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EP4452337A1 - Circular polyribonucleotides encoding antifusogenic polypeptides - Google Patents

Circular polyribonucleotides encoding antifusogenic polypeptides

Info

Publication number
EP4452337A1
EP4452337A1 EP22859564.1A EP22859564A EP4452337A1 EP 4452337 A1 EP4452337 A1 EP 4452337A1 EP 22859564 A EP22859564 A EP 22859564A EP 4452337 A1 EP4452337 A1 EP 4452337A1
Authority
EP
European Patent Office
Prior art keywords
polyribonucleotide
days
polypeptide
sequence
antifusogenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22859564.1A
Other languages
German (de)
English (en)
French (fr)
Inventor
Gines Diego MIRALLES
Jesper Gromada
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Flagship Pioneering Innovations VI Inc
Original Assignee
Flagship Pioneering Innovations VI Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Flagship Pioneering Innovations VI Inc filed Critical Flagship Pioneering Innovations VI Inc
Publication of EP4452337A1 publication Critical patent/EP4452337A1/en
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—Special targeting system for viral vectors
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • compositions and methods for delivering an antifusogenic polypeptide e.g., to treat or prevent a viral infection.
  • compositions and methods for producing, purifying, and using circular RNA encoding an antifusogenic polypeptide are provided.
  • the invention features a circular polyribonucleotide that includes a polyribonucleotide cargo encoding the antifusogenic polypeptide.
  • the polyribonucleotide cargo includes an expression sequence encoding the antifusogenic polypeptide.
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide of Table 1 . In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of Table 1 . In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -324.
  • the circular polyribonucleotide includes a splice junction joining a 5’ exon fragment and a 3’ exon fragment.
  • the polyribonucleotide cargo includes an IRES operably linked to the expression sequence encoding the antifusogenic polypeptide.
  • the circular polyribonucleotide may further include a spacer region between the IRES and the 3’ exon fragment or the 5’ exon fragment.
  • the spacer region may be at least 5 ribonucleotides in length.
  • the spacer region may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000, or more ribonucleotides in length.
  • the spacer region is from 5 to 500 ribonucleotides in length.
  • the spacer region may include a polyA, a polyA-C, polyA-U, or polyA-G sequence.
  • the spacer region may be a random sequence.
  • the circular polyribonucleotide is at least 500 ribonucleotides in length.
  • the circular polyribonucleotide may be at least 500, 600, 700, 800, 900, 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or more polyribonucleotides.
  • the circular polyribonucleotide is from 500 to 20,000 ribonucleotides in length.
  • a linear polyribonucleotide including, from 5’ to 3’, (A) a 3' intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo encoding the antifusogenic polypeptide; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' intron fragment.
  • the polyribonucleotide cargo includes an expression sequence encoding the antifusogenic polypeptide.
  • the polyribonucleotide cargo includes an IRES operably linked to the expression sequence encoding the antifusogenic polypeptide (e.g., a polypeptide of Table 1 ).
  • the circular polyribonucleotide may further include a spacer region between the IRES and the 3’ exon fragment or the 5’ exon fragment.
  • the circular polyribonucleotide may further include a spacer region between one or more of (A), (B), (C), (D), (E), (F), and (G).
  • the spacer region may be at least 5 ribonucleotides in length.
  • the spacer region may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more ribonucleotides in length.
  • the spacer region is from 5 to 500 ribonucleotides in length.
  • the spacer region may include a polyA, a polyA-C, polyA-U, or polyA-G sequence.
  • the spacer region may be a random sequence.
  • the circular polyribonucleotide lacks an IRES. In some embodiments, the circular polyribonucleotide lacks one or both of a 5’ cap and a polyA sequence.
  • the circular polyribonucleotide comprises a protein translation initiation site.
  • the protein translation initiation site comprises a Kozak sequence.
  • the linear polyribonucleotide is at least 500 ribonucleotides in length.
  • the linear polyribonucleotide may be at least 500, 600, 700, 800, 900, 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or more polyribonucleotides.
  • the linear polyribonucleotide is from 500 to 20,000 ribonucleotides in length.
  • featured is a DNA vector encoding a polyribonucleotide (e.g., a linear or circular polyribonucleotide) as described herein.
