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the circular 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. In some embodiments, the circular polyribonucleotide may be negatively or positively charged.
The modified nucleotides, which may be incorporated into the polyribonucleotide, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide 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. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, 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 circular polyribonucleotide is expected to reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'-0-(1 - thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine (a-thio-cytidine), 5'-0-(1-thiophosphate)- guanosine, 5'-0-(1-thiophosphate)-uridine, or 5'-0-(1-thiophosphate)-pseudouridine).
Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.
In some embodiments, a circular polyribonucleotide may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into circular 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, 1-(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-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1 H,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'- methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1 -beta-D- arabinofuranosylcytosine, N4-octadecyl-1 -beta-D-arabinofuranosylcytosine, N4- palmitoyl-1-(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. For example, 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) may or may not be uniformly modified in the circular polyribonucleotide, or in a given predetermined sequence region thereof. In some embodiments, the circular polyribonucleotide includes a pseudouridine. In some embodiments, the circular polyribonucleotide includes an inosine, which may aid in the immune system characterizing the circular polyribonucleotide as endogenous versus viral RNAs. The incorporation of 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.
In some embodiments, all nucleotides in a polyribonucleotide (or in a given sequence region thereof) are modified. In some embodiments, 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).
Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in a circular polyribonucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the circular polyribonucleotide, such that the function of the circular polyribonucleotide is not substantially decreased. A modification may also be a non-coding region modification. The circular 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 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).
Production of circular polyribonucleotides
The disclosure provides methods for producing circular polyribonucleotides complexed with a targeting moiety, including, e.g., recombinant technology or chemical synthesis. For example, a DNA molecule used to produce an RNA circle can include a DNA sequence of a naturally occurring original 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.
The circular polyribonucleotides may be prepared according to any available technique, including, but not limited to chemical synthesis and enzymatic synthesis. In some embodiments, a linear primary construct or linear RNA may be cyclized or concatenated to create a circRNA described herein. 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. For example, a splint ligase, such as a SplintR® ligase, can be used for splint ligation. According to this method, a single stranded polynucleotide (splint), such as a single-stranded DNA or RNA, 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. In some embodiments, a DNA or RNA ligase may be used in the synthesis of the circular polynucleotides. As a non-limiting example, the ligase may be a circ ligase or circular ligase.
In another example, 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 circRNA includes an active ribozyme sequence capable of ligating the 5' end of the linear polyribonucleotide to the 3' end of the linear polyribonucleotide. 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). In another example, a linear polyribonucleotide may be cyclized or concatenated by using at least one non-nucleic acid moiety. For example, 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 in order to cyclize or concatenate the linear polyribonucleotide. In another example, 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. The non-nucleic acid moieties may be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage or a cleavable linkage. As another non-limiting example, the non-nucleic acid moiety is a ligation moiety. As yet another non-limiting example, 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.
In another example, linear polyribonucleotides may be cyclized or concatenated by self-splicing. In some embodiments, the linear polyribonucleotides may include loop E sequence to self-ligate. In another embodiment, the linear polyribonucleotides may include a self-circularizing intron, e.g., a 5' and 3’ slice junction, or a self-circularizing catalytic intron such as a Group I, Group II or Group III Introns. Nonlimiting examples of 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.
In another example, a linear polyribonucleotide 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. The one or more linear polyribonucleotides may be cyclized or concatenated by intermolecular forces or intramolecular forces. Non-limiting examples of 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.
In another example, the linear polyribonucleotide 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, thereby causing a linear polyribonucleotide to cyclize or concatenate. In another example, 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 ligated using various methods known in the art such as, but not limited to, protein ligation. Non-limiting examples of 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.
In yet another example, 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.
Methods of making the circular polyribonucleotides described herein are described in, for example, Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); in Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012).
Various methods of synthesizing circular polyribonucleotides are also described elsewhere (see, e.g., US Patent No. US6210931 , US Patent No. US5773244, US Patent No. US5766903, US Patent No. US5712128, US Patent No. US5426180, US Publication No. US20100137407, International Publication No. W01992001813, International Publication No. W02010084371 , and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015); the contents of each of which are herein incorporated by reference in their entirety).
In some embodiments, the circular polyribonucleotide is purified, e.g., free ribonucleic acids, linear or nicked RNA, DNA, proteins, etc. are removed. In some embodiments, the circular polyribonucleotides may be purified by any known method commonly used in the art. Examples of nonlimiting purification methods include, column chromatography, gel excision, size exclusion, etc.
Formulations
In some embodiments of the present disclosure a circular polyribonucleotide described herein may be formulated in composition, e.g., a composition for delivery to a cell, a plant, an invertebrate animal, a non-human vertebrate animal, or a human subject, e.g., an agricultural, veterinary, or pharmaceutical composition. In some embodiments, the circular polyribonucleotide is formulated in a pharmaceutical composition. In some embodiments, a composition includes a circular polyribonucleotide and a diluent, a carrier, an adjuvant, or a combination thereof. In a particular embodiment, a composition includes a circular polyribonucleotide described herein and a carrier or a diluent free of any carrier. In some embodiments, a composition including a circular polyribonucleotide with a diluent free of any carrier is used for naked delivery of the circular polyribonucleotide to a subject.
Pharmaceutical 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). Non-limiting 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.
Although the descriptions of pharmaceutical 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. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient 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.
In some embodiments, the reference criterion for the amount of circular polyribonucleotide molecules present in the 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 pharmaceutical preparation.
In some embodiments, the reference criterion for the amount of linear polyribonucleotide molecules present in the 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, 700 pg/ml, 800 pg/ml, 900 pg/ml, 1 mg/ml, 1.5 mg/ml, or 2 mg/ml of linear polyribonucleotide molecules.
In some embodiments, the reference criterion for the amount of linear polyribonucleotide molecules present in the 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 pharmaceutical preparation.
In some embodiments, the reference criterion for the amount of nicked polyribonucleotide molecules present in the 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 pharmaceutical preparation.
In some embodiments, the reference criterion for the amount of combined nicked and linear polyribonucleotide molecules present in the 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 pharmaceutical preparation. In some embodiments, a pharmaceutical preparation is an intermediate pharmaceutical preparation of a final circular polyribonucleotide drug product. In some embodiments, a pharmaceutical preparation is a drug substance or active pharmaceutical ingredient (API). In some embodiments, a pharmaceutical preparation is a drug product for administration to a subject.
In some embodiments, a preparation of circular polyribonucleotides is (before, during or after the reduction of linear RNA) 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. In some embodiments, 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. In some embodiments, the pharmaceutical formulation disclosed herein is a stable liquid pharmaceutical formulation. In some embodiments, the pharmaceutical formulation disclosed herein includes protamine or a protamine salt (e.g., protamine sulfate). Delivery Agents A composition or pharmaceutical composition provided herein may include one or more delivery agents, wherein the delivery agents may increase cellular delivery, functional delivery, or endosomal escape of the circular polyribonucleotide. The delivery agent may be used in place of or in combination with lipofectamine to increase transfection of a circular polyribonucleotide into cell. In some embodiments, the circular polyribonucleotide is administered to a subject without the use of lipofectamine. In some embodiments, the circular polyribonucleotide described herein may be formulated in a composition, wherein the composition includes a ribonuclease inhibitor. In some embodiments, the ribonuclease inhibitor is selected from RNseOUTTM recombinant ribonuclease inhibitor, RNasinTM ribonuclease inhibitor, and SUPERase-InTM RNase inhibitor. The ribonuclease inhibitor may be in any amount suitable for delivery of the composition; for example, the ribonuclease inhibitor may be in an amount of from 0.05 U/mL to 1 U/mL, where one unit is defined as the amount of ribonuclease inhibitor required to inhibit the activity of 5 ng of ribonuclease A by 50%, and activity is measured by the inhibition of hydrolysis of cytidine 2´,3´-cyclic monophosphate by ribonuclease A. In some embodiments, the circular polyribonucleotide described herein may be formulated in a composition, wherein the composition includes a calcium cation, magnesium cation, manganese cation, strontium cation, or any combination thereof. For example, the composition may include calcium chloride, calcium acetate, calcium carbonate, calcium citrate, calcium gluconate, calcium lactate, and calcium phosphate. The calcium, manganese, or strontium cation may be in any amount suitable for delivery of the composition. In some embodiments, the calcium, manganese, or strontium cation may be included at a concentration of from 0.01 mM to 500 mM (e.g., 0.01 mM to 400 mM, 0.01 mM to 300 mM, 0.01 mM to 200 mM, 0.01 mM to 100 mM, 0.01 mM to 1 mM, 0.01 mM to 0.1 mM, 0.1 mM to 500 mM, 1 mM to 500 mM, 10 mM to 500 mM, 100 mM to 500 mM, 200 mM to 500 mM, 300 mM to 500 mM, or 400 mM to 500 mM). In some embodiments, the calcium, manganese, or strontium cation may be included at a concentration of from 0.1 mM to 100 mM (e.g., 0.1 mM to 90 mM, 0.1 mM to 70 mM, 0.1 mM to 50 mM, 0.1 mM to 30 mM, 0.1 mM to 10 mM, 0.1 mM to 1 mM, 1 mM to 100 mM, 10 mM to 100 mM, 30 mM to 100 mM, 50 mM to 100 mM, 70 mM to 100 mM, or 90 mM to 100 mM). In some embodiments, the circular polyribonucleotide described herein may be formulated in a composition, wherein the composition includes an endosomal escape agent. In some embodiments, the endosomal escape agent includes chloroquine, amantadine, ammonium chloride, 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), UNC-108, or any combination thereof. The endosomal escape agent may be in any amount suitable for delivery of the composition. For example, the chloroquine concentration in the composition may be from 10 µM to 1 M (e.g., 10 µM to 10 mM, 10 µM to 1 mM, 10 µM to 100 µM, 100 µM to 1 M, 1 mM to 1 M, or 10 mM to 1 M) . In some embodiments, the endosomal escape agent concentration is from 10 µM to 100 µM (e.g., 10 µM to 90 µM, 10 µM to 70 µM, 10 µM to 50 µM, 10 µM to 30 µM, 30 µM to 100 µM, 50 µM to 100 µM, or 70 µM to 100 µM). In some embodiments, the circular polyribonucleotide described herein may be formulated in a composition, wherein the composition includes globular protein. In some embodiments, the globular protein is albumin. In some embodiments, the composition includes human serum albumin. In some embodiments, the composition includes bovine serum albumin (BSA). The BSA may be in any amount suitable for delivery of the composition. In some embodiments, the composition includes one or more delivery agents described herein (e.g., in any amount described herein). In some embodiments, the composition may include endosomal escape agent and a calcium, strontium, or manganese cation (e.g., in any amounts described herein). In some embodiments, the composition may include a calcium, strontium, or manganese cation and a ribonuclease inhibitor (e.g., in any amounts described herein). In some embodiments, the composition may include a ribonuclease inhibitor and endosomal escape agent (e.g., in any amounts described herein). In some embodiments, the composition includes albumin and endosomal escape agent (e.g., in any amounts described herein). In some embodiments, the composition includes albumin and endosomal escape agent (e.g., in any amounts described herein). In some embodiments, the composition includes albumin and a ribonuclease inhibitor (e.g., in any amounts described herein). In some embodiments, the composition may include calcium chloride, chloroquine, and a ribonuclease inhibitor (e.g., in any amounts described herein). In some embodiments, the composition may include a calcium, manganese, or strontium cation, endosomal escape agent, a ribonuclease inhibitor, and albumin (e.g., in any amounts described herein). Preservatives A composition or pharmaceutical composition provided herein can include material for a single administration, or can include material for multiple administrations (e.g., a “multidose” kit). The polyribonucleotide can be present in either linear or circular form. The composition or pharmaceutical composition can include one or more preservatives such as thiomersal or 2-phenoxyethanol. Preservatives can be used to prevent microbial contamination during use. 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. Polyribonucleotides can be susceptible to RNase that can be abundant in ambient environment. Compositions provided herein can include reagents that inhibit RNase activity, thereby preserving the polyribonucleotide from degradation. In some cases, the composition or pharmaceutical composition includes any RNase inhibitor known to one skilled in the art. Alternatively or additionally, the polyribonucleotide, and cell-penetrating agent and/or pharmaceutically acceptable 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. In some cases, 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. Salts In some cases, a composition or pharmaceutical composition provided herein includes one or more salts. For controlling the tonicity, a physiological salt such as sodium salt can be included a composition provided herein. Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, or the like. In some cases, the 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. Buffers/pH A composition or pharmaceutical composition provided herein can include one or more buffers, such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (e.g., with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers, in some cases, are included in the 5-20 mM range. A composition or pharmaceutical composition provided herein can have a pH from about 5.0 to about 8.5, from about 6.0 to about 8.0, from about 6.5 to about 7.5, or from about 7.0 to about 7.8. The composition or pharmaceutical composition can have a pH of about 7. The polyribonucleotide can be present in either linear or circular form. Detergents/surfactants A composition or pharmaceutical composition provided herein can include one or more detergents and/or surfactants, depending on the intended administration route, e.g., polyoxyethylene sorbitan esters surfactants (commonly referred to as “Tweens”), e.g., polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, e.g., octoxynol-9 (Triton X-100, or t- octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as “SPANs”), such as sorbitan trioleate (Span 85) and sorbitan monolaurate, an octoxynol (such as octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide (“CTAB”), or sodium deoxycholate. The one or more detergents and/or surfactants can be present only at trace amounts. In some cases, 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. Diluents In some embodiments, a composition of the disclosure includes a circular polyribonucleotide and a diluent. In some embodiments, a composition of the disclosure includes a linear polyribonucleotide and a diluent. A diluent can be a non-carrier excipient. A non-carrier excipient serves as a vehicle or medium for a composition, such as a circular polyribonucleotide as described herein. A non-carrier excipient serves as a vehicle or medium for a composition, such as a linear 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. 