  • a polyribonucleotide e.g., a linear or circular polyribonucleotide
  • an antifusogenic polypeptide e.g., a polypeptide of Table 1
  • the method includes providing a circular, a linear polyribonucleotide, or the DNA vector as described herein to the cell under conditions suitable to express the antifusogenic polypeptide.
  • featured is a method of producing a circular polyribonucleotide from a linear polyribonucleotide as described herein.
  • the method includes providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to produce the circular polyribonucleotide.
  • a pharmaceutical composition that includes the circular polyribonucleotide, the linear polyribonucleotide, or the DNA vector of any of the above embodiments, and a diluent, carrier, or excipient.
  • a method of expressing the antifusogenic polypeptide in a subject.
  • the method includes 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/
  • 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 the antifusogenic 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, 3,000 ng/mL, or more) of the antifus
  • the method treats or prevents a viral infection in the subject.
  • the pharmaceutical composition may be administered to the subject in an amount and for a duration sufficient to treat or prevent a viral infection.
  • the pharmaceutical composition may be administered to the subject to reduce the risk of a viral infection.
  • the method treats or prevents a human immunodeficiency virus (HIV) infection.
  • HIV human immunodeficiency virus
  • the method treats or prevents a coronavirus infection (e.g., a betacoronavirus infection, e.g., SARS-CoV-2 infection, such as a SARS-CoV-2 infection that produces symptoms of COVID-19).
  • a coronavirus infection e.g., a betacoronavirus infection, e.g., SARS-CoV-2 infection, such as a SARS-CoV-2 infection that produces symptoms of COVID-19.
  • the method treats or prevents a Hepatitis C Virus (HCV) infection.
  • HCV Hepatitis C Virus
  • a circular polynucleotide encoding the antifusogenic polypeptide (e.g., a polypeptide of Table 1 ) is used for reducing viral entry.
  • a circular polyribonucleotide that includes a polyribonucleotide cargo encoding multiple antifusogenic polypeptides.
  • the polyribonucleotide cargo may include expression sequences encoding the antifusogenic polypeptides.
  • the antifusogenic polypeptides are directed to the same virus. Alternatively, the antifusogenic polypeptides may be directed to more than one virus.
  • any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.
  • the term “about” refers to a value that is within ⁇ 10% of a recited value.
  • 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 phyto glycogen 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 phyto glycogen 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 differentiated reti
  • circular polyribonucleotide As used herein, the terms “circular polyribonucleotide,” “circular RNA,” and “circRNA” 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.
  • 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, tetrahydrofurfuryl 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,
  • 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 (e.g., an antifusogenic polypeptide).
  • 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. It is understood that all the present disclosure contemplates fragments of any antifusogenic polypeptide disclosed herein.
  • the term “Fc domain” refers to a polypeptide chain that includes at least a hinge domain and second and third antibody constant domains (CH2 and CH3) or functional fragments thereof (e.g., fragments that that capable of dimerizing and binding to an Fc receptor).
  • the Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD (e.g., IgG).
  • the Fc domain can be an IgG subtype (e.g., lgG1 , lgG2a, lgG2b, lgG3, or lgG4) (e.g., lgG1 ).
  • Fc domain does not include any portion of an immunoglobulin that can act as an antigen-recognition region, e.g., a variable domain or a complementarity determining region (CDR).
  • Fc domains in the conjugates as described herein can contain one or more changes from a wild-type Fc domain sequence (e.g., 1 -10, 1 -8, 1 -6, 1 -4 amino acid substitutions, additions, or deletions) that alter the interaction between an Fc domain and an Fc receptor. Examples of suitable changes are known in the art.
  • numbering of amino acid residues in the IgG or Fc domain is according to the EU numbering system for antibodies, also called the Kabat EU index, as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991
  • GC content refers to the percentage of guanine (G) and cytosine (C) in a nucleic acid sequence.
  • the formula for calculation of the GC content is (G+C) I (A+G+C+U) x 100% (for RNA) or (G+C) / (A+G+C+T) x 100% (for DNA).
  • uridine content refers to the percentage of uridine (U) in a nucleic acid sequence.
  • U the percentage of uridine
  • thymidine content refers to the percentage of thymidine (T) in a nucleic acid sequence.
  • the formula for calculation of the thymidine content is T / (A+G+C+T) x 100%.
  • 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.
  • the term “intron fragment” refers to a portion of an intron, where a first intron fragment and a second intron fragment together form an intron, such as a catalytic intron.
  • An intron fragment may be a 5’ portion of an intron (e.g., a 5’ portion of a catalytic intron) or a 3’ portion of an intron (e.g., a 3’ portion of a catalytic intron), such that the 5’ intron fragment and the 3’ intron fragment, together, form a functional intron, such as a functional intron capable of catalytic self-splicing.
  • the term intron fragment is meant to refer to an intron split into two portions.
  • the term intron fragment is not meant to state, imply, or suggest that the two portion or halves are equal in length.
  • the term intron fragment is used synonymously with the term split-intron and may be used instead of the term “half-intron.”
  • 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 through, for example, splint ligation, or chemical, enzymatic, ribozyme- or splicing- catalyzed circularization methods.
  • 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.
  • RNA As used herein, the terms “nicked RNA,” “nicked linear polyribonucleotide,” and “nicked linear polyribonucleotide molecule” are used interchangeably and mean a polyribonucleotide molecule having a 5’ and 3’ end that results from nicking or degradation of a circular RNA.
  • 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.
  • a polyribonucleotide sequence that recites thymine (T) is understood to represent uracil (U).
  • 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 and polyA sequence refer to an untranslated, contiguous region of a nucleic acid molecule of at least 5 nucleotides in length and consisting of adenosine residues.
  • a polyA sequence 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 sequence is located 3’ to (e.g., downstream of) an open reason frame (e.g., an open reading frame encoding a polypeptide), and the polyA sequence is 3’ to a termination element (e.g., a stop codon) such that the polyA is not translated.
  • a polyA sequence is located 3’ to a termination element and a 3’ untranslated region.
  • the elements of a nucleic acid are “operably connected” or “operably linked” 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 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. Embodiments of polynucleotides include isolated and purified DNA/RNA molecules, synthetic DNA/RNA molecules, and synthetic DNA/RNA analogs.
  • Embodiments of polynucleotides may include one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s), 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'-
  • 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 are 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 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 contain amine -modified groups, such as amino allyl 1-dUTP (aa-dUTP) and aminohexylacrylamide-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 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.
  • the term “prevent” means to reduce the likelihood of developing a disease, disorder, or condition (e.g., a viral infection, e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV), 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.
  • 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., between 10 and 45 amino acids in length, that is present at the N-terminus of a polypeptide sequence of a nascent protein which targets the polypeptide sequence to the secretory pathway.
  • 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, 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.
  • the term "subject" refers to an organism, such as an animal, plant, or microbe.
  • the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian).
  • the subject is a human.
  • the subject is a non-human mammal.
  • the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, bison, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit).
  • a non-human primate e.g., monkeys, apes
  • ungulate e.g., cattle, buffalo, bison, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys
  • carnivore e.g., dog, cat
  • rodent e.g., rat, mouse
  • lagomorph e.g., rabbit
  • the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).
  • avian taxa Galliformes e.g., chickens, turkeys, pheasants, quail
  • Anseriformes e.g., ducks, geese
  • Paleaognathae e.g., ostriches, emus
  • Columbiformes e.g., pigeons, doves
  • Psittaciformes e.g., par
  • the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc.
  • the subject is an invertebrate agricultural pest or an invertebrate that is parasitic on an invertebrate or vertebrate host.
  • the subject is a plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte.
  • the subject is a eukaryotic alga (unicellular or multicellular).
  • the subject is a plant of agricultural or horticultural importance, such as row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses.
  • an antifusogenic polypeptide refers to a polypeptide, such as a polypeptide of between 10 and 200 amino acids, which inhibits viral fusion-associated events such as viral entry or viral fusion.
  • An antifusogenic polypeptide includes, for example, a polypeptide of Table 1 .
  • An antifusogenic polypeptide includes a polypeptide as well as any biologically active fragments thereof (e.g., a fragment of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids).
  • An antifusogenic polypeptide includes, for example, a polypeptide that targets HIV, SARS-CoV-2, HCV, or RSV.
  • an antifusogenic polypeptide includes a polypeptide having at least 70%, e.g., at least 80%, e.g., at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -324.
  • An antifusogenic polypeptide also refers to a polynucleotide (e.g., polyribonucleotide, e.g., circular polyribonucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1 ) or a biologically active fragment thereof.