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 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. In some embodiments, the circular polyribonucleotide may be delivered as a naked delivery formulation, such as including a diluent. A naked delivery formulation delivers a circular polyribonucleotide, to a cell without the aid of a carrier and without modification or partial or complete encapsulation of the circular polyribonucleotide, capped polyribonucleotide, or complex thereof. A naked delivery formulation is a formulation that is free from a carrier and wherein the circular 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 circular polyribonucleotide. In some embodiments, a circular 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 protein, small molecule, a particle, a polymer, or a biopolymer. A circular polyribonucleotide without covalent modification that binds a moiety that aids in delivery to a cell does not contain a modified phosphate group. For example, a circular 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. In some embodiments, a naked delivery formulation is free of any or all of: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. In some embodiments, 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, 1,2-Dioleoyl-3-Trimethylammonium- Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2- (oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N—(N\N'- Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HC1), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N- (1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein (LDL), high- density lipoprotein (HDL), or globulin. In certain embodiments, a naked delivery formulation includes a non-carrier excipient. In some embodiments, a non-carrier excipient includes an inactive ingredient that does not exhibit a cell- penetrating effect. In some embodiments, a non-carrier excipient includes a buffer, for example PBS. In some embodiments, a non-carrier excipient is a solvent, a non-aqueous solvent, 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. In some embodiments, a naked delivery formulation includes a diluent. A diluent may be a liquid diluent or a solid diluent. In some embodiments, a diluent is an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of 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, propylene glycol, trehalose, or sucrose. Carriers In some embodiments, a composition of the disclosure includes a circular polyribonucleotide and a carrier. In some embodiments, a composition of the disclosure includes a linear polyribonucleotide and a carrier. In certain embodiments, a composition includes a circular polyribonucleotide as described herein in a vesicle or other membrane-based carrier. In certain embodiments, a composition includes a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier. In other embodiments, a composition includes the circular polyribonucleotide in or via a cell, vesicle or other membrane-based carrier. In other embodiments, a composition includes the linear polyribonucleotide in or via a cell, vesicle or other membrane-based carrier. In one embodiment, a composition includes the circular polyribonucleotide in liposomes or other similar vesicles. In one embodiment, a composition includes the linear 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 a relatively 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). Vesicles can be made from several different 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). Although 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, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference. In certain embodiments, a composition of the disclosure includes a circular polyribonucleotide and lipid nanoparticles, for example lipid nanoparticles described herein. In certain embodiments, a composition of the disclosure includes a linear polyribonucleotide and lipid nanoparticles. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a circular polyribonucleotide molecule as described herein. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a linear 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 an important 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 (PLNs), 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. For a review, see, e.g., Li et al.2017, Nanomaterials 7, 122; doi:10.3390/nano7060122. Additional non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride- modified phytoglycogen or glycogen-type material), protein carriers (e.g., a protein covalently linked to the circular polyribonucleotide or a protein covalently linked to the linear polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or transfection reagent). Non-limiting examples of 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, 1,2- Dioleoyl-3-Trimethylammonium-Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N—(N\N'-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HC1), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). Non-limiting examples of protein carriers include human serum albumin (HSA), low-density lipoprotein (LDL), high- density lipoprotein (HDL), or globulin. Exosomes can also be used as drug delivery vehicles for a circular RNA composition or preparation described herein. Exosomes can be used as drug delivery vehicles for a linear polyribonucleotide composition or preparation described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; doi.org/10.1016/j.apsb.2016.02.001. Ex vivo differentiated red blood cells can also be used as a carrier for a circular RNA composition or preparation described herein. Ex vivo differentiated red blood cells can also be used as a carrier for a linear polyribonucleotide composition or preparation described herein. See, e.g., International Patent Publication Nos. WO2015/073587; WO2017/123646; WO2017/123644; WO2018/102740; WO2016/183482; WO2015/153102; WO2018/151829; WO2018/009838; Shi et al.2014. Proc Natl Acad Sci USA.111(28): 10131–10136; US Patent 9,644,180; Huang et al.2017. Nature Communications 8: 423; Shi et al.2014. Proc Natl Acad Sci USA.111(28): 10131–10136. Fusosome compositions, e.g., as described in International Patent Publication No. WO2018/208728, can also be used as carriers to deliver a circular polyribonucleotide molecule described herein. Fusosome compositions, e.g., as described in WO2018/208728, can also be used as carriers to deliver a linear polyribonucleotide molecule described herein. Virosomes and virus-like particles (VLPs) can also be used as carriers to deliver a circular polyribonucleotide molecule described herein to targeted cells. Virosomes and virus-like particles (VLPs) can also be used as carriers to deliver a linear polyribonucleotide molecule described herein to targeted cells. Plant nanovesicles and plant messenger packs (PMPs), e.g., as described in International Patent Publication Nos. WO2011/097480, WO2013/070324, WO2017/004526, or WO2020/041784 can also be used as carriers to deliver the circular RNA composition or preparation described herein. Plant nanovesicles and plant messenger packs (PMPs) can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein. Lipid reconstructed plant messenger packs (LPMPs), e.g., as described in International Patent Publication Nos. WO2021/041301, WO2023/069498, or WO2023/122080 can also be used as carriers to deliver the circular RNA composition or preparation described herein. Lipid reconstructed plant messenger packs (LPMPs) can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein. Lipid reconstructed natural messenger packs (LNMPs), e.g., as described in International Patent Publication Nos. WO2024/102434 can also be used as carriers to deliver the circular RNA composition or preparation described herein. Lipid reconstructed natural messenger packs (LNMPs) can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein. Bacteria- derived lipid compositions, e.g., as described in International Patent Publication Nos. WO2023/096858 can also be used as carriers to deliver the circular RNA composition or preparation described herein. Bacteria-derived lipid compositions can also be used as carriers to deliver a linear polyribonucleotide composition or preparation described herein. Microbubbles can also be used as carriers to deliver a circular polyribonucleotide molecule described herein. Microbubbles can also be used as carriers to deliver a linear polyribonucleotide molecule described herein. See, e.g., US7115583; Beeri, R. et al., Circulation.2002 Oct 1;106(14):1756- 1759; Bez, M. et al., Nat Protoc.2019 Apr; 14(4): 1015–1026; Hernot, S. et al., Adv Drug Deliv Rev.2008 Jun 30; 60(10): 1153–1166; Rychak, J.J. et al., Adv Drug Deliv Rev.2014 Jun; 72: 82–93. In some embodiments, microbubbles are albumin-coated perfluorocarbon microbubbles. The carrier including the circular 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 circular polyribonucleotide into a cell. Deposition of the circular polyribonucleotide into certain cell types may favor different particle sizes. For example, the particle size may be optimized for deposition of the circular polyribonucleotide into antigen presenting cells. The particle size may be optimized for deposition of the circular polyribonucleotide into dendritic cells. Additionally, the particle size may be optimized for depositions of the circular polyribonucleotide into draining lymph node cells. Lipid Nanoparticles The 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, in some embodiments, 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 WO2019217941; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol). Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example those described in Table 4 of WO2019217941, which is incorporated by reference—e.g., a lipid- containing nanoparticle can include one or more of the lipids in Table 4 of WO2019217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941, incorporated by reference. In some embodiments, conjugated lipids, when present, can include one or more of PEG- diacylglycerol (DAG) (such as l-(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-glycero-3- phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing. In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in WO2009/127060 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. In some embodiments, 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. For example, in some embodiments, 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. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1. In some embodiments, 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. Generally, the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg/ml to about 30 mg/mL. Some non-limiting example of 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)) described herein includes,
Figure imgf000115_0001
In some embodiments an LNP including Formula (i) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000115_0002
In some embodiments an LNP including Formula (ii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000115_0003
In some embodiments an LNP including Formula (iii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000116_0001
In some embodiments an LNP including Formula (v) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000116_0002
In some embodiments an LNP including Formula (vi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000116_0003
In some embodiments an LNP including Formula (viii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000117_0001
In some embodiments an LNP including Formula (ix) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000117_0002
wherein X1 is O, NR1, or a direct bond, X2 is C2-5 alkylene, X3 is C(=O) or a direct bond, R1 is H or Me, R3 is C1-3 alkyl, R2 is C1-3 alkyl, or R2 taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2 form a 4-, 5-, or 6-membered ring, or X1 is NR1, R1 and R2 taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R2 taken together with R3 and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1 is C2-12 alkylene, Y2 is selected from
Figure imgf000117_0003
(in either orientation), (in either orientation), (in either orientation), n is 0 to 3, R4 is C1-15 alkyl, Z1 is C1-6 alkylene or a direct bond,
Figure imgf000117_0004
(in either orientation) or absent, provided that if Z1 is a direct bond, Z2 is absent; R5 is C5-9 alkyl or C6-10 alkoxy, R6 is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7 is H or Me, or a salt thereof, provided that if R3 and R2 are C2 alkyls, X1 is O, X2 is linear C3 alkylene, X3 is C(=0), Y1 is linear Ce alkylene, (Y2 )n-R4 is
Figure imgf000117_0005
, R4 is linear C5 alkyl, Z1 is C2 alkylene, Z2 is absent, W is methylene, and R7 is H, then R5 and R6 are not Cx alkoxy. In some embodiments an LNP including Formula (xii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000118_0001
In some embodiments an LNP including Formula (xi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000118_0003
In some embodiments an LNP includes a compound of Formula (xiii) and a compound of Formula (xiv).
Figure imgf000118_0002
In some embodiments an LNP including Formula (xv) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000119_0001
In some embodiments an LNP including a formulation of Formula (xvi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells.
Figure imgf000119_0002
In some embodiments, 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)) described herein is made by one of the following reactions:
Figure imgf000120_0001
In some embodiments an LNP including Formula (xxi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxi) is an LNP described by WO2021113777 (e.g., a lipid of Formula (1) such as a lipid of Table 1 of WO2021113777).
Figure imgf000120_0002
wherein each n is independently an integer from 2-15; L1 and L3 are each independently -OC(O)-* or - C(O)O-*, wherein “*” 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, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkyl sulfonyl, and alkyl sulfonealkyl; and R2 is selected from a group consisting of:
Figure imgf000121_0001
In some embodiments an LNP including Formula (xxii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxii) is an LNP described by WO2021113777 (e.g., a lipid of Formula (2) such as a lipid of Table 2 of WO2021113777).
Figure imgf000121_0002
wherein each n is independently an integer from 1-15; R1 and R2 are each independently selected from a group consisting of:
Figure imgf000122_0001
R3 is selected from a group consisting of:
Figure imgf000123_0001
. In some embodiments an LNP including Formula (xxiii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxiii) is an LNP described by WO2021113777 (e.g., a lipid of Formula (3) such as a lipid of
Figure imgf000123_0002
wherein X is selected from -O-, -S-, or -OC(O)-*, wherein * indicates the attachment point to R1; R1 is selected from a group consisting of:
Figure imgf000123_0003
and R2 is selected from a group consisting of:
Figure imgf000124_0001
In some embodiments, a composition described herein (e.g., a nucleic acid (e.g., a circular polyribonucleotide) or a protein) is provided in an LNP that includes an ionizable lipid. In some embodiments, 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 US9,867,888 (incorporated by reference herein in its entirety). In some embodiments, 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 WO2015/095340 (incorporated by reference herein in its entirety). In some embodiments, 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). In some embodiments, 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 WO2010/053572 (incorporated by reference herein in its entirety). In some embodiments, 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-lH- cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020/106946 (incorporated by reference herein in its entirety). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, 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 from 40 to 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA (e.g., a circular polyribonucleotide)) described herein, encapsulated within or associated with the lipid nanoparticle. In some embodiments, 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. In some embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP including a cationic lipid. In some embodiments, the lipid nanoparticle may include a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, 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 WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016/0311759; I of US20150376115 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-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 US2017/0119904; I or II of WO2017/117528; A of US2012/0149894; A of US2015/0057373; A of WO2013/116126; A of US2013/0090372; A of US2013/0274523; A of US2013/0274504; A of US2013/0053572; A of W02013/016058; A of W02012/162210; 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, IIA, IIB, IIC, IID, or III-XXIV of US2014/0308304; of US2013/0338210; I, II, III, or IV of W02009/132131; A of US2012/01011478; I or XXXV of US2012/0027796; XIV or XVII of US2012/0058144; of US2013/0323269; I of US2011/0117125; I, II, or III of US2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011/0076335; I or II of US2006/008378; I of US2013/0123338; I or X-A-Y-Z of US2015/0064242; XVI, XVII, or XVIII of US2013/0022649; I, II, or III of US2013/0116307; I, II, or III of US2013/0116307; I or II of US2010/0062967; I-X of US2013/0189351; I of US2014/0039032; V of US2018/0028664; I of US2016/0317458; I of US2013/0195920; 5, 6, or 10 of US10,221,127; III-3 of WO2018/081480; I-5 or I-8 of WO2020/081938; 18 or 25 of US9,867,888; A of US2019/0136231; II of WO2020/219876; 1 of US2012/0027803; OF-02 of US2019/0240349; 23 of US10,086,013; cKK-E12/A6 of Miao et al (2020); C12-200 of WO2010/053572; 7C1 of Dahlman et al (2017); 304-O13 or 503-O13 of Whitehead et al; TS- P4C2 of US9,708,628; I of WO2020/106946; I of WO2020/106946; and (1), (2), (3), or (4) of WO2021/113777. Exemplary lipids further include a lipid of any one of Tables 1-16 of WO2021/113777. Further exemplary lipids are described in International Patent Publication Nos. WO2023/183616, WO2023/091490, WO2023/091787, and WO2024/049979, which are incorporated herein by reference in their entirety, In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28,3 l- tetraen-l9-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3- nonyldocosa-l3, l6-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety). Exemplary 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 (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), l8-l-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl- phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The 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, in certain embodiments, 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 stereate, 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 WO2017/099823 or US patent publication US2018/0028664, the contents of which is incorporated herein by reference in their entirety. In some embodiments, 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. In some embodiments, the non- cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, 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). In some embodiments, the lipid nanoparticles do not include any phospholipids. In some aspects, the lipid nanoparticle can further include a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53-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. In some embodiments, the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4 '-hydroxy)-buty1 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. In some embodiments, the component providing membrane integrity, such as a sterol, can include 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle. In some embodiments, 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. 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. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)- conjugated lipid. Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(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-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl- methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5,885,6l3, US6,287,59l, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, 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. In some embodiments, 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 (l-[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)-2000]. In some embodiments, 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:
Figure imgf000128_0001