  • the terms “treat” and “treating” refer to a prophylactic or therapeutic treatment of a viral infection, e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV, 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 viral infection, stabilizing (i.e., not worsening) the state of the viral infection, or preventing the spread of the viral infection as compared to the state or the condition of the viral infection in the absence of the therapeutic treatment.
  • 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.
  • a "vector” means a piece of 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 drawing showing the protein domains or regions of various Coronavirus S proteins and sequences of the HR1 and HR2 regions.
  • FIG. 2 is a schematic drawing showing various antifusogenic polypeptides and sequences derived from the HR2 region of SARS CoV-2.
  • FIG. 3 is a schematic drawing showing exemplary embodiments of multi ORF antifusogenic polypeptide constructs.
  • FIGS. 4A and 4B are graphs showing inhibitory efficacy on fusion using Omicron and Delta pseudo viruses.
  • FIG. 4A shows % inhibition of Delta and Omicron using either HR2 Full length or HR2 Full length with a HiBiT tag.
  • FIG. 4B shows relative expression of the antifusogenic polypeptide as compared to a mock control.
  • FIG. 5 is a graph showing in vivo expression of HR2 full length antifusogenic polypeptides with and without HiBiT tag at 6 hours and 24 hours.
  • FIGS. 6A and 6B are graphs showing in vitro neutralization of pseudovirus of the Wuhan and Omicron strains of SARS CoV-2 using the HR2A polypeptide.
  • FIG. 6A shows neutralization of the Wuhan strain of SARS CoV-2 using HR2A.
  • FIG. 6B shows neutralization of the Omicron strain of SARS CoV-2 using HR2A.
  • FIGS. 7A and 7B are graphs showing the inhibition rate (%) of SARS CoV-2 Pseudovirus Omicron BA4 and BA.5 (FIG. 7A) or SARS CoV-1 Pseudovirus (FIG. 7B) strains.
  • FIGS. 8A-8D are graphs showing the inhibition rate (%) of SARS CoV-2 Pseudovirus using full length HR2 (HR2Complete).
  • FIG. 8A shows inhibition of Wuhan strain.
  • FIG. 8B shows inhibition of Omicron BA.4 and BA. 5 strain.
  • FIG. 8C shows inhibition of Omicron BA.1 strain.
  • FIG. 8D shows inhibition of SARS CoV-1 Pseudovirus.
  • FIG. 9 is a schematic drawing showing construct designs and sequences for various HIV antifusogenic polypeptides.
  • FIGS. 10A and 10B are a graph (FIG. 10A) and a table (FIG. 10B) showing expression of various HIV antifusogenic polypeptides from circular RNA.
  • FIGS. 11 A and 11B are graphs showing expression of various HIV antifusogenic polypeptides from circular RNA (FIG. 11 A) or plasmid DNA (FIG. 11 B)
  • FIG. 12 is a table showing expression of various HIV antifusogenic polypeptides from circular RNA.
  • the present invention features compositions containing a circular polyribonucleotide (circular RNA) encoding an antifusogenic polypeptide and methods of use thereof.
  • Circular polyribonucleotides described herein are particularly useful for delivering a polynucleotide cargo encoding an antifusogenic polypeptide to a target cell.
  • the circular polyribonucleotide may include a polyribonucleotide cargo encoding a polypeptide of Table 1 .
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of Table 1 .
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -324.
  • the circular polyribonucleotide may be produced from a precursor, such as a linear deoxyribonucleotide, a circular deoxyribonucleotide, or a circular polyribonucleotide.
  • a circular polyribonucleotide may include, for example, a splice junction joining a 5’ exon fragment and a 3’ exon fragment.
  • the linear RNA molecules described herein a polyribonucleotide encoding an antifusogenic polypeptide.
  • the linear RNA molecules include, from 5’ to 3’, (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo encoding an antifusogenic polypeptide (e.g., polyribonucleotide cargo encoding an IRES operably linked to an expression sequence encoding an antifusogenic polypeptide); (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment.
  • the catalytic intron fragments and splice sites may then allow the linear polyribonucleotide to self-splice, thus forming a circular polyribonucleotide encoding an antifusogenic polypeptide.
  • the circular polyribonucleotide can be formulated as a composition (e.g., a pharmaceutical composition) for administration to a subject, e.g., a human subject.
  • a pharmaceutical composition may be administered in one or more doses of the composition.
  • the composition may be administered to the subject to treat or prevent a viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV).
  • a viral infection e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV.
  • a circular polyribonucleotide is produced from a linear polyribonucleotide (e.g., by self-splicing compatible ends of the linear polyribonucleotide).
  • a linear polyribonucleotide is transcribed from a deoxyribonucleotide template (e.g., a vector, a linearized vector, or a cDNA).
  • the disclosure features deoxyribonucleotides, linear polyribonucleotides, and circular polyribonucleotides and compositions thereof useful in the production of circular polyribonucleotides encoding an antifusogenic polypeptide.
  • the present invention features a template deoxyribonucleotide for making a circular RNA as described herein.
  • the deoxyribonucleotide includes the following, operably linked in a 5’-to-3’ orientation: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo encoding an antifusogenic polypeptide; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment.
  • the deoxyribonucleotide includes further elements, e.g., outside of or between any of elements (A), (B), (C), (D), (E), (F), or (G).
  • any of the elements (A), (B), (C), (D), (E), (F), or (G) is separated from each other by a spacer sequence, as described herein.
  • the deoxyribonucleotide is, for example, a circular DNA vector, a linearized DNA vector, or a linear DNA (e.g., a cDNA, e.g., produced from a DNA vector).
  • the deoxyribonucleotide further includes an RNA polymerase promoter operably linked to a sequence encoding a linear RNA described herein.
  • the RNA polymerase promoter is heterologous to the sequence encoding the linear RNA.
  • the RNA polymerase promoter is a T7 promoter, a T6 promoter, a T4 promoter, a T3 promoter, an SP6 virus promoter, or an SP3 promoter.
  • the deoxyribonucleotide includes a multiple-cloning site (MCS).
  • MCS multiple-cloning site
  • the deoxyribonucleotide is used to produce circular RNA with the size range of about 100 to about 20,000 nucleotides.
  • the circular RNA is at least 100, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400, 1 ,500, 1 ,600 1 ,700, 1 ,800, 1 ,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000 nucleotides in size.
  • the circular RNA is no more than 20,000, 15,000 10,000, 9,000, 8,000, 7,000, 6,000, 5,000 or 4,000 nucleotides in size. Linear Polyribonucleotides
  • the present invention also features linear polyribonucleotides encoding an antifusogenic polypeptide.
  • the linear polyribonucleotide may be used to create a circular polyribonucleotide, e.g., by ligating or splicing (e.g., self-splicing) the linear polyribonucleotide to produce the circular polyribonucleotide.
  • the linear polyribonucleotide includes the following, operably linked in a 5’-to-3’ orientation: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo encoding an antifusogenic polypeptide; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment.
  • the linear polyribonucleotide includes further elements, e.g., outside of or between any of elements (A), (B), (C), (D), (E), (F), or (G). For example, any of elements (A), (B), (C), (D), (E), (F), or (G) may be separated by a spacer sequence, as described herein.
  • RNA encoding an antifusogenic polypeptide by performing transcription in a cell-free system (e.g., in vitro transcription) using a deoxyribonucleotide (e.g., a vector, linearized vector, or cDNA) encoding an antifusogenic polypeptide provided herein as a template (e.g., a vector, linearized vector, or cDNA provided herein with an RNA polymerase promoter positioned upstream of the region that codes for the linear RNA).
  • a deoxyribonucleotide e.g., a vector, linearized vector, or cDNA
  • a template e.g., a vector, linearized vector, or cDNA provided herein with an RNA polymerase promoter positioned upstream of the region that codes for the linear RNA.
  • a deoxyribonucleotide template is transcribed to a produce a linear RNA containing the components described herein.
  • the linear polyribonucleotide produces a splicing-compatible polyribonucleotide, which may be self-spliced in order to produce a circular polyribonucleotide.
  • the linear polyribonucleotide is from 50 to 20,000, 100 to 20,000, 200 to 20,000, 300 to 20,000 (e.g., 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400, 1 ,500, 1 ,600, 1 ,700, 1 ,800, 1 ,900, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11 ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) ribonucleotides in length.
  • the linear polyribonucleotide is, e.g., at least 500, at least 1 ,000, at least 2,000, at least 3,000, at least 4,000, or at least 5,000 ribonucleotides in length.