In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, 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 WO2019051289A9, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, 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. For example, 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. Preferably, 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. In some other embodiments, 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% non-cationic 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 composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, 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. In some embodiments, 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. In some embodiments, the lipid particle includes ionizable lipid / non-cationic- lipid / sterol / conjugated lipid at a molar ratio of 50:10:38.5: 1.5. In an aspect, the disclosure provides a lipid nanoparticle formulation including phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine. In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately, or the additional compounds can be included in the lipid nanoparticles of the invention. In other words, the lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof. In some embodiments, the LNPs include biodegradable, ionizable lipids. In some embodiments, the LNPs include (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate, also called 3- ((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,l2Z)-octadeca- 9,l2-dienoate) or another ionizable lipid. See, e.g., lipids of WO2019/067992, WO/2017/173054, WO2015/095340, and WO2014/136086, as well as references provided therein. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH. In some embodiments, the average LNP diameter of the LNP formulation may be from 10s of nm to 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about l mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm. A LNP may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a LNP may be from about 0.10 to about 0.20. The zeta potential of a LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV. The efficiency of encapsulation of a protein and/or nucleic acid describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and/or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%. A LNP may optionally include one or more coatings. In some embodiments, a LNP may be formulated in a capsule, film, or table having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density. Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020/061457, WO2021/113777, and WO2021226597, each of which is incorporated herein by reference in its entirety. Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021). doi.org/10.1038/s41578-021-00358-0, which is incorporated herein by reference in its entirety (see, for example, exemplary lipids and lipid derivatives of Figure 2 of Hou et al.). In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2‐dilinoleyl‐4‐dimethylaminoethyl‐[1,3]‐dioxolane (DLin‐KC2‐DMA) or dilinoleylmethyl‐4‐dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety. LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, both incorporated by reference, and are useful for delivery of circular polyribonucleotides and linear polyribonucleotides described herein. Additional specific LNP formulations useful for delivery of nucleic acids (e.g., circular polyribonucleotides, linear polyribonucleotides) are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO. In embodiments, a polyribonucleotide encoding at least a portion of a polypeptide described herein is formulated in an LNP, wherein: (a) the LNPs include a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, and (b) the LNPs have a mean particle size of from 80 nm to 160 nm. Exemplary dosing of polyribonucleotide (e.g., a circular polyribonucleotide) LNP may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg/kg (RNA). In some embodiments, a dose of a polyribonucleotide composition described herein is from 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg. Kits In some aspects, the disclosure provides a kit. In some embodiments, the kit includes (a) a circular polyribonucleotide complexed with a targeting moiety or a pharmaceutical composition described herein, and optionally (b) informational material. The informational material may be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the pharmaceutical composition or circular polyribonucleotide for the methods described herein. The pharmaceutical composition or circular polyribonucleotide may include material for a single administration (e.g., single dosage form), or may include material for multiple administrations (e.g., a “multidose” kit). The informational material of the kits is not limited in its form. In one embodiment, the informational material may include information about production of the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product, molecular weight of the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods for administering a dosage form of the pharmaceutical composition. In one embodiment, the informational material relates to methods for administering a dosage form of the circular polyribonucleotide. In addition to a dosage form of the pharmaceutical composition and circular polyribonucleotide described herein, the kit may include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, a flavoring agent (e.g., a bitter antagonist or a sweetener), a fragrance, a dye or coloring agent, for example, to tint or color one or more components in the kit, or other cosmetic ingredient, and/or a second agent for treating a condition or disorder described herein. Alternatively, the other ingredients may be included in the kit, but in different compositions or containers than a pharmaceutical composition or circular polyribonucleotide described herein. In such embodiments, the kit may include instructions for admixing a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein and the other ingredients, or for using a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein together with the other ingredients. In some embodiments, the components of the kit are stored under inert conditions (e.g., under Nitrogen or another inert gas such as Argon). In some embodiments, the components of the kit are stored under anhydrous conditions (e.g., with a desiccant). In some embodiments, the components are stored in a light blocking container such as an amber vial. A dosage form of a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein may be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein be substantially pure and/or sterile. When a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit. The kit may include one or more containers for the composition containing a dosage form described herein. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the pharmaceutical composition or circular polyribonucleotide may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the dosage form of a pharmaceutical composition or nucleic acid molecule (e.g., a circular polyribonucleotide) described herein is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms of a pharmaceutical composition or circular polyribonucleotide described herein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a dosage form described herein. The containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight. The kit optionally includes a device suitable for use of the dosage form, e.g., a syringe, pipette, forceps, measured spoon, swab (e.g., a cotton swab or wooden swab), or any such device. The kits of the invention may include dosage forms of varying strengths to provide a subject with doses suitable for one or more of the initiation phase regimens, induction phase regimens, or maintenance phase regimens described herein. Alternatively, the kit may include a scored tablet to allow the user to administered divided doses, as needed. Methods of Use In some embodiments, a circular polyribonucleotide complexed with a targeting moiety is used for the treatment or prevention of a disease or condition in a subject. In some embodiments, a circular polyribonucleotide complexed with a targeting moiety by way of irradiating the components of the complex with light is used for the treatment or prevention of a disease or condition in a subject. For example, a circular polyribonucleotide as described herein may be administered to a subject (e.g., in a pharmaceutical composition). In some embodiments, the complex is irradiated prior to administration to a subject. In some embodiments the complex is irradiated after administration to a subject. In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the subject is a non- human mammal. In embodiments, the subject is a non-human mammal is 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). In embodiments, 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). In embodiments, the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusk. In some embodiments, the disclosure provides a method of modifying a subject by providing to the subject a composition or formulation described herein. In some embodiments, the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes or a eukaryotic or prokaryotic cell including a nucleic acid described herein. In some embodiments, the disclosure provides a method of treating a disease or condition in a subject in need thereof by providing to the subject a composition or formulation described herein. In some embodiments, the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes a eukaryotic or prokaryotic cell including a nucleic acid described herein. In some embodiments, the polyribonucleotide is provided in an amount and for a duration sufficient to treat a disease or condition in a subject, e.g., in need thereof. Methods of Making The present disclosure includes a method of forming covalent attachments between one or more binding moieties and the circular polyribonucleotide upon irradiation with light by way of one or more photoreactive crosslinking agents included within each of the one or more binding moieties, wherein each of the one or more binding moieties is conjugated (e.g., directly, chemically-covalently conjugated, etc.) to one or more targeting moieties. In some embodiments, a method of covalently attaching one or more binding moieties, each including one or more photoreactive crosslinking agents, to the circular polyribonucleotide includes irradiating any of the complexes described herein with light. In some embodiments, a wavelength of the irradiated light is from 350-370 (e.g., 350, 351, 352, 353.354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, or 370) nm. In some embodiments, the complex is irradiating with light at the wavelength for 1 to 120 (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes. In some embodiments, the method of covalently attaching one or more binding moieties, each including one or more photoreactive crosslinking agents, to the circular polyribonucleotide by irradiating any of the complexes described herein with light further includes a method of removing the covalent attachments between one or more binding moieties, each including one or more photoreactive crosslinking agents, and the circular polyribonucleotide includes irradiating the complex with light at a second wavelength. In some embodiments, the second wavelength of the irradiated light for removing the covalent attachment is 300-320 (e.g., 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320) nm. In some embodiments, the complex is irradiating with light at the second wavelength for 1 to 120 (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120) minutes. In some embodiments, the complex is irradiated prior to administration to a cell or tissue. In some embodiments, the complex is irradiated after administration to a cell or tissue. In some embodiments, the complex is irradiated prior to administration to a subject. In some embodiments, the complex is irradiated after administration to a subject. Methods of Delivery In Vivo The present invention includes methods of in vivo delivery of circular polyribonucleotides and compositions thereof. In some embodiments, a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety includes parenterally administering the circular polyribonucleotide or composition thereof to a subject. In some embodiments, a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety includes parenterally administering the circular polyribonucleotide or composition thereof to a subject, wherein the circular polyribonucleotide is a translation incompetent circular polyribonucleotide. In some embodiments, a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety includes parenterally administering the circular polyribonucleotide or composition thereof to a subject, wherein the complex is irradiated prior to administration to the subject. In some embodiments, a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety includes parenterally administering the circular polyribonucleotide or composition thereof to a subject, wherein the complex is irradiated after administration to the subject. In some embodiments, a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety includes parenterally administering the circular polyribonucleotide or composition thereof to a subject, wherein the circular polyribonucleotide is a translation incompetent circular polyribonucleotide. In embodiments, the circular polyribonucleotide complexed with a targeting moiety is in an amount effective to elicit a biological response in the subject. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety is in an amount effective to have a biological response in the subject. In some embodiments, a method of in vivo delivery as described herein is a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety or composition thereof to a cell or tissue of a subject including parenterally administering to the cell or tissue the circular polyribonucleotide complexed with a targeting moiety or composition thereof. In some embodiments, a method of in vivo delivery as described herein is a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety or composition thereof to a cell or tissue of a subject including parenterally administering to the cell or tissue the circular polyribonucleotide complexed with a targeting moiety or composition thereof, wherein the circular polyribonucleotide is a translation incompetent circular polyribonucleotide. In some embodiments, a method of in vivo delivery as described herein is a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety or composition thereof to a cell or tissue of a subject including parenterally administering to the cell or tissue the circular polyribonucleotide complexed with a targeting moiety or composition thereof, wherein the complex is irradiated prior to administration to the cell or tissue. In some embodiments, a method of in vivo delivery as described herein is a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety or composition thereof to a cell or tissue of a subject including parenterally administering to the cell or tissue the circular polyribonucleotide complexed with a targeting moiety or composition thereof, wherein the complex is irradiated after administration to the cell or tissue. In some embodiments, a method of in vivo delivery as described herein is a method of in vivo delivery of a circular polyribonucleotide complexed with a targeting moiety or composition thereof to a cell or tissue of a subject including parenterally administering to the cell or tissue the circular polyribonucleotide complexed with a targeting moiety or composition thereof, wherein the circular polyribonucleotide is a translation incompetent circular polyribonucleotide. In some embodiments, the administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is conducted using any delivery method described herein. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is administered to the subject via intravenous injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof includes, but is not limited to, prenatal administration, neonatal administration, postnatal administration, oral, by injection (e.g., intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous and intramuscular), by ophthalmic administration, and by intranasal administration. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is prenatal administration. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is neonatal administration. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is postnatal administration. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is oral. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intravenous injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraarterial injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraperitoneal injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intradermal injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration. In some embodiments, the compositions are parenterally administered and include a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is prenatal administration and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is neonatal administration and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is postnatal administration and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is oral and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intravenous injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraarterial injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraperitoneal injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intradermal injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration and includes a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration and includes a carrier. In some embodiments, the compositions are parenterally administered and include a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is prenatal administration and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is neonatal administration and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is postnatal administration and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is oral and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intravenous injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraarterial injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraperitoneal injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intradermal injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration and includes a diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration and includes a diluent. In some embodiments, the compositions are parenterally administered and include a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is prenatal administration and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is neonatal administration and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is postnatal administration and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is oral and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intravenous injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraarterial injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraperitoneal injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intradermal injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration and includes a parenterally acceptable diluent. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration and includes a parenterally acceptable diluent. In some embodiments, the compositions are parenterally administered and lack a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is prenatal administration and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is neonatal administration and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is postnatal administration and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is oral and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intravenous injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraarterial injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intraperitoneal injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intradermal injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by subcutaneous injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by intramuscular injection and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is by ophthalmic administration and lacks a carrier. In some embodiments, the parenteral administration of the circular polyribonucleotide complexed with a targeting moiety or composition thereof is intranasal administration and lacks a carrier. In some embodiments, a use of a circular polyribonucleotide complexed with a targeting moiety in the manufacture of a parenteral composition is for delivering to a cell or tissue of a subject. In some embodiments, the parenteral composition is formulated for intravenous, intramuscular, ophthalmical or topical administration. In some embodiments, the parenteral composition is a pharmaceutical composition further including a pharmaceutically acceptable excipient. In some embodiments, the parenteral composition includes a carrier. In some embodiments, the parenteral composition includes a parenterally acceptable diluent and is free of any carrier. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety in the manufacture of a paternal composition is irradiated with light prior to delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition forms a complex with the target and the circular polyribonucleotide or the target is detectable at least 5 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety is detectable at least 7, 8, 9, or 10 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety is detectable 7 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety is detectable 8 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety is detectable 9 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety is detectable 10 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition is present at least five days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present at least 6, 7, 8, 9, or 10 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 6 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 7 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 8 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 9 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety the parenteral composition is present 10 days after delivery. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition is an unmodified circular polyribonucleotide. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition has a quasi-double-stranded secondary structure. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition lacks a poly-A sequence, lacks a replication element, lacks a free 3’ end, or lacks an RNA polymerase recognition motif, or any combination thereof. In some embodiments, the circular polyribonucleotide of the parenteral composition lacks a poly-A sequence. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition lacks a replication element. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition lacks a free 3’ end. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition lacks an RNA polymerase recognition motif. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition is a translation incompetent circular polyribonucleotide. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety further includes an expression sequence. In some embodiments, the circular polyribonucleotide complexed with a targeting moiety of the parenteral composition includes a termination element or an IRES, or the combination thereof. In some embodiments, the polypeptide, when expressed in the cell is functional. Upon delivery of the circular polyribonucleotide to a cell by the targeting moiety complexed to the circular polyribonucleotide, the polypeptide encoded by the circular polyribonucleotide is expressed and folded resulting in a polypeptide capable of performing a function and/or therapeutic use. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a livestock cell. In some embodiments, the tissue is a connective tissue. In some embodiments, the tissue is a muscle tissue. In some embodiments, the tissue is a nervous tissue. In some embodiments, the tissue is an epithelial tissue. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a pet. In some embodiments, the subject is a live-stock animal. In some embodiments, the parenteral nucleic acid delivery system as disclosed herein (e.g., the nucleic acid compositions in the methods of delivery as described above) is used as a medicament or a pharmaceutical. A parenteral nucleic acid delivery system as disclosed herein can be used in a method of treatment of a human or animal body by therapy. A parenteral nucleic acid delivery system as disclosed herein can be used in the manufacture of a medicament or a pharmaceutical. A parenteral nucleic acid delivery system as disclosed herein can be used in the manufacture of a medicament or a pharmaceutical for treating a human or animal body by therapy. Methods of Dosing A method of dosing to produce a level of circular complexed with a targeting moiety in a cell after providing the cell with at least two doses or compositions of circular polyribonucleotide is disclosed herein. A method of dosing to produce a level of circular polyribonucleotide complexed with a targeting moiety in a subject (e.g., a mammal, e.g., a human) after providing (e.g., administering to) the subject with at least two doses or compositions of circular polyribonucleotide is disclosed herein. The composition includes a circular polyribonucleotide complexed with a targeting moiety as described herein. A method of dosing can include administering two or more doses of a composition of circular polyribonucleotides, e.g., over short time period or over an extended period. In some embodiments, the composition containing a circular polyribonucleotide further includes a pharmaceutically acceptable carrier or excipient. The circular polyribonucleotide encodes a polypeptide, which can be expressed in a cell, e.g., following administration. The polypeptide when expressed in a cell is a function polypeptide e.g., it is capable of carrying out a biological function. The methods described herein may include administering a first dose of the pharmaceutical composition in an amount sufficient to produce a serum concentration of at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL, or more) of a polypeptide in the subject. In some embodiments, the method may further include administering a second dose of the pharmaceutical composition. The method may further include administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more doses of the pharmaceutical composition. In some embodiments, a subsequent dose helps maintain a serum concentration of at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL, or more) of a polypeptide in the subject. In some embodiments, a subsequent dose is administered before the serum concentration drops below 500 ng/mL of a polypeptide in the subject. In some embodiments, multiple doses are provided to produce a level of the composition or express a level of the 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 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. In some embodiments, the second dose is administered at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer) after the first dose of the pharmaceutical composition. In some embodiments, the second dose is administered from 1 hour to 1 year (e.g., from 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day to one week, e.g., two days, three days, four days, five days, six days, or one week, e.g., from one week to one month, e.g., two weeks, three weeks, or one month, e.g., from one month to one year, e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year) after the first dose of the pharmaceutical composition. In some embodiments, the second dose is administered from 1 days to 180 days (e.g., from 1 day to 90 days, from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to 7 days, from 2 days to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days, from 3 days to 90 days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3 days to 7 days, from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days to 14 days, from 4 days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days, from 5 days to 14 days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6 days to 30 days, from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to 45 days, from 7 days to 30 days, from 7 days to 14 days, from 14 days to 90 days, from 14 days to 45 days, from 14 days to 30 days, from 21 days to 90 days, from 21 days to 60 days, from 21 days to 45 days, from 21 days to 30 days, from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45 days, from 45 to 180 days, from 45 to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to 60 days, from 60 to 180 days, from 60 to 120 days, from 60 to 100 days, from 60 to 90 days, from 90 to 100 days, from 90 to 120 days, or from 90 to 180 days) after the first dose of the pharmaceutical composition. In some embodiments, the third dose is administered at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer) after the second dose of the pharmaceutical composition. In some embodiments, the third dose is administered from 1 hour to 1 year (e.g., from 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day to one week, e.g., two days, three days, four days, five days, six days, or one week, e.g., from one week to one month, e.g., two weeks, three weeks, or one month, e.g., from one month to one year, e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year) after the second dose of the pharmaceutical composition. In some embodiments, the third dose is administered from 1 days to 180 days (e.g., from 1 day to 90 days, from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to 7 days, from 2 days to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days, from 3 days to 90 days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3 days to 7 days, from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days to 14 days, from 4 days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days, from 5 days to 14 days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6 days to 30 days, from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to 45 days, from 7 days to 30 days, from 7 days to 14 days, from 14 days to 90 days, from 14 days to 45 days, from 14 days to 30 days, from 21 days to 90 days, from 21 days to 60 days, from 21 days to 45 days, from 21 days to 30 days, from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45 days, from 45 to 180 days, from 45 to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to 60 days, from 60 to 180 days, from 60 to 120 days, from 60 to 100 days, from 60 to 90 days, from 90 to 100 days, from 90 to 120 days, or from 90 to 180 days) after the second dose of the pharmaceutical composition. In some embodiments, the second dose is administered before a serum concentration of an polypeptide is less than about 500 ng/mL in serum of the subject. In some embodiments, the method maintains a serum concentration of at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL, or more) of polypeptide in the subject, e.g., for at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer). A method of administering multiple doses of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide) includes providing two or more compositions over a period of time, to a cell, tissue or subject (e.g., a mammal). According to certain embodiments, multiple doses of a composition of a nucleic acid molecule described herein may be administered to a subject over a defined time course. The methods according to this aspect of the invention include sequentially administering to a subject multiple doses of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide) (e.g., in a pharmaceutical or veterinary composition). As used herein, “sequentially administering” means that each dose of composition of a nucleic acid molecule described herein is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). In some embodiments, the present invention provides methods which include sequentially administering to the subject a single initial dose of a composition of a nucleic acid molecule described herein, followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition. The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of a composition of a nucleic acid molecule described herein. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen; 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 nucleic acid molecule described herein, and in certain embodiments, may differ from one another in terms of frequency of administration. In certain embodiments, the amount of a composition of a nucleic acid molecule described herein 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. In certain embodiments, 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”). In certain embodiments, 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 nucleic acid molecule described herein which is administered to a subject prior to the administration of the very next dose in the sequence with no intervening doses. In certain embodiments, 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. In certain embodiments, 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 include administering to a subject any number of secondary and/or tertiary doses of a composition of a nucleic acid molecule described herein. For example, in certain embodiments, only a single secondary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the subject. Likewise, in certain embodiments, only a single tertiary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the subject. In certain embodiments, 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. In some embodiments, the method includes 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). In some embodiments, the method further includes providing (e.g., administering) a third composition, fourth composition, fifth composition, sixth composition, seventh composition, eighth composition, ninth composition, tenth composition, or more. In some embodiments, additional compositions are provided for the duration of the life of the cell. In some embodiments, additional compositions are provided (e.g., administered) while the cell, tissue or subject obtains a benefit from the composition. In some embodiments, a first composition in a multiple dosing regimen includes a first amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, a second composition in a multiple dosing regimen includes a second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, 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 includes a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition varies by no more than 1%, 5%, 10%, 15%, 20%, or 25% of an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition is no more than 1%, 5%, 10%, 15%, 20%, or 25% less than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1-fold to 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular 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 nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., a composition administered after a first composition) is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1-fold to 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular 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 nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., a composition administered after a first composition) is 0.1-fold, 1-fold, 5- fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., after a first composition of an amount of nucleic acid molecule (e.g., circular polyribonucleotide)) is from 0.1-fold to 1000-fold higher or lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., after a first composition of an amount of nucleic acid molecule (e.g., circular polyribonucleotide)) is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher or lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. For example, a first composition includes 1-fold nucleic acid molecule (e.g., circular polyribonucleotide), a second composition includes 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition, and a third composition includes 0.2-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition. In some embodiments, the second composition includes at least 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to an amount of nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, the first composition includes a higher amount of the nucleic acid molecule (e.g., circular polyribonucleotide) than the second composition. In some embodiments, the first composition includes a higher amount of the nucleic acid molecules (e.g., circular polyribonucleotides) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition. In some embodiments, the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding a polypeptide, which 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 nucleic acid molecule (e.g., circular polyribonucleotide) encoding a polypeptide, which are administered in a multiple dosing regimen as described herein, are different compositions. In some embodiments, the same compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding the same polypeptide. In some embodiments, the different compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding different polypeptides, or a combination thereof. In some embodiments, in a multiple dosing regimen, the method of administering the nucleic acid molecule (e.g., circular polyribonucleotide) provided herein includes administering to a subject in need thereof the nucleic acid molecule for multiple times (multiple doses), e.g., at least 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. In some embodiments, in a multiple dosing regimen, the method provided herein includes administering to a subject in need thereof the nucleic acid molecule for at least 3 times, with an interval of about 7 days. In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses) provided herein, a level of the polypeptide (e.g., a plasma 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. In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses) provided herein, a level of the polypeptide (e.g., a plasma polypeptide level) 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 polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose). In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e.g., at least 3 doses) provided herein with an interval of about 7 days, a level of the polypeptide (e.g., a plasma polypeptide level) 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 polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose). Examples The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their invention. Example 1. Complexation of one or more carbohydrates to a circular polyribonucleotide This example demonstrates the complexation of a carbohydrate targeting moiety to a circular polyribonucleotide. A non-naturally occurring circular RNA complexed with a carbohydrate targeting moiety was produced. As shown in the following example, a trivalent GalNAc moiety was complexed to a circular RNA. A binding moiety (X), consisting of a 2’O-methyl RNA (23-mer (ACGGAUUUUAAGUCCGUAGCGUC SEQ ID NO: 84) or 17-mer (UUUGUCGCCUUCGUAGG SEQ ID NO: 85)) was linked via a triethylene glycol linker (L) to a trivalent GalNAc targeting moiety (B). The 23- mer had 100% complementarity to a portion of the 5’ exon fragment and 3’ exon fragment remaining after circularization of the polyribonucleotide (E1/E2). The 17-mer had 100% complementarity to a portion of the ORF encoding a Gaussia luciferase polypeptide. The trivalent GalNAc moiety conjugated to a 23-mer and 17-mer made up of 2’O-methyl RNA by way of a triethylene glycol linker was mixed with a circular polyribonucleotide in the presence of 25 mM HEPES pH 7.0, 237 mM NaCl, 4.6 mM KCl, 1.4 mM Na2HPO4, 5 mM MgCl2, and 4.7 mM glucose in a ratio of either 1:1 or 1:3 circular polyribonucleotide to oligomer conjugated to the GalNAc moiety by of a linker. The oligomer conjugated to the triethylene glycol (TEG) linker conjugated to the trivalent GalNAc moiety was then annealed to the circular polyribonucleotide in a thermocycler by heating at 75°C for 2 minutes, cooling to 25°C for 2 minutes, and repeating one time. The resulting circular polyribonucleotide complexed to a GalNAc targeting moiety was stored at room temperature. These conditions resulted in the circular polyribonucleotide as described in the schematic in FIG.3. Example 2. Effect of one or more targeting moieties on the expression of a polypeptide encoded by a circular polyribonucleotide in vitro This example demonstrates the effect one or more targeting moieties complexed with a circular polyribonucleotide encoding a polypeptide has on the expression of the polypeptide encoded by the circular polyribonucleotide in vitro. A circularized polyribonucleotide encoding a luciferase polypeptide was complexed to an oligomer conjugated to a TEG linker which was conjugated to a targeting moiety of a GalNAc moiety, cholesterol, or tocopherol was prepared, as described in Example 1. The oligomer conjugated to each targeting moiety by way of a linker was annealed to the region of the circular polyribonucleotide containing the portion of the 5’ exon fragment and 3’ exon fragment remaining after circularization of the polyribonucleotide (E1/E2) or just the 5’ exon fragment remaining after circularization (E1). This resulted in a circular polyribonucleotide (eRNA) complexed to one or more of: (a) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to E1 by way of a TEG linker (A); (b) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to E1/E2 by way of a TEG linker (B); (c) an oligomer conjugated to a GalNAc targeting moiety at the 5’ end of the oligomer bound to E1/E2 by way of a TEG linker (C); (d) an oligomer conjugated to a cholesterol targeting moiety at the 3’ end of the oligomer bound to E1/E2 by way of a TEG linker (I); (e) an oligomer conjugated to a cholesterol targeting moiety at the 3’ end of the oligomer bound to E1 by way of a TEG linker (J); (f) an oligomer conjugated to a tocopherol targeting moiety at the 5’ end of the oligomer bound to E1/E2 by way of a TEG linker (Q); and/or (g) an oligomer conjugated to a tocopherol targeting moiety at the 5’ end of the oligomer bound to E1 by way of a TEG linker (R). 