  • the invention features a circular polyribonucleotide including an expression sequence encoding an antifusogenic polypeptide.
  • the polyribonucleotide includes an IRES operably linked to an expression sequence encoding an antifusogenic polypeptide.
  • the circular polyribonucleotide may include a splice junction, e.g., joining a 5’ exon fragment and a 3’ exon fragment.
  • the circular polyribonucleotide may include any one or more of the elements described herein.
  • the circular polyribonucleotide includes any feature or any combination of features as disclosed in International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
  • the circular polynucleotide further includes a polyribonucleotide cargo.
  • the polyribonucleotide cargo includes an expression (or coding) sequence, a non-coding sequence, or a combination of an expression (coding) sequence and a non-coding sequence.
  • the polyribonucleotide cargo includes an expression (coding) sequence encoding a polypeptide.
  • the polyribonucleotide includes an IRES operably linked to an expression sequence encoding a polypeptide.
  • the IRES is located upstream of the expression sequence. In some embodiments, the IRES is located downstream of the expression sequence.
  • the circular polyribonucleotide further includes a spacer region between the IRES and the 3’ exon fragment or the 5’ exon fragment.
  • the spacer region may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length ribonucleotides in length.
  • the 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.
  • the spacer region includes a polyA sequence.
  • the spacer region includes a polyA-C sequence. In some embodiments, the spacer region includes a polyA-G sequence. In some embodiments, the spacer region includes a polyA-T sequence. In some embodiments, the spacer region includes a random sequence. In some embodiments, the first annealing region and the second annealing region are joined, thereby forming a circular polyribonucleotide.
  • the circular RNA is produced by a deoxyribonucleotide template or a linear RNA described herein. In some embodiments, the circular RNA is produced by any of the methods described herein.
  • the circular polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1 ,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides
  • the circular polyribonucleotide is between 500 nucleotides and 20,000 nucleotides, between 1 ,000 and 20,000 nucleotides, between 2,000 and 20,000 nucleotides, or between 5,000 and 20,000 nucleotides. In some embodiments, the circular polyribonucleotide is between 500 nucleotides and 10,000 nucleotides, between 1 ,000 and 10,000 nucleotides, between 2,000 and 10,000 nucleotides, or between 5,000 and 10,000 nucleotides.
  • the circular polyribonucleotide may include certain characteristics that distinguish it from linear RNA.
  • the circular polyribonucleotide is less susceptible to degradation by exonuclease as compared to linear RNA.
  • the circular polyribonucleotide is more stable than a linear RNA, especially when incubated in the presence of an exonuclease.
  • the increased stability of the circular polyribonucleotide compared with linear RNA makes circular polyribonucleotide more useful as a cell transforming reagent to produce polypeptides and can be stored more easily and for longer than linear RNA.
  • the stability of the circular polyribonucleotide treated with exonuclease can be tested using methods standard in art which determine whether RNA degradation has occurred (e.g., by gel electrophoresis). Moreover, unlike linear RNA, the circular polyribonucleotide is less susceptible to dephosphorylation when the circular polyribonucleotide is incubated with phosphatase, such as calf intestine phosphatase.
  • the circular polyribonucleotides described herein and compositions or pharmaceutical compositions thereof may be used in therapeutic and veterinary methods of dosing to produce a level of circular polyribonucleotide, a level of binding to a target, or a level of protein in a plurality of cells after providing the plurality with at least two doses of circular polyribonucleotide.
  • the circular polyribonucleotide is non-immunogenic in a mammal, e.g., a human.
  • the circular polyribonucleotide is capable of replicating or replicates in a cell from an aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof.
  • an aquaculture animal fish, crabs, shrimp, oysters etc.
  • a mammalian cell e.g., a cell from a pet or zoo animal (cats, dogs
  • the invention includes a cell that includes the circular polyribonucleotide described herein, wherein the cell is a cell from an aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (horses, cows, pigs, chickens etc.), a human cell, a cultured cell, a primary cell or a cell line, a stem cell, a progenitor cell, a differentiated cell, a germ cell, a cancer cell (e.g., tumorigenic, metastatic), a non-tumorigenic cell (normal cells), a fetal cell, an embryonic cell, an adult cell, a mitotic cell, a non-mitotic cell, or any combination thereof.