1 pmol of the complex including the circular polyribonucleotide and the targeting moiety was transfected into mouse hepatocyte cells and incubated for 24-48 hours at 37 °C under 5% CO2. After incubation, a luciferase assay was performed to assess polypeptide expression in the cells in comparison to a circular polyribonucleotide that was not complexed to a targeted ligand. Results of these experiments are shown in FIGS.4 and 5. These experiments demonstrate the principle that expression of the polypeptide encoded by the circular polyribonucleotide could be achieved. In order for expression to occur, the oligomer conjugated to the circular polyribonucleotide had to be annealed to the circular polyribonucleotide in order for there to be some enhancement of expression. Furthermore, it was observed that certain combinations of targeting moieties led to enhanced expression in comparison to complexes having only one targeting moiety. Various linker lengths were also tested for oligomers that were annealed to the ORF to identify if a longer linker length was required for enhanced expression of the polypeptide encoded by the circular polyribonucleotide. A circularized polyribonucleotide encoding a luciferase polypeptide was complexed to an oligomer conjugated to a TEG linker or PEG24 linker which was conjugated to a targeting moiety of a GalNAc moiety as described in Example 1. The oligomer conjugated to each targeting moiety by way of a linker was annealed to the ORF, which encoded a luciferase polypeptide. This resulted in a circular polyribonucleotide (eRNA) complexed to one or more of: (a) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to ORF by way of a TEG linker (FA and FB); (b) an oligomer conjugated to a GalNAc targeting moiety at the 5’ end of the oligomer bound to ORF by way of a TEG linker (FC and FD); or (c) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to ORF by way of a PEG24 linker (FE24). 1 pmol of the complex including the circular polyribonucleotide and the targeting moiety was transfected into mouse hepatocyte cells and incubated for 24-48 hours at 37 °C under 5% CO2. After incubation, a luciferase assay was performed to assess polypeptide expression in the cells in comparison to a circular polyribonucleotide that was not complexed to a targeted ligand. Results of this experiment are shown in FIG.22. This experiment showed that complexes where the oligomer was annealed to the ORF resulted in enhanced expression of the polypeptide in comparison to circular polyribonucleotides that were not complexed to a targeting moiety, but required a longer linker be used. Example 3. Delivery of polypeptide into cells using various targeting moieties and various formulation conditions in vitro and in vivo This example demonstrates the effect of the various delivery agents have on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with various targeting agents. Ribonuclease Inhibitor and Calcium Ions In Vitro These experiments demonstrate the effect that ribonuclease inhibitors and CaCl2 have on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with multiple targeting agents. A circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with one or more of: (a) an oligomer conjugated to a GalNAc targeting moiety at the 3’ end of the oligomer bound to E1 by way of a TEG linker (B); (b) an oligomer conjugated to a GalNAc targeting moiety at the 5’ end of the oligomer bound to E1/E2 by way of a TEG linker (C); (c) an oligomer conjugated to a cholesterol targeting moiety at the 3’ end of the oligomer bound to E1/E2 by way of a TEG linker (I); (d) an oligomer conjugated to a by way of a linker (FA, FB, FC, and FD), or (d) an oligomer conjugated to a GalNAc moiety by way of specifically a PEG24 linker (FE24) using the methods described in Example 1. A ribonuclease inhibitor and/or CaCl2 were then added to the solution including the complex including the circular polyribonucleotide and the targeting moiety. The resulting mixture was subsequently transfected into mouse hepatocyte cells, incubated, and luminescence was measured as described in Example 2 to understand the effect of the delivery agents on the polypeptide expression (FIG.6 and FIG.7). Albumin This experiment demonstrates the effect of albumin on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with multiple targeting agents. A circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer conjugated to a GalNAc moiety by way of a linker and an oligomer conjugated to tocopherol by way of a linker (BQ), as described in Example 1. Bovine serum albumin (BSA) was added in a 1:1 ratio to the circular RNA-targeting moiety complex, and the resulting mixture was subsequently transfected into mouse hepatocyte cells, incubated, and subsequently the luminescence was measured as described in Example 2. As seen in FIG.8A, BSA enhanced expression of a polypeptide encoded on the circular polyribonucleotide complexed with the two targeting moieties in comparison to the circular polyribonucleotide complexed with the two targeting moieties that were not mixed with BSA. Endosomal Escape Agent in Combination with Calcium Ions and/or Ribonuclease Inhibitors These experiments demonstrate the effect of the endosomal escape agent chloroquine alone and in combination with CaCl2 on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with multiple targeting agents. A circular polyribonucleotide encoding luciferase was complexed with an oligomer conjugated to a GalNAc moiety by way of a linker and an oligomer conjugated to tocopherol by way of a linker (BQ), as described in Example 1. Chloroquine at a concentration of 60 µM, CaCl2 at a concentration of 20 mM, and/or a ribonuclease inhibitor at a concentration of 0.29 U/mL were added to the solution including the complex including the circular polyribonucleotide and the targeting moiety. The resulting mixture was subsequently transfected into mouse hepatocyte cells, incubated, and expression of the polypeptide measured as described in Example 2 in comparison to the circular polyribonucleotide that was not complexed to a targeting moiety and in comparison to the circular polyribonucleotide in complex with the targeting moiety but not in the presence of the chloroquine and/or CaCl2. Results of these experiments are provided in FIG.8B (without a ribonuclease inhibitor) and FIG.12A (with a ribonuclease inhibitor). These experiments demonstrated that the presence of chloroquine resulted in enhanced expression of the polypeptide encoded by the circular polyribonucleotide. This enhanced expression was even further enhanced by addition of CaCl2 and/or ribonuclease inhibitor. Delivery Agents In Vivo This experiment demonstrates the effect of various delivery agents have on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with a cholesterol targeting moiety in a mouse. In a first experiment, the effect of the presence of an endosomal escape agent and/or calcium ion on polypeptide expression was studied in vivo. A circular polyribonucleotide encoding a luciferase polypeptide was complexed with an oligomer conjugated to cholesterol by way of a linker (J), as described in Example 1, and was subsequently mixed with CaCl2 at a concentration of 1.8 mM or 20 mM and/or 60 µM chloroquine. The resulting mixtures were injected into CD1 mice intradermally, and the mice were imaged to detect polypeptide expression after 5 hours. Results of this experiments are provided in FIG 12B. This experiment demonstrated that in vivo the combination of 20 mM CaCl2 and chloroquine resulted in the most enhanced expression. In another experiment, the effect chloroquine, ribonuclease inhibitor and CaCl2 have on the expression of a polypeptide encoded by a circular polyribonucleotide in combination with a cholesterol targeting moiety in a mouse was studied. A circular polyribonucleotide encoding a luciferase polypeptide was complexed with an oligomer conjugated to cholesterol by way of a linker as described in Example 1. 75 µg of the complex was mixed with 1 U/µL of a ribonuclease inhibitor (e.g., RNasin® ribonuclease inhibitor), CaCl2 at a final concentration of 1.8 mM, chloroquine at a final concentration of 600 µM, mouse serum albumin (MSerumAlbumin) at a ratio of 1:3 complex to BSA, 25 µg of lipid nanoparticles (LNP), or a combination thereof as indicated in FIG.15. The resulting mixtures were injected two times intradermally in CD1 mice. Mice were injected intraperitoneally with d-luciferin over a period of 29 days, and the whole body of the mouse was imaged for luminescence at the indicated time points to assess expression of the polypeptide encoded by the circular polyribonucleotide (FIG.15). As seen in FIG.15, ribonuclease inhibitor, CaCl2, and chloroquine singly and in combination with BSA, affected polypeptide expression. Example 4. Delivery of circular polyribonucleotide encoding a polypeptide into adipocytes and in vivo using various targeting ligands This example demonstrates the ability of various targeting ligands including cholesterol, tocopherol, and/or an aptamer to deliver a circular polyribonucleotide encoding a polypeptide to adipocytes or in mice. A circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with: (a) an oligomer conjugated to cholesterol by way of a linker (J and I), (b) an oligomer conjugated to a GalNAc moiety by way of a linker (A), (c) an oligomer conjugated to tocopherol by way of a linker (Q), (d) an oligomer conjugated to cholesterol by way of a linker and an oligomer conjugated to tocopherol by way of a linker (IQ and JQ), (d) two oligomers that each were conjugated to cholesterol by way of a linker (IJ), or (e) two oligomers that each were conjugated to tocopherol by way of a linker (QR), as described in Example 1. CaCl2 (20 mM) was added to the solution including the complex including the circular polyribonucleotide and the targeting moiety. The resulting mixture was subsequently transfected into cryopreserved human adipocyte cells, incubated for a period of 1-2 days, and subsequently expression of the polypeptide was measured as described in Example 2. Results of this experiment are shown in FIG. 9A. This experiment resulted in enhanced expression of the polypeptide when two targeting moieties were complexed with the circular polyribonucleotide in comparison to when only one targeting moiety was complexed with the circular polyribonucleotide. In another experiment, a circular polyribonucleotide encoding a luciferase polypeptide was complexed with: (a) an oligomer conjugated to cholesterol by way of a linker and an oligomer conjugated to tocopherol (Chol+Toco), or (b) an oligomer conjugated by way of linker to an aptamer (Aptamer 7), as described in Example 1, in order to compare the resulting polypeptide expression to a circular polyribonucleotide that is not complexed with a targeting moiety but is administered with lipid nanoparticles (LNP). The mixtures were injected subcutaneously into CD1 mice as described in Example 3. The mice were imaged to detect polypeptide expression after 6 hours, 24 hours, and 48 hours. Results of this experiment are shown in FIG.9B. This experiment demonstrated sustained expression of the polypeptide over a period of 48 hours. Particularly, this result was achieved when two targeting moieties were complexed with the circular polyribonucleotide. Example 5. Optimization of calcium concentration for the delivery of a circular polyribonucleotide encoding a polypeptide in vitro and in vivo This example identifies the optimal range of CaCl2 concentration for the delivery of a circular polyribonucleotide encoding a polypeptide conjugated to a targeting moiety for in vitro and in vivo systems. A circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer conjugated to cholesterol by way of a linker as described in Example 1. CaCl2 was added after the annealing and cooling step to the solution including the complex at a final concentration of 0.3 mM, 0.9 mM, 2.7 mM, 8.0 mM, 10 mM, or 20 mM. The resulting mixture was subsequently transfected into mouse hepatocyte cells, incubated, and expression of the polypeptide was measured as described in Example 2. Results of this experiment are shown in FIG.11A. The CaCl2 concentration was shown to increase polypeptide expression until reaching 20 mM at which point the CaCl2 no longer correlated with increased polypeptide expression. The effect of calcium concentration on polypeptide expression was also studied in vivo. The complex including the circular polyribonucleotide and the targeting moiety was incubated with 0.6 mM, 1.8 mM, 5.4 mM, or 16.2 mM CaCl2 for a period of 10 minutes and then injected intradermally into BALB/c mice. The mice were imaged to detect polypeptide expression after 72 hours and compared to polypeptide expression resulting from administering a circular polyribonucleotide with an LNP. Results of this experiment are shown in FIG.11B. Similar to the in vitro results, these results showed there was an optimal concentration of CaCl2 that resulted in higher polypeptide expression. Generally, the concentration of CaCl2 of from about 0.6 mM to about 1.8 mM resulted in enhanced expression of the polypeptide. Example 6. Use of various targeting moieties for the delivery of a circular polyribonucleotide encoding a polypeptide in vivo over time This example demonstrates the effect of the use of various targeting ligands on the in vivo delivery of a circular polyribonucleotide encoding a polypeptide as monitored by expression of the polypeptide in vivo over time. Targeting Agents used: biotinylated mannose bound to avidin, cholesterol, and tocopherol A circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed to an oligomer conjugated to tocopherol by way of a linker and an oligomer conjugated to cholesterol by way of a linker, or was complexed with an oligomer conjugated to a linker which was conjugated to an avidin molecule which was complexed to one or two biotinylated mannose residues bound to an avidin molecule. The complexes including the circular polyribonucleotide and tocopherol and cholesterol targeting moieties were generated as described in Example 1. The complexes including the circular polyribonucleotide and the avidin bound to the biotinylated mannose were generated by mixing 1 pmol of circular polyribonucleotide and 1 pmol of a biotinylated oligomer in HEPES buffered saline. The oligomer was then annealed to the circular polyribonucleotide in a thermocycler by heating at 75 °C for 2 minutes, cooling to 25 °C for 2 minutes, and repeating one time. To this mixture, biotinylated mannose was added and mixed and then avidin was added in a 1:1 ratio with the circular polyribonucleotide. The solution was then left to equilibrate for 15 minutes at 20 oC. The circular RNA-targeting moiety complexes were subsequently injected intramuscularly into C57BL/J6 mice and imaged to assess polypeptide expression after 4-5 hours, 24 hours, and 72 hours from the dorsal view or the ventral view. Results of these experiments are shown in FIG.14A (dorsal view) and FIG.14B (ventral view). These experiments demonstrated that over time, the circular polyribonucleotide complexed to the targeting moiety had greater expression over time of the polyribonucleotide cargo in comparison to LNP’s which showed reduced expression of the polyribonucleotide cargo over time. Targeting Agents used: aptamer, cholesterol, and tocopherol A circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer conjugated to a cholesterol by way of a linker and an oligomer conjugated to a tocopherol by way of a linker, or an oligomer conjugated to an adipocyte-targeting aptamer (Aptamer MA33) by way of a linker using the methods described in Example 1. After 20 minutes of equilibration, 50 µL of the circular RNA-targeting moiety complexes were subsequently injected subcutaneously into CD1 mice and imaged to assess polypeptide expression, as described in Example 3, 6 hours, 24 hours, and 48 hours after injection. Results of this experiment are shown in FIG.16. The results of this experiment show that the use of two targeting moieties, in this instance cholesterol and tocopherol, resulted in enhanced expression of the polyribonucleotide cargo in comparison to a circular polyribonucleotide which was not annealed to any targeting moiety or complexed with only a single targeting moiety. Example 7. Expression of SARS-CoV-2 polypeptide in vivo encoded by circular polyribonucleotides complexed to a targeting moiety This example demonstrates the use of various targeting ligands to successfully express a SARS- CoV-2 RBD polypeptide. A circular polyribonucleotide encoding a SARS-CoV-2 RBD polypeptide was complexed with: (a) an oligomer bound to cholesterol by way of a TEG linker; or (b) an oligomer bound to an avidin, by way of a biotinylated TEG linker, bound to three biotinylated mannose molecules; using the methods described in Examples 1 and 6. Chloroquine was added after the annealing and cooling step to the solution including the complex. The resulting mixture was injected intradermally into CD1 mice. After 6 hours, serum was harvested from the mice and subsequently expression of the RBD polypeptide in the serum was measured. Results of this experiment are shown in FIG.13. The amount of RBD measured in circulating serum was greater when a targeting moiety was complexed with the circular polyribonucleotide in comparison to when the circular polyribonucleotide was not complexed with a targeting moiety. Furthermore, the amount of RBD measured in the serum was greatest when the avidin bound to the biotinylated mannose was used as the targeting moiety. Example 8. Use of avidin bound to a biotinylated aptamer, biotinylated antibody, or biotinylated small molecule to deliver the circular polyribonucleotide into a cell This example demonstrates the use of various targeting ligands successfully leads to the internalization of the circular polyribonucleotide into the cell. A circular polyribonucleotide was complexed with two biotinylated oligomers each of which were bound to an avidin bound to biotinylated DEC205 antibody using the methods described in Example 1. The biotinylated DEC205 antibody was mixed with the circular polyribonucleotide in a ratio of either 1:3 (DEC205 Ab 1:3) or 1:6 (DEC205 Ab 1:6). Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to biotinylated DEC205 aptamer using the methods described in Example 1. The biotinylated DEC205 aptamer was mixed with the circular polyribonucleotide in a ratio of either 1:3 (DEC205 APT 1:3) or 1:6 (DEC205 APT 1:6). Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated Transferrin R (Trf) aptamer using the methods described in Example 1. The biotinylated DEC205 antibody and biotinylated Trf aptamer were mixed in a 1:1 ratio with each other and in a ratio of one circular polyribonucleotide to three targeting moieties (DEC205 Ab+Trf APT 3:3). Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 aptamer and biotinylated Transferrin R (Trf) aptamer using the methods described in Example 1. The biotinylated DEC205 aptamer and biotinylated Trf aptamer were mixed in a 1:1 ratio with each other and in a ratio of one circular polyribonucleotide (eRNA) to three targeting moieties (DEC205 APT+Trf APT 3:3). Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated phosphatidylserine (PS) using the methods described in Example 1. The biotinylated DEC205 antibody and biotinylated PS were mixed in a 1:1 ratio with each other and in a ratio of one circular polyribonucleotide (eRNA) to three targeting moieties (DEC205 Ab+PS 3:3). Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to biotinylated PS using the methods described in Example 1. The biotinylated PS and the circular polyribonucleotide (eRNA) were mixed in a ratio of 1:6 circular polyribonucleotide to PS (PS 1:6) or 1:3 circular polyribonucleotide to PS (PS 1:3). Following generation of the complexes including the targeting moieties and the circular polyribonucleotide an avidin-conjugated FITC was added to the mixture. After equilibration, the circular RNA complexes were added to plated RAW264.7 macrophages, internalized for 4 hours, and then cells were removed and analyzed via flow cytometry in order to measure the percent of cells that internalized the circular polyribonucleotide (FIG.17A), the mean amount of cells that internalized the circular polyribonucleotide (FIG.17B), and specifically the mean amount of cells that internalized the complexes having both the DEC205 antibody and PS targeting moieties, in comparison to a lipofectamine control (MM+Av). The results of this experiment show that the uptake of circular polyribonucleotide by the cell is increased when a targeting ligand is used in comparison to when a targeting ligand is not annealed to the circular polyribonucleotide. Furthermore, these experiments clearly show the circular polyribonucleotide annealed to the targeting ligand are internalized by the cell and not merely associated with the cell surface. Lastly, it was observed that using a lipid targeting moiety in combination with an antibody targeting moiety resulted in a synergistic uptake of the circular polyribonucleotide by the cell. Example 9. Use of avidin bound to a biotinylated antibody, antibody, carbohydrate, or small molecule to deliver the circular polyribonucleotide into a cell This example demonstrates using various biotinylated targeting moieties bound to avidin which is conjugated by way of a linker to an oligomer that binds to the circular polyribonucleotide to successfully deliver the circular polyribonucleotide into a cell. A circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated Transferrin R (Trf) aptamer as described in Example 8. Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated PS as described in Example 8. Another circular polyribonucleotide was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated mannose using the methods described in Example 1. Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated DEC205 antibody using the methods described in Example 1. Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated mannose using the methods described in Example 1. Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated Trf aptamer using the methods described in Example 1. Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated PS as described in Example 8. Another circular polyribonucleotide was complexed with an oligomer bound to an avidin bound to biotinylated Trf antibody using the methods described in Example 1. The biotinylated Trf antibody was mixed in a ratio of 1 circular polyribonucleotide (eRNA) to 6 targeting moieties (Trf Ab 1:6). The circular RNA complexes were added to RAW264.7 macrophages plated onto #1.5 thickness glass coverslips and incubated for 4 hours before washing cells with Hank’s Buffered Saline solution containing Hoechst 33342 nuclear stain. Live cells were analyzed by confocal fluorescence microscopy which showed that the circular polyribonucleotide annealed to the targeting moiety was successfully internalized by the cells into the endosomes of the cells. The dPCR was performed to measure the amount of the circular polyribonucleotide complexes inside the cells and to further confirm that the circular polyribonucleotides were in fact internalized by the cell and not associated merely with the cell surface. Results of this experiment are shown in FIG.18. Example 10. The effect of using avidin to bind a biotinylated targeting moiety to conjugate the targeting moiety to the moiety that binds the circular polyribonucleotide on polypeptide expression This example demonstrates using an avidin to bind a biotinylated targeting ligand and conjugate it to the moiety that binds the circular polyribonucleotide encoding a polypeptide modestly affects the expression of the polypeptide. A circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated Transferrin R (Trf) aptamer as described in Example 8. Another circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated PS as described in Example 8. Another circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with two oligomers each of which were bound to an avidin bound to a mixture of biotinylated DEC205 antibody and biotinylated mannose using the methods described in Example 1, wherein the DEC205 antibody and biotinylated mannose were mixed in a 1:1 ratio and in a ratio of 1 circular polyribonucleotide to 3 targeting moieties. Another circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer bound to an avidin bound to biotinylated DEC205 antibody using the methods described in Example 1, wherein the DEC205 antibody and circular polyribonucleotide where mixed in a ratio of one circular polyribonucleotide to three antibodies. Another circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer bound to an avidin bound to biotinylated PS using the methods described in Example 1, wherein the mannose and circular polyribonucleotide where mixed in a ratio of one circular polyribonucleotide to three PS molecules. Another circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer bound to an avidin bound to biotinylated mannose using the methods described in Example 1, wherein the mannose and circular polyribonucleotide where mixed in a ratio of 1 circular polyribonucleotide to 3 mannose molecules. Another circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with an oligomer bound to cholesterol by way of a linker and an oligomer bound to tocopherol by way of a linker using the methods described in Example 1. The circular polyribonucleotide complexes were added to RAW264.7 macrophage cells and incubated for 48 hours. The complex including the DEC205 antibody as the targeting moiety (DEC205 Ab) and the complex including mannose were also administered in the presence of chloroquine (CQ). Luminescence assays were performed using a Pierce™ Firefly Luciferase Flash Assay Kit (ThermoFisher) to assess the amount of protein expression. Results of this experiment are shown in FIG. 19. Addition of the chloroquine to the complex mixtures resulted in increased polypeptide expression. To investigate if the annealed oligomers and ligands were hampering polypeptide expression, a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed to: (a) two biotinylated oligomers, (b) two biotinylated oligomers that were each independently bound to and avidin bound to biotinylated Trf antibodies; or (c) two biotinylated oligomers that were each independently bound to and avidin bound to biotinylated DEC205 antibodies. The circular polyribonucleotide complexes were added to HeLa cells and incubated for 24 hours. Luminescence assays were performed using a Pierce™ Firefly Luciferase Flash Assay Kit (ThermoFisher) to assess the amount of protein expression. Results of this experiment are provided in FIG.20. The use of an avidin in the complex modestly affected the expression of the polypeptide. In another experiment, the ability for a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) complexed to a biotinylated oligomer bound to avidin bound to one or more biotinylated Trf antibodies to be internalized by the cell and result in polypeptide expression was studied. Circular polyribonucleotide complexes were prepared with complexes to: (a) a biotinylated oligomer bound to avidin, (b) a biotinylated oligomer bound to avidin bound to a singular biotinylated Trf antibody, and (c) a biotinylated oligomer bound to avidin bound to three biotinylated Trf antibodies. Each of these complexes were transfected into HeLa cells and the resulting fluorescence was assessed using confocal imaging. The results showed that the presence of the Trf antibody as a targeting moiety resulted in enhanced internalization of the circular polyribonucleotide in comparison the complex lacking the Trf antibody. In another experiment, a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) was complexed with a biotinylated oligomer which was bound to an avidin. The complex was then mixed in with a biotinylated Trf antibody in a ratio of 1:1, 1:3, or 1:6 circular polyribonucleotide to Trf antibody to generate the circular polyribonucleotide targeting moiety complex. The resulting complex was transfected into HeLa cells in the presence of calcium and in either the presence or absence of 100x molar excess of transferrin. The resulting luminescence in the cells was measured to assess the polypeptide expression Results of this experiment are provided in FIG.21. The results showed that the Trf targeting antibody enhances expression in HeLa cells in a receptor-specific manner as expression of the polypeptide decreased as the excess transferrin competed for binding. Example 11. Covalent attachment of an oligomer binding moiety to complementary, linear RNA by way of a photoreactive crosslinking agent This example demonstrates the covalent attachment of complementary, linear RNA to an oligomer binding moiety by way of a photoreactive crosslinking agent included within the binding moiety. A binding moiety (X), named E1_cnvK-biotin, consisting of an oligomer including a methylated RNA 23-mer (mAmCmUmUmGmAmAmCmCmCmA[CNVK]mAmCmGmAmCmCmGmUmUmUmA SEQ ID NO: 86) and a 3-cyanovinylcarbazole nucleoside (cvnK) photoreactive crosslinking agent was linked to a biotin targeting moiety. The binding moiety had 96% complementarity to the complement, linear oligomer termed oligomimic. The sequence name, ID number, sequence, linker, position, and target moiety for each binding moiety used in this and the following Examples are listed in Table 3. Table 3. Binding Moiety Name, Sequence, Linker, and Target Moiety Used in Examples
Figure imgf000156_0001
Figure imgf000157_0001
10 µM of E1_cnvK-biotin was annealed to 10 µM of the oligomimic in a thermocycler by heating at 75°C for 2 minutes, cooling to 25°C for 2 minutes, and repeated one time. The annealing buffer was HEPES buffered saline 1x (HBS) containing 5 mM MgCl2 and 280 mM NaCl. E1_cnvK-biotin was annealed to the oligomimic or annealed then transferred to a cuvette and irradiated with light having a wavelength of 366 nm for 20 minutes to covalently attach E1_cnvK-biotin to the oligomimic. High performance liquid chromatography was used to measure the retention time of each sample at an operating temperature of 70 °C. Results of this experiment are shown in FIG.24. This experiment shows clear separation of retention times for the oligomer binding moiety, E1_cnvK-biotin, and oligomimic, each having a single peak at ~12.2 minutes and ~5.9 minutes, respectively. The annealed complex shows dissociation into its components as observed by their retention times overlapping with those of the oligomer binding moiety and the oligomimic. The irradiated complex shows a distinct retention time of ~10 minutes that is between those of the oligomer binding moiety and the oligomimic, indicating that the covalent attachment by way of irradiation stabilized the complex at conditions that denatured the annealed complex. Example 12. Effect of the binding region of the circular polyribonucleotide and covalent attachment of an oligomer by way of irradiation on the expression of a polypeptide encoded by a circular polyribonucleotide in vitro This example demonstrates the effect of the binding region of a circular polyribonucleotide encoding a polypeptide and covalent attachment, by way of irradiation, of an oligomer to the circular polyribonucleotide on the expression of the polypeptide in vitro. A non-naturally occurring circular RNA encoding a luciferase polypeptide and complexed with a dimeric fluorescent TAT (dfTAT) peptide, a cell-penetrating peptide, was further complexed with a cholesterol targeting moiety. A binding moiety (X), consisting of: (a) RNA 23-mer of a first sequence ([5-Cy5]ACUUGAACCCA[CNVK]ACGACCGUUUA SEQ ID NO: 88) named CHOL-E1_CK; (b) RNA 23-mer of a second sequence (ACGGAUUUUA[CNVK]GUCCGUAGCGUC[3-Cy5] SEQ ID NO: 89) named CHOL-E2/E1_CK; or (c) RNA 23-mer of a third sequence (ACUUGAACCCACACGACCGUUUA SEQ ID NO: 90) named Chol J was linked via a triethylene glycol linker (L) to a cholesterol targeting moiety (B). CHOL-E1_CK and CHOL-E2/E1_CK each include one 3-cyanovinylcarbazole nucleoside (CNVK) photoreactive crosslinking agent. CHOL-E1_CK includes one Cy5 fluorescent label at the 5’ end, and CHOL-E2/E1_CK includes one Cy5 fluorescent label at the 3’ end. CHOL-E1_CK is same sequence as Chol J, however, with one CNVK photoreactive crosslinking agent replacing a cytosine nucleotide at an internal position. CHOL-E1_CK had 96% complementarity to just a portion of the 5’ exon fragment remaining after circularization of the polyribonucleotide (E1). CHOL-E2/E1_CK had 96% complementarity to a portion of the 5’ exon fragment and 3’ exon fragment remaining after circularization (E1/E2). Chol J had 100% complementarity to just a portion of the 5’ exon fragment remaining after circularization (E1). The oligomer conjugated to the triethylene glycol (TEG) linker conjugated to the cholesterol moiety was annealed to the circular polyribonucleotide complexed to a dfTAT peptide in a thermocycler by heating at 75°C for 2 minutes, cooling to 25°C for 2 minutes, and repeated one time. The annealing buffer was HBS containing 5 mM MgCl2 and 280 mM NaCl. The resulting circular polyribonucleotide and dfTAT peptide complex further complexed to a cholesterol targeting moiety was stored at room temperature. The resulting circular polyribonucleotide and dfTAT peptide complex further complexed to the cholesterol targeting moiety was then covalently attached to the oligomer by irradiating the complex with light of a wavelength of 366 nm for various lengths of time.0.01 pmol of the complex including the circular polyribonucleotide and the cholesterol targeting moiety was transfected into human embryonic kidney cells and incubated for 24 hours at 37 °C under 5% CO2. This resulted in a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) and complexed with a dfTAT peptide further complexed CHOL-E1_CK, described earlier in Example 12 as including one CNVK photoreactive crosslinking agent, conjugated to a TEG linker conjugated to a cholesterol targeting moiety by way of: (a) annealing; (b) annealing then irradiating for 1 minute; (c) annealing then irradiating for 5 minutes; or (d) annealing then irradiating for 30 minutes. After incubation with the cells for 24 hours, a luciferase assay was performed to assess polypeptide expression in the cells in comparison to cells transfected with no eRNA complexed with a dfTAT peptide, the eRNA complexed with a dfTAT peptide, the eRNA complexed with a dfTAT peptide further complexed with Chol J conjugated to a TEG linker conjugated to a cholesterol targeting moiety, and the eRNA complexed with a dfTAT peptide delivered with lipofectamine (lipo). All controls were a result of the previously mentioned protocol in Example 12 but were not irradiated. Results of this experiment are shown in FIG.25B. This experiment demonstrates the principle that expression of the polypeptide encoded by the circular polyribonucleotide could be achieved after irradiation to covalently conjugate the oligomer binding moiety to the circular polyribonucleotide via a photoreactive crosslinking agent. This experiment also resulted in enhanced expression of the polypeptide when the complex was annealed then irradiated in comparison to when the complex was only annealed. This experiment also demonstrated enhanced expression of the polypeptide when the complex was annealed then irradiated in comparison to when the complex containing Chol J, described earlier in Example 12 to not contain a photoreactive crosslinking agent, was only annealed. Particularly, these results were achieved when the complexes were irradiated for 30 minutes. In another experiment, the previously mentioned protocol in Example 12 resulted in a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) and complexed with a dfTAT peptide further complexed with CHOL-E2/E1_CK, described earlier in Example 12 as including one CNVK photoreactive crosslinking agent, conjugated to a TEG linker conjugated to a cholesterol target by way of: (a) annealing only; (b) annealing then irradiating for 1 minute; (c) annealing then irradiating for 5 minutes; or (d) annealing then irradiating for 30 minutes. After incubation with the cells for 24 hours, a luciferase assay was performed to assess polypeptide expression in the cells and compared to the results of the prior experiment in Example 12 (FIG.25B). Results of this experiment are shown in FIG.25A. This experiment demonstrated enhanced expression occurs after irradiation of the complex when the binding region of the circular polyribonucleotide only contains a portion of E1 rather than a portion of E1/E2. Particularly, this result was achieved when the complexes were irradiated for 30 minutes. Example 13. Optimization of the location and number of photoreactive crosslinking agents within an oligomer for the expression of a polypeptide encoded by a circular polyribonucleotide in vitro This example demonstrates the effect of the location and number of photoreactive crosslinking agents within an oligomer and the covalent attachment, by way of irradiation, of the oligomer to a circular polyribonucleotide encoding a polypeptide on the expression of the polypeptide in vitro. The non-naturally occurring circular RNA encoding a luciferase polypeptide of Example 12 was complexed with a cholesterol targeting moiety. A binding moiety (X), consisting of: (a) CHOL-E1_CK, as described in Example 12; (b) Chol J, as described in Example 12; (c) an RNA 23-mer of a fourth sequence (ACUUGAACCC[CNVK]CACGACCGUUUA SEQ ID NO: 91) termed PS1D; (d) an RNA 23-mer of a fifth sequence (ACUUG[CNVK]ACCCACACG[CNVK]CCGUUUA SEQ ID NO: 92) termed PS2E; (e) an RNA 23-mer of a sixth sequence (ACGGAUUUUA[CNVK]GUCCGUAGCGUC SEQ ID NO: 93) termed PS1A; or (f) an RNA 23-mer of a seventh sequence (ACGG[CNVK]UUUUAAGUCCGU[CNVK]GCGUC SEQ ID NO: 94) termed PS2B was linked via a TEG linker (L) to a cholesterol targeting moiety (J) or tocopherol targeting moiety (Q). PS1D and PS2E each have a cholesterol targeting moiety, and PS1A and PS2B each have a tocopherol targeting moiety. The sequence name, ID number, sequence, position, linker, and target moiety for each binding moiety are listed in Table 3. All oligomers previously listed contain at least one 3- cyanovinylcarbazole nucleoside (CNVK) photoreactive crosslinking agent except for Chol J. PS1D is the same sequence as Chol J, however, with one CNVK photoreactive crosslinking agent replacing an adenosine nucleotide at an internal position. PS2E is the same sequence as Chol J, however, with two CNVK photoreactive crosslinking agents each replacing an adenosine nucleotide at an internal position. PS1A is the same sequence as CHOL-E2/E1_CK but without the Cy5 fluorescent label. PS2B is the same sequence as PS1A, however, with a second CNVK photoreactive crosslinking agent replacing an adenosine nucleotide at an internal position. In this example, the binding moiety is also Chol J linked via a TEG linker (L) to a tocopherol targeting moiety (Q) or to both a cholesterol and tocopherol targeting moiety. PS1D had 96% complementarity to just a portion of the E1. PS2E had 91% complementarity to just a portion of E1. PS1A had 96% complementarity to a portion of E1/E2. PS2B had 91% complementarity to a portion of E1/E2. The oligomer conjugated to the TEG linker conjugated to the one or more targeting moieties was then annealed to the circular polyribonucleotide in a thermocycler by heating at 75°C for 30 seconds, heating at 75°C for 1 minute and 30 seconds, cooling to 25°C for 2 minutes, and repeating one time. The annealing buffer was HBS containing 5 mM MgCl2 and 280 mM NaCl. The resulting circular polyribonucleotide complexed to the one or more targeting moieties was stored at room temperature. The resulting circular polyribonucleotide complexed to the one or more targeting moieties was then covalently attached to the oligomer by irradiating the complex with light of a wavelength of 366 nm for 30 minutes.1 pmol of the complex including the circular polyribonucleotide and the one or more targeting moieties was transfected into human embryonic kidney cells and incubated for 24 hours at 37 °C under 6% CO2. This resulted in a circular polyribonucleotide encoding a luciferase polypeptide (eRNA) complexed to a TEG linker (L) and a cholesterol targeting moiety (J) by way of an oligomer binding moiety of one of the following: (a) CHOL-E1_CK, as described in Example 12; (b) PS1D; (c) PS2E; (d) PS1A; or (e) PS2B. After incubation for 48 hours, a luciferase assay was performed to assess polypeptide expression in the cells in comparison to cells transfected with the eRNA complexed with Chol J complexed to a cholesterol targeting moiety (J) by a TEG linker (L), the eRNA complexed with Chol J complexed to a tocopherol targeting moiety (Q) by a TEG linker (L), and the eRNA complexed with Chol J complexed to both a cholesterol targeting moiety (J) and tocopherol targeting moiety (Q) by a TEG linker (L). All controls were also a result of the previously mentioned protocol in Example 13 but were not irradiated. Results of this experiment are shown in FIGS.26A and 26B. This experiment demonstrated the enhanced expression of the polypeptide for a complex containing one or more photoreactive crosslinking agents that was annealed then irradiated is dependent on the binding region of the circular polyribonucleotide, as enhanced expression after irradiation was only achieved for the complex including either CHOL-E1_CK, PS1D, or PS2E that each bind to just a portion of E1. This experiment also resulted in enhanced expression of the polypeptide after irradiation when the complex included PS1D as compared to when the complex included PS2E and when the complex included CHOL- E1_CK. This experiment also demonstrated enhanced expression of the polypeptide when the complex that includes one or more photoreactive crosslinking agents was annealed then irradiated in comparison to when the complex does not include photoreactive crosslinking agents and was only annealed despite containing two targeting moieties. Particularly, these results were achieved when the complexes were irradiated for 30 minutes. Ordered Embodiments 1. A complex comprising A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5. 