  • the cell is modified to include the
  • the circular polyribonucleotide includes sequences for expression products. In some embodiments, the circular polyribonucleotide includes a binding site for binding to a target. In some embodiments, the circular polyribonucleotide is provided to a plurality of cells via any a dosing regimen described herein. In some embodiments, the circular polyribonucleotide as described herein induces a response or response level in a subject. In some embodiments, the expression products encoded by the sequences included in the circular polyribonucleotide are expressed in one or more of cells in the plurality of cells.
  • the circular polyribonucleotide has a half-life of at least that of a linear counterpart, e.g., linear expression sequence, or linear polyribonucleotide. In some embodiments, the circular polyribonucleotide has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more.
  • the circular polyribonucleotide has a half-life or persistence in a cell for at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer.
  • the circular polyribonucleotide has a half-life or persistence in a cell for from about 1 hour to about 60 days, e.g., about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days.
  • the circular polyribonucleotide has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell post division. In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a dividing cell for at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer.
  • 10 minutes e.g., at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,
  • the circular polyribonucleotide has a half-life or persistence in a dividing cell of from about 10 minutes to about 60 days, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 60 days.
  • the circular polyribonucleotide modulates a cellular function, e.g., transiently, or long term.
  • the cellular function is stably altered, such as a modulation that persists for at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer.
  • the cellular function is stably altered, such as a modulation that persists for from about 1 hour to about 60 days, e.g., from about 1 hour to about 30 days, e.g., for at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 60 days.
  • polynucleotides e.g., circular polyribonucleotides
  • the polynucleotides described herein may include any one or more of the elements described herein and an expression sequence encoding an antifusogenic polypeptide.
  • the disclosure provides circular polyribonucleotides that encode at least one expression sequence encoding an antifusogenic polypeptide.
  • the antifusogenic polypeptide inhibits viral entry. In some embodiments, the antifusogenic polypeptide inhibits viral fusion.
  • the antifusogenic polypeptide is a polypeptide or a variant thereof including an amino acid sequence selected from a sequence of Table 1 .
  • the antifusogenic polypeptide is a polypeptide including a contiguous stretch of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids of a sequence in Table 1 .
  • the antifusogenic polypeptide is a polypeptide including a contiguous stretch of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the amino acids of a sequence in Table 1 .
  • the antifusogenic polypeptide is a polypeptide including a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of Table 1 .
  • the antifusogenic polypeptide is a variant of a sequence in Table 1 that includes no more than one, two, three, four, five, six, seven, eight, nine, or ten mutations (e.g., substitutions, deletions, or insertions).
  • the circular polyribonucleotide includes an expression sequence encoding more than one antifusogenic polypeptide.
  • the circular polyribonucleotide encoding more than one fusion protein reduces the chance for viral escape.
  • the antifusogenic polypeptide targets one or more (e.g., two, three, four, or five) viruses.
  • Viruses that may be targeted by the antifusogenic polypeptide include, but are not limited to, all strains of viruses listed in Table 1 .
  • the virus does not infect humans.
  • the virus infects humans.
  • the virus is human immunodeficiency virus (HIV) (e.g., the antifusogenic polypeptide inhibits viral entry of HIV).
  • HIV is HIV-1 .
  • the HIV is a strain of HIV-1 (e.g., HIV-1 HE, HIV-1 HUB, HIV-1 MN, HIV-1 NDK, HIV-1 NL4-3, HIV-1 RF, or HIV-1 SF2).
  • the HIV is HIV-2.
  • the HIV is a strain of HIV-2 (e.g., HIV-2EHO or HIV-2ROD).
  • the HIV is an HIV pseudovirion.
  • the antifusogenic polypeptide prevents HIV viral fusion by specifically binding to HIV glycoprotein 120 (gp120). In some embodiments, the antifusogenic polypeptide prevents binding of gp120 to the cluster of differentiation 4 (CD4) co-receptor. In some embodiments, the antifusogenic polypeptide reduces the affinity of gp120 for a co-receptor (e.g., C-C chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 4 (CXCR4)). In some embodiments, the antifusogenic polypeptide prevents binding of gp120 to a co-receptor (e.g., CCR5 and CXCR4).