2. The complex of embodiment 1, wherein n is an integer from 2 to 20. 3. The complex of embodiment 1, wherein n is an integer from 1 to 5. 4. The complex of any one of embodiments 1-3, wherein z is an integer from 1 to 3. 5. The complex of embodiment 4, wherein z is 1. 6. The complex of embodiment 4, wherein z is 2. 7. The complex of any one of embodiments 1-6, wherein Xn(L-B)z comprises Xn(L-B1)z and Xn(L- B2)z, where B1 is a first targeting moiety and B2 is a second targeting moiety. 8. The complex of any one of embodiments 1-7, wherein Xn(L-B)z comprises Xn(L1-B)z and Xn(L2- B)z, where L1 is a first linker and L2 is a second linker. 9. The complex of any one of embodiments 1-8, wherein Xn(L-B)z comprises X1-(L-B)z and X2-(L- B)z, where X1 is a first moiety that binds specifically to a first region of A and X2 is a second moiety that binds specifically to a second region of A. 10. The complex of any one of embodiments 1-9, wherein Xn(L-B)z comprises B1-L- X-L-B2, where B1 is a first targeting moiety and B2 is a second targeting moiety. 11. The complex of any one of embodiments 1-10, wherein Xn(L-B)z comprises B-L1-X-L2-B, where L1 is a first linker and L2 is a second linker. 12. The complex of any one of embodiments 1-11, wherein the targeting moiety comprises a small molecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid, or a combination thereof. 13. The complex of embodiment 12, wherein the targeting moiety comprises a small molecule. 14. The complex of embodiment 13, wherein the small molecule is selected from folic acid, urea, α-mannose, high mannose, ursodeoxycholic acid, an endosomal escape agent, or lithocholic acid. 15. The complex of embodiment 12, wherein the targeting moiety comprises a polypeptide. 16. The complex of embodiment 15, wherein the polypeptide is a cell-penetrating peptide. 17. The complex of embodiment 15 or embodiment 16, wherein the polypeptide is selected from ASSLNIA, M12, RGD, melittin, LPS-binding protein (LBP) peptide, an adipose-homing peptide, or an endolytic peptide. 18. The complex of embodiment 15, wherein the polypeptide is an antibody or a target-binding fragment thereof. 19. The complex of embodiment 18, wherein the antibody or target-binding fragment thereof is selected from a monoclonal antibody or target-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, or a tandem scFv (taFv). 20. The complex of embodiment 18 or embodiment 19, wherein the antibody or target-binding fragment thereof is selected from an anti-FcRn antibody, an anti-MR antibody, an anti-CD205 antibody, an anti-CD169 antibody, an anti-CD14 antibody, an anti-CD36 antibody, an anti-CD5 antibody, an anti - CD71 antibody, an anti-CD38 antibody, or an anti-prohibin antibody. 21. The complex of embodiment 15, wherein the polypeptide is a nanobody. 22. The complex of embodiment 21, wherein the nanobody is selected from an anti-transferrin nanobody, an anti-HER2 nanobody, or an anti-EGFR nanobody. 23. The complex of embodiment 12, wherein the targeting moiety comprises a carbohydrate. 24. The complex of embodiment 23, wherein the carbohydrate comprises a saccharide, disaccharide, or polysaccharide. 25. The complex of embodiment 23 or embodiment 24, wherein the carbohydrate comprises mannose, galactose, or glucose. 26. The complex of any one of embodiments 23-25, wherein the carbohydrate comprises GalNAc or mannose 6-phosphate. 27. The complex of embodiment 26, wherein the carbohydrate comprises a mono-, di-, tri-, or tetra-GalNAc. 28. The complex of embodiment 27, wherein the carbohydrate is tri-GalNAc. 29. The complex of embodiment 12, wherein the targeting moiety comprises a lipid. 30. The complex of embodiment 29, wherein the lipid comprises a fatty acid. 31. The complex of embodiment 30, where the fatty acid is a saturated, monounsaturated, or polyunsaturated fatty acid. 32. The complex of embodiment 30 or embodiment 31, wherein the fatty acid is a branched or unbranched chain comprising from 4 to 40 main-chain carbon atoms. 33. The complex of any one of embodiments 30-32, wherein the fatty acid comprises squalene, stearic acid, oleic acid, palmitic acid, linoleic acid, stearic acid, lauric acid, docosahexanoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, α- tocopherol, α-tocopherol succinate, or a retinoic acid. 34. The complex of embodiment 33, wherein the fatty acid comprises DCA. 35. The complex of embodiment 33, wherein the fatty acid comprises DHA. 36. The complex of embodiment 33, wherein the fatty acid comprises myristic acid. 37. The complex of embodiment 29, wherein the lipid comprises a steroid or sterol selected from cholesterol, tocopherol, ursodeoxycholic acid, or lithocholic acid. 38. The complex of embodiment 37, wherein the steroid or sterol is cholesterol. 39. The complex of embodiment 37, wherein the steroid or sterol is tocopherol. 40. The complex of embodiment 29, wherein the lipid comprises a fat-soluble vitamin selected from vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof. 41. The complex of embodiment 29, wherein the lipid comprises a phospholipid. 42. The complex of embodiment 41, wherein the phospholipid is selected from phosphocholine (PC), PC-docosahexaenoic acid (PC-DHA), PC-docosanoic acid (PC-DCA), PC-eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC-retinoic acid (PC-RA), or PC-α-tocopherol succinate (PC-TS). 43. The complex of embodiment 12, wherein the targeting moiety comprises an oligonucleotide. 44 The complex of embodiment 43, wherein the targeting moiety comprises an aptamer. 45. The complex of any one of embodiments 1-44, wherein the linker is a bond. 46. The complex of any one of embodiments 1-44, wherein the linker comprises 1 to 250 backbone atoms, wherein the backbone atoms are selected from C, N, O, and S. 47. The complex of any one of embodiments 1-44, wherein the linker comprises an oligonucleotide. 48. The complex of any one of embodiments 1-44, wherein the linker comprises a polypeptide. 49. The complex of any one of embodiments 1-44, wherein the linker comprises at least one PEG unit. 50. The complex of embodiment 49, wherein the PEG is a PEG2-PEG10,000. 51. The complex of any one of embodiments 1-44, wherein the linker comprises at least one TEG unit. 52. The complex of embodiment 51, wherein the TEG is a TEG2-TEG10,000. 53. The complex of any one of embodiments 1-44, wherein the linker is an enzymatically cleavable linker. 54. The complex of any one of embodiments 1-50, wherein the linker has a length of at least 0.1 nm, optionally wherein the linker has a length of from 0.1 nm to 20 nm. 55. The complex of any one of embodiments 1-54, wherein the linker is further conjugated to avidin. 56. The complex of embodiment 55, wherein the avidin binds from 1 to 4 targeting moieties. 57. The complex of embodiment 55 or embodiment 56, wherein the avidin binds from 1 to 4 targeting moieties, wherein the targeting moieties are conjugated to at least one biotin compound. 58. The complex of any one of embodiments 1-57, wherein X is an oligonucleotide. 59. The complex of embodiment 58, wherein the oligonucleotide comprises a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. 60. The complex of embodiment 58 or embodiment 59, wherein the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety. 61. The complex of embodiment 58 or embodiment 59, wherein the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety. 62. The complex of embodiment 58 or 59, wherein the oligonucleotide is from 5 to 100 nucleotides in length. 63. The complex of any one of embodiments 58-61, wherein the circular polynucleotide comprises one or more binding regions each comprising from 5 to 200 ribonucleotides, wherein each binding region binds to an oligonucleotide. 64. The complex of any one of embodiments 58-63, wherein each binding region comprises at least 50%, 60%, 70%, 80%, 90%, or 100% complementarity to the oligonucleotide. 65. The complex of any one of embodiments 1-57, wherein X is a polypeptide. 66. The complex of embodiment 65, wherein the polypeptide comprises an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521-B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi-Argonaute-Zwille domain, or intrinsically disordered region. 67. The complex of embodiment 65 or embodiment 66, wherein the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. 68. The complex of embodiment 65 or embodiment 66, wherein the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety. 69. The complex of any one of embodiments 65-68, wherein the circular polyribonucleotide comprises one or more protein binding regions each comprising from 5 to 200 ribonucleotides, wherein each protein binding region binds to a polypeptide. 70. The complex of any one of embodiments 1-69, wherein the circular polyribonucleotide further comprises at least one coding region. 71. The complex of embodiment 70, wherein the at least one coding region comprises an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide. 72. The complex of embodiment 71, wherein the polypeptide is expressed in the cell. 73. The complex of embodiment 72, wherein the polypeptide expressed in the cell is functional. 74. The complex of any one of embodiments 1-73, wherein the circular polyribonucleotide comprises one or more binding regions each comprising from 5 to 200 ribonucleotides, wherein each binding region binds to X. 75. The complex of embodiment 74, wherein the binding region is contained within or overlaps in- part with an expression sequence or with a spacer region. 76. The complex of embodiment 74 or embodiment 75, wherein the binding region is not contained within nor does it overlap in-part with an IRES. 77. The complex of any one of embodiments 74-76 wherein: (i) the 3’ end of the binding region is at least 5 ribonucleotides from 5’ end of the IRES; (ii) the 3’ end of the binding region is from 5 to 200 ribonucleotides from 5’ end of the IRES; (iii) the 5’ end of the binding region is at least 5 ribonucleotides from 3’ end of the IRES; or (iv) the 5’ end of the binding region is from 5 to 200 ribonucleotides from 3’ end of the IRES. 78. The complex of any one of embodiments 1-73, wherein the circular polyribonucleotide comprises the following elements, arranged in the following order: (i) a first spacer region; (ii) at least one coding region comprising an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide; (iii) optionally a second spacer region; and (iv) a binding region comprising from 5 to 200 ribonucleotides, wherein the binding region binds to X. 79. The complex of any one of embodiments 1-77, wherein the circular polyribonucleotide comprises the following elements, arranged in the following order: (i) a first spacer region; (ii) a target binding region comprising at least one aptamer or at least ribozyme sequence; (iii) optionally a second spacer region; and (iv) a binding region comprising from 5 to 200 ribonucleotides, wherein the binding region binds to X. 80. The complex of embodiment 78 or 79, wherein the first spacer region comprises 10 to 500 ribonucleotides. 81. The complex of any one of embodiments 78-80, wherein the second spacer region comprises 10-500 ribonucleotides. 82. The complex of embodiment 78, wherein the binding region is contained within or overlaps in- part with the expression sequence. 83. The complex of any one of embodiments 78-82, wherein the binding region is contained within or overlaps the first spacer region. 84. The complex of any one of embodiments 78-82, wherein the binding region is contained within or overlaps the second spacer region. 85. The complex of any one of embodiments 78-84, wherein the first spacer and second spacer are adjacent to one another. 86. The complex of embodiment 85, wherein the binding region overlaps the first and second spacer regions. 87. The complex of embodiment 78, wherein the binding region is not contained within nor does it overlap in-part with the expression sequence or with the first or second spacer region. 88. The complex of any one of embodiments 1-87, wherein the circular polyribonucleotide comprises at least 1,000 ribonucleotides. 89. The complex of embodiment 86, wherein the circular polyribonucleotide comprises at least 3,000 ribonucleotides. 90. The complex of any one of embodiments 1-87, wherein the circular polyribonucleotide comprises from 1,000 to 20,000 ribonucleotides. 91. The complex of any one of embodiments 1-90, wherein the circular polyribonucleotide encodes a polypeptide. 92. The complex of any one of embodiments 1-91, wherein each X comprises a photoreactive crosslinking agent. 93. A complex comprising A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A and comprises a photoreactive crosslinking agent, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5. 94. The complex of embodiment 92 or 93, wherein X is an oligonucleotide. 95. The complex of embodiment 94, wherein the oligonucleotide comprises a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. 96. The complex of embodiment 94 or 95, wherein the photoreactive crosslinking agent is attached to the 5’ end of the oligonucleotide. 97. The complex of any one of embodiments 94-96, wherein the photoreactive crosslinking agent is attached to the 3’ end of the oligonucleotide. 98. The complex of any one of embodiments 94-97, wherein the photoreactive crosslinking agent comprises 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a phenylselenide, a furan, or an abasic site. 99. The complex of embodiment 98, wherein the carbazole is 3-cyanovinylcarbazole, 4- methylpyranocarbazole, or pyranocarbazole. 100. The complex of embodiment 99, wherein the coumarin is 7-hydroxycoumarin. 101. The complex of any one of embodiments 94-100, wherein the photoreactive crosslinking agent is a photoreactive nucleotide analog. 102. The complex of embodiment 101, wherein the photoreactive nucleotide analog is located at an internal position within the oligonucleotide. 103. The complex of embodiment 101 or 102, wherein the 3’ end of the oligonucleotide has at least 1 nucleotide from the photoreactive nucleotide analog. 104. The complex of embodiment 103, wherein the 3’ end of the oligonucleotide has from 1 to 10 nucleotides from the photoreactive nucleotide analog. 105. The complex of any one of embodiments 101-104, wherein the 5’ end of the oligonucleotide has at least 1 nucleotide from the photoreactive nucleotide analog. 106. The complex of embodiment 105, wherein the 5’ end of the oligonucleotide has from 1 to 10 nucleotides from the photoreactive nucleotide analog. 107. The complex of any one of embodiments 101-106, wherein the photoreactive nucleotide analog crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. 108. The complex of any one of embodiments 94-107, wherein the oligonucleotide comprises a plurality of photoreactive crosslinking agents. 109. The complex of embodiment 108, wherein each of the plurality of photoreactive crosslinking agents comprises 5-bromo-2’-deoxyuridine (BrdU), a carbazole, a psoralen, a coumarin, 4’-thiouridine, a diazirine, a pheylselenide, a furan, or an abasic site. 110. The complex of embodiment 109, wherein the carbazole is 3-cyanovinylcarbazole, 4- methylpyranocarbazole, or pyranocarbazole. 111. The complex of embodiment 109, wherein the coumarin is 7-hydroxycoumarin. 112. The complex of any one of embodiments 108-111, wherein each of the plurality of photoreactive crosslinking agents is a photoreactive nucleotide analog. 113. The complex of embodiment 112, wherein each photoreactive nucleotide analog is located at an internal position within the oligonucleotide. 114. The complex of embodiment 112 or 113, wherein the 3’ end of the oligonucleotide has at least 2 nucleotides from the nearest photoreactive nucleotide analog. 115. The complex of embodiment 114, wherein the 3’ end of the oligonucleotide has from 1 to 10 nucleotides from the nearest photoreactive nucleotide analog. 116. The complex of any one of embodiments 112-115, wherein the 5’ end of the oligonucleotide has at least 2 nucleotides from the nearest photoreactive nucleotide analog. 117. The complex of embodiment 116, wherein the 5’ end of the oligonucleotide has from 2 to 10 nucleotides from the nearest photoreactive nucleotide analog. 118. The complex of any one of embodiments 112-117, wherein at least one of the photoreactive nucleotide analogs is attached to the 3’ end of the oligonucleotide. 119. The complex of any one of embodiments 112-118, wherein at least one of the photoreactive nucleotide analogs is attached to the 5’ end of the oligonucleotide. 120. The complex of any one of embodiments 112-119, wherein the oligonucleotide comprises 1 to 10 nucleotides between each of the photoreactive nucleotide analogs. 121. The complex of any one of embodiments 112-120, wherein each of the photoreactive nucleotide analogs crosslinks to a complementary ribonucleotide or a ribonucleotide located 1 base upstream or 1 base downstream of the complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. 122. The complex of any one of embodiments 94-121, wherein the oligonucleotide comprises an aptamer. 123. The complex of any one of embodiments 94-122, wherein the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety. 124. The complex of any one of embodiments 94-123, wherein the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety. 125. The complex of any one of embodiments 94-124, wherein the oligonucleotide is from 5 to 100 nucleotides in length. 126. The complex of any one of embodiments 94-125, wherein the circular polynucleotide comprises a binding region that anneals to the oligonucleotide. 127. The complex of embodiment 126, wherein the binding region is from 5 to 200 ribonucleotides. 128. The complex of embodiments 126 or 127, wherein the binding region comprises at least 70% complementarity to the oligonucleotide. 129. The complex of any one of embodiments 126-127, wherein the binding region has zero or one mismatch with the oligonucleotide. 130. The complex of embodiment 92 or 93, wherein X is a polypeptide. 131. The complex of embodiment 130, wherein the photoreactive crosslinking agent is a photoreactive amino acid analog. 132. The complex of 131, wherein the photoreactive amino acid analog is located at an internal position within the polypeptide. 133. The complex of embodiment 131 or 132, wherein the N-terminus of the polypeptide has at least 1 amino acids from the photoreactive amino acid analog. 134. The complex of any one of embodiments 131-133, wherein the N-terminus of the polypeptide has from 1 to 10 amino acids from the photoreactive amino acid analog. 135. The complex of any one of embodiments 131-134, wherein the C-terminus of the polypeptide has at least 1 amino acid from the photoreactive amino acid analog. 136. The complex of any one of embodiments 131-134, wherein the C-terminus of the polypeptide has from 1 to 10 amino acids from the photoreactive amino acid analog. 137. The complex of embodiment 136, wherein the photoreactive amino acid analog is attached to the N-terminus of the polypeptide. 138. The complex of embodiment 137, wherein the photoreactive nucleotide analog is attached to the C-terminus of the polypeptide. 139. The complex of any one of embodiments 131-138, wherein the photoreactive amino acid analog crosslinks to a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. 140. The complex of any one of embodiment 131-139, wherein the photoreactive amino acid analog is an alkyl diazirene-based, arylazide-based, benzophenone-based unnatural amino acid, or N-ε- [2-(furan-2-yl)ethoxy]carbonyl-lysine. 141. The complex of embodiment 140, wherein the polypeptide comprises a plurality of photoreactive crosslinking agents. 142. The complex of embodiment 141, wherein each of the plurality of photoreactive crosslinking agents is a photoreactive amino acid analog. 143. The complex of embodiment 142, wherein at least one photoreactive amino acid analog is located at an internal position within the polypeptide. 144. The complex of embodiment 142 or 143, wherein the N-terminus of the polypeptide has at least 2 amino acids from the nearest photoreactive amino acid analog. 145. The complex of embodiment 144, wherein the N-terminus of the polypeptide has from 2 to 10 amino acids from the nearest photoreactive amino acid analog. 146. The complex of any one of embodiments 142-145, wherein the C-terminus of the polypeptide has at least 2 amino acids from the nearest photoreactive amino acid analog. 147. The complex of embodiment 146, wherein the C-terminus of the polypeptide has from 2 to 10 amino acids from the nearest photoreactive amino acid analog. 148. The complex of any one of embodiments 142-147, wherein at least one of the photoreactive amino acid analogs is attached to the N-terminus of the polypeptide. 149. The complex of any one of embodiments 142-148, wherein at least one of the photoreactive amino acid analogs is attached to the C-terminus of the polypeptide. 150. The complex of any one of embodiments 142-149, wherein the polypeptide comprises 1 to 10 amino acids between each of the photoreactive amino acid analogs. 151. The complex of any one of embodiments 142-150, wherein each of the photoreactive amino acids crosslinks to a complementary ribonucleotide within the circular polyribonucleotide upon photoirradiation. 152. The complex any one of embodiments 142-151, wherein each photoreactive amino acid analog is an alkyl diazirene-based, arylazide-based, benzophenone-based unnatural amino acid, or N-ε- [2-(furan-2-yl)ethoxy]carbonyl-lysine. 153. The complex of any one of embodiments 130-152, wherein the polypeptide comprises an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521-B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi- Argonaute-Zwille domain, or intrinsically disordered region. 154. The complex of any of embodiments 130-153, wherein the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. 155. The complex of any one of embodiments 130-154, wherein the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety. 156. The complex of any one of embodiments 130-155, wherein the circular polyribonucleotide comprises one or more protein binding regions each comprising from 5 to 200 ribonucleotides, wherein each protein binding region binds to a polypeptide. 157. A complex comprising A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically and is covalently attached to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5. 158. The complex of embodiment 157, wherein X is an oligonucleotide. 159. The complex of embodiment 158, wherein the oligonucleotide comprises a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid. 160. The complex of any one of embodiments 157-159, wherein the oligonucleotide comprises an aptamer. 161. The complex of any one of embodiments 157-160, wherein the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety. 162. The complex of any one of embodiments 157-161, wherein the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety. 163. The complex of any one of embodiments 157-162, wherein the oligonucleotide is from 5 to 100 nucleotides in length. 164. The complex of any one of embodiments 157-163, wherein the circular polynucleotide comprises a binding region that anneals to the oligonucleotide. 165. The complex of embodiment 164, wherein the binding region is from 5 to 200 ribonucleotides. 166. The complex of embodiment 164 or 165, wherein the binding region comprises at least 70% complementarity to the oligonucleotide. 167. The complex of any one of embodiments 164-166, wherein the binding region has zero or one mismatch with the oligonucleotide. 168. The complex of embodiment 157, wherein X is a polypeptide. 169. The complex of embodiment 158, wherein the polypeptide comprises an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521-B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi-Argonaute-Zwille domain, or intrinsically disordered region. 170. The complex of embodiment 168 or 169, wherein the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 polypeptides, wherein each polypeptide is complexed to at least one targeting moiety. 171. The complex of any one of embodiments 168-170, wherein the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety. 172. The complex of any one of embodiments 168-171, wherein the circular polyribonucleotide comprises one or more protein binding regions each comprising from 5 to 200 ribonucleotides, wherein each protein binding region binds to a polypeptide. 173. A pharmaceutical composition comprising a circular polyribonucleotide of any one of embodiments 1-172 and one or more delivery agents. 174. The pharmaceutical composition of embodiment 173, wherein the delivery agent is selected from calcium, magnesium, manganese, or strontium. 175. The pharmaceutical composition of embodiment 173 or 174, wherein the delivery agent is an endosomal escape agent. 176. The pharmaceutical composition of embodiment 175, wherein the endosomal escape agent comprises chloroquine, amantadine, ammonium chloride, 4-bromobenzaldehyde N-(2,6- dimethylphenyl)semicarbazone (EGA), UNC-108, or any combination thereof. 177. The pharmaceutical composition of any one of embodiments 173-176, wherein the delivery agent is a globular protein. 178. The pharmaceutical composition of embodiment 177, wherein the globular protein is albumin. 179. The pharmaceutical composition of any one of embodiments 173-178, wherein the delivery agent is ribonuclease inhibitor. 180. A method of delivering a circular polyribonucleotide to a cell, the method comprising contacting the cell with a complex or pharmaceutical composition of any one of embodiments 1-172. 181. The method of embodiment 180, wherein the cell is a eukaryotic cell. 182. The method of embodiment 181, wherein the cell is a mammalian cell. 183. The method of embodiment 182, wherein the cell is a human cell. 184. The method of any one of embodiments 180-183, wherein the circular polyribonucleotide is delivered to the cell ex-vivo. 185. The method of any one of embodiments 180-184 wherein the cell is administered to a subject after the delivery of the circular polyribonucleotide to the cell. 186. The method of embodiment 185, wherein administration of the cell to the subject treats a disease, disorder, or condition in the subject. 187. A method of delivering a circular polyribonucleotide to a subject, the method comprising administering to the subject a complex or pharmaceutical composition of any one of embodiments 1-98. 188. A method of treating a disease, disorder, or condition in a subject, the method comprising administering to the subject a complex or pharmaceutical composition of any one of embodiments 1-172. 189. A method of inducing an immune response in a subject, the method comprising administering to the subject a complex or pharmaceutical composition of any one of embodiments 1-172. 190. The method of any one of embodiments 180-189, wherein the complex or pharmaceutical composition is administered intramuscularly, subcutaneously, intravenously, intraperitoneally, topically, or orally. 191. The method of any one of embodiments 180-190, wherein the subject is a mammal. 192. The method of embodiment 191, wherein the subject is a human. 193. The method of embodiment 192, wherein the subject is a non-human mammal. 194. The method of embodiment 193, wherein the non-human mammal is a cow, a sheep, a goat, a pig, a dog, a horse, or a cat. 195. The method of any one of embodiments 180-194, wherein the subject is a bird. 196. The method of embodiment 195, wherein the bird is a hen, a rooster, a turkey, or a parrot. 197. A method of covalently attaching X to A comprising irradiating the complex of any one of embodiments 92-156 with light. 198. A method of forming a covalent complex comprising: (a) forming a complex comprising A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A and comprises a photoreactive crosslinking agent, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5; and (b) irradiating the complex with light. 199. The method of embodiment 197 or 198, wherein a wavelength of the irradiated light is from 350-370 nm. 200. The method of any one of embodiments 197-199, wherein the complex is irradiating for 1 to 120 minutes. 201. The method of any one of embodiments 197-200, further comprising irradiating the complex with light at a second wavelength to remove the covalent attachment. 202. The method of embodiment 201, wherein the second wavelength of the irradiated light is from 300-320 nm. 203. The method of embodiment 201 or 202, wherein the complex is irradiated at the second wavelength for 1 to 120 minutes. 204. The method of any one of embodiments 197-203, further comprising contacting a cell with the complex to deliver the complex to the cell. 205. The method of embodiment 204, wherein the complex is irradiated prior to contacting. 206. The method of embodiment 204, wherein the complex is irradiated after contacting. 207. The method of any one of embodiments 204-206, wherein the cell is a eukaryotic cell. 208. The method of embodiment 207, wherein the eukaryotic cell is a mammalian cell. 209. The method of embodiment 208, wherein the mammalian cell is a human cell. 210. The method of any one of embodiments 204-209, wherein the complex is delivered to the cell ex vivo. 211. The method of any one of embodiments 204-209, wherein the complex is delivered to the cell in vivo. 212. The method of any one of embodiments 204-211, wherein the complex is administered to a subject. 213. The method of embodiment 212, wherein the complex is irradiated prior to administration. 214. The method of embodiment 212, wherein the complex is irradiated after administration. 215. The method of any one of embodiments 204-214, wherein contacting the cell treats a disease or disorder in the subject. 216. A covalent complex produced by the method of any one of embodiments 198-215. Other Embodiments Various modifications and variations of the described compositions, methods, and uses of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

CLAIMS 1. A complex comprising A and Xn(L-B)z, wherein A is a circular polyribonucleotide, each X is independently a moiety that binds specifically to a region of A, each L is independently a linker, each B is independently a targeting moiety, wherein n is an integer from 1 to 20, and wherein z is an integer from 1 to 5.
2. The complex of claim 1, wherein n is an integer from 2 to 20.
3. The complex of claim 1, wherein n is an integer from 1 to 5.
4. The complex of any one of claims 1-3, wherein z is an integer from 1 to 3.
5. The complex of claim 4, wherein z is 1.
6. The complex of claim 4, wherein z is 2.
7. The complex of any one of claims 1-6, wherein Xn(L-B)z comprises Xn(L-B1)z and Xn(L-B2)z, where B1 is a first targeting moiety and B2 is a second targeting moiety.
8. The complex of any one of claims 1-7, wherein Xn(L-B)z comprises Xn(L1-B)z and Xn(L2-B)z, where L1 is a first linker and L2 is a second linker.
9. The complex of any one of claims 1-8, wherein Xn(L-B)z comprises X1-(L-B)z and X2-(L-B)z, where X1 is a first moiety that binds specifically to a first region of A and X2 is a second moiety that binds specifically to a second region of A.
10. The complex of any one of claims 1-9, wherein Xn(L-B)z comprises B1-L- X-L-B2, where B1 is a first targeting moiety and B2 is a second targeting moiety.
11. The complex of any one of claims 1-10, wherein Xn(L-B)z comprises B-L1-X-L2-B, where L1 is a first linker and L2 is a second linker.
12. The complex of any one of claims 1-11, wherein the targeting moiety comprises: (a) a small molecule, optionally, folic acid, urea, α-mannose, high mannose, ursodeoxycholic acid, an endosomal escape agent, or lithocholic acid; or (b) a polypeptide, optionally, a cell-penetrating peptide, ASSLNIA, M12, RGD, melittin, LPS- binding protein (LBP) peptide, an adipose-homing peptide, an endolytic peptide, an antibody or a target- binding fragment thereof, a nanobody; or (c) a carbohydrate, optionally, a saccharide, a disaccharide, a polysaccharide, mannose, galactose, glucose, mannose 6-phosphate, or GalNAc, optionally, a mono-GalNAc, a di-GalNAc, a tri- GalNAc, or a tetra-GalNAc; or (d) a lipid, optionally, a fatty acid, optionally, a saturated, monounsaturated, or polyunsaturated fatty acid, or a branched or unbranched chain comprising from 4 to 40 main-chain carbon atoms, a steroid or sterol, optionally, cholesterol, tocopherol, ursodeoxycholic acid, or lithocholic acid, a fat-soluble vitamin, optionally, vitamin A, vitamin D, vitamin E, vitamin K, or an analog or metabolite thereof, a phospholipid, optionally, phosphatidylcholine, phosphocholine (PC), PC-docosahexaenoic acid (PC- DHA), PC-docosanoic acid (PC-DCA), PC-eicosapentaenoic acid (PC-EPA), PC-lithocholic acid (PC-LA), PC-retinoic acid (PC-RA), or PC-α-tocopherol succinate (PC-TS); or (e) a nucleic acid, optionally, an oligonucleotide or an aptamer; or (f) a combination thereof.
13. The complex of claim 12, wherein the fatty acid comprises squalene, stearic acid, oleic acid, palmitic acid, linoleic acid, stearic acid, lauric acid, docosahexanoic acid (DHA), docosanoic acid (DCA), eicosapentaenoic acid (EPA), octadecanoic acid, myristic acid, anadamide, α-tocopherol, α-tocopherol succinate, or a retinoic acid.
14. The complex of any one of claims 1-13, wherein the linker: (i) is a bond; or (ii) comprises 1 to 250 backbone atoms, wherein the backbone atoms are selected from C, N, O, and S; or (iii) comprises an oligonucleotide; or (iv) comprises a polypeptide; or (v) comprises at least one PEG unit, optionally, PEG2-PEG10,000; or (vi) comprises at least one TEG unit, optionally, TEG2-TEG10,000; or (vii) is an enzymatically cleavable linker.
15. The complex of claim 14, wherein the linker has a length of at least 0.1 nm, optionally wherein the linker has a length of from 0.1 nm to 20 nm.
16. The complex of claim 14 or 15, wherein the linker is further conjugated to avidin.
17. The complex of claim 16, wherein the avidin binds from 1 to 4 targeting moieties, optionally wherein the targeting moieties are conjugated to at least one biotin compound.
18. The complex of any one of claims 1-17, wherein X is an oligonucleotide, optionally wherein the oligonucleotide comprises a ribonucleic acid, a deoxyribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a glycol nucleic acid.
19. The complex of claim 18, wherein the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 oligonucleotides, wherein each oligonucleotide is complexed to at least one targeting moiety.
20. The complex of claim 19, wherein the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 oligonucleotides wherein each oligonucleotide is complexed to at least one targeting moiety.
21. The complex of claim 18, wherein the oligonucleotide is from 5 to 100 nucleotides in length.
22. The complex of any one of claims 18-21, wherein the circular polynucleotide comprises one or more binding regions each comprising from 5 to 200 ribonucleotides, wherein each binding region binds to an oligonucleotide.
23. The complex of any one of claims 18-22, wherein each binding region comprises at least 50%, 60%, 70%, 80%, 90%, or 100% complementarity to the oligonucleotide.
24. The complex of any one of claims 1-17, wherein X is a polypeptide.
25. The complex of claim 24, wherein the polypeptide comprises an RNA recognition motif wherein the RNA recognition motif is selected from a K homology domain, zinc finger motif, Pumilio homology domain, pentatricopeptide repeat domain, pseudouridine synthase and archaeosine transglycosylase domain, THUMP domain, YT521-B homology domain, double stranded RNA binding domain, helicase domain, cold shock domain, S1 domain, Sm domain, La motif, Piwi-Argonaute-Zwille domain, or intrinsically disordered region.
26. The complex of claim 24 or 25, wherein the circular polyribonucleotide is bound to from 1 to 100, 1 to 50, 1 to 20, or 1 to 10 polypeptides, wherein each polypeptide is complexed to at least one targeting moiety.
27. The complex of claim 24 or 25, wherein the circular polyribonucleotide is bound to from 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, 5 to 10, 10 to 100, 10 to 50, or 10 to 20 polypeptides wherein each polypeptide is complexed to at least one targeting moiety.
28. The complex of any one of claims 25-27, wherein the circular polyribonucleotide comprises one or more protein binding regions each comprising from 5 to 200 ribonucleotides, wherein each protein binding region binds to a polypeptide.
29. The complex of any one of claims 1-28, wherein the circular polyribonucleotide further comprises at least one coding region.
30. The complex of claim 29, wherein the at least one coding region comprises an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide.
31. The complex of any one of claims 1-30, wherein the circular polyribonucleotide comprises one or more binding regions each comprising from 5 to 200 ribonucleotides, wherein each binding region binds to X.
32. The complex of claim 31, wherein the binding region is contained within or overlaps in-part with an expression sequence or with a spacer region.
33. The complex of claim 31 or 32, wherein the binding region is not contained within nor does it overlap in-part with an IRES.
34. The complex of any one of claims 31-33 wherein: (i) the 3’ end of the binding region is at least 5 ribonucleotides from 5’ end of the IRES; (ii) the 3’ end of the binding region is from 5 to 200 ribonucleotides from 5’ end of the IRES; (iii) the 5’ end of the binding region is at least 5 ribonucleotides from 3’ end of the IRES; or (iv) the 5’ end of the binding region is from 5 to 200 ribonucleotides from 3’ end of the IRES.
35. The complex of any one of claims 1-30, wherein the circular polyribonucleotide comprises the following elements, arranged in the following order: (i) a first spacer region; (ii) at least one coding region comprising an internal ribosomal entry site (IRES) operably linked to an expression sequence encoding a polypeptide; (iii) optionally a second spacer region; and (iv) a binding region comprising from 5 to 200 ribonucleotides, wherein the binding region binds to X.
36. The complex of any one of claims 1-34, wherein the circular polyribonucleotide comprises the following elements, arranged in the following order: (i) a first spacer region; (ii) a target binding region comprising at least one aptamer or at least ribozyme sequence; (iii) optionally a second spacer region; and (iv) a binding region comprising from 5 to 200 ribonucleotides, wherein the binding region binds to X.
37. The complex of claim 35 or 36, wherein the first spacer region comprises 10 to 500 ribonucleotides.
38. The complex of any one of claims 35-37, wherein the second spacer region comprises 10-500 ribonucleotides.
39. The complex of claim 35, wherein the binding region is contained within or overlaps in-part with the expression sequence.
40. The complex of any one of claims 35-39, wherein (i) the binding region is contained within or overlaps the first spacer region; or (ii) the binding region is contained within or overlaps the second spacer region.
41. The complex of any one of claims 35-40, wherein the first spacer and second spacer are adjacent to one another.
42. The complex of claim 41, wherein the binding region overlaps the first and second spacer regions.
43. The complex of claim 35, wherein the binding region is not contained within nor does it overlap in-part with the expression sequence or with the first or second spacer region.
44. The complex of any one of claims 1-43, wherein (i) the circular polyribonucleotide comprises at least 1,000 ribonucleotides; or (ii) the circular polyribonucleotide comprises from 1,000 to 20,000 ribonucleotides 45. The complex of claim 42, wherein the circular polyribonucleotide comprises at least 3,000 ribonucleotides. 46. The complex of any one of claims 1-45, wherein the circular polyribonucleotide encodes a polypeptide. 47. The complex of any one of claims 1-46, wherein each X comprises a photoreactive crosslinking agent.
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