  • a co-receptor e.g., CCR5 and CXCR4
  • the antifusogenic polypeptide prevents HIV viral fusion by specifically binding to HIV glycoprotein 41 (gp41 ). In some embodiments, the antifusogenic polypeptide inhibits entry of the HIV viral core into the cell.
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide (e.g., a polypeptide that inhibits viral entry of HIV) of any one of SEQ ID NOs: 1 , 5, 11 , 13-18, 22-38, 40-46, 49-56, 58, 59, 61 , 66-78, 80-85, 87-91 , 95-100, 112, 113, 153, 163, 64, 168, 169, 171 -184, 211 -215, 242, 243, 247, 255, 256, 258, 261 , 261 , 265, 267, 271 , 272, 274, 276, 286, 287, or 312-324.
  • a polypeptide e.g., a polypeptide
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of any one of SEQ ID NOs: 1 , 5, 11 , 13-18, 22-38, 40-46, 49-56, 58, 59, 61 , 66-78, 80-85, 87-91 , 95-100, 112, 113, 153, 163, 64, 168, 169, 171 -184, 211 -215, 242, 243, 247, 255, 256, 258, 261 , 261 , 265, 267, 271 , 272, 274, 276, 286, 287, or 312-324.
  • the virus is hepatitis virus (e.g., the antifusogenic polypeptide inhibits viral entry of a hepatitis virus).
  • the hepatitis virus is hepatitis A virus (HAV).
  • the hepatitis virus is hepatitis B virus (HBV).
  • the hepatitis virus is hepatitis C virus (HCV).
  • the hepatitis virus is hepatitis D virus (HDV).
  • the hepatitis virus is hepatitis E virus (HEV).
  • the hepatitis virus is duck hepatitis virus (DHV).
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide (e.g., a polypeptide that inhibits viral entry of a hepatitis virus such as HCV) of any one of SEQ ID NOs: 104, 109, 112, 113, 141 -145, 158-160, or 284.
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of any one of SEQ ID NOs: 104, 109, 112, 113, 141 -145, 158-160, or 284.
  • the virus is a coronavirus, such as a betacoronavirus (e.g., the antifusogenic polypeptide inhibits viral entry of a coronavirus).
  • the betacoronavirus is SARS-CoV type-1 (SARS-CoV-1 ).
  • the betacoronavirus is SARS-CoV type 2 (SARS-CoV-2).
  • the betacoronavirus is a pseudotyped virus expressing the SARS-CoV-2 spike protein.
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide (e.g., a polypeptide that inhibits viral entry of a betacoronavirus such as SARS-CoV-1 or SARS-CoV-2) of any one of SEQ ID NOs: 6-9, 119-123, 165- 167, or 288-311 .
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of any one of SEQ ID NOs: 6-9, 119-123, 165-167, or 288-311 .
  • the virus is respiratory syncytial virus (RSV) (e.g., the antifusogenic polypeptide inhibits viral entry of RSV).
  • RSV respiratory syncytial virus
  • the RSV is RSV subtype A (RSV A).
  • RSV is RSV subtype B (RSVB).
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide (e.g., a polypeptide that inhibits viral entry of RSV) of any one of SEQ ID NOs: 11 , 13, 39, 112, 113, 153, 185-188, 216-219, 244, 246, 247- 249, 251 , 252, 257, 259, 264, 266, 268, 270, 273, 282, or 285.
  • a polypeptide e.g., a polypeptide that inhibits viral entry of RSV
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of any one of SEQ ID NOs: 11 , 13, 39, 112, 113, 153, 185-188, 216-219, 244, 246, 247-249, 251 , 252, 257, 259, 264, 266, 268, 270, 273, 282, or 285.
  • influenza e.g., seasonal influenza, pandemic influenza, influenza A, influenza H1 N1 subtype, influenza B, influenza C, influenza D.
  • influenza is influenza A.
  • influenza B In some embodiments, influenza is influenza C.
  • influenza D In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding a polypeptide (e.g., a polypeptide that inhibits viral entry of influenza) of any one of SEQ ID NOs: 1 , 4, 11 , 245, 247, 253, 262, 269, 275, 277, 278, 279, 280, 281 , or 283.
  • the polyribonucleotide cargo includes an expression sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of any one of SEQ ID NOs: 1 , 4, 11 , 245, 247, 253, 262, 269, 275, 277, 278, 279, 280, 281 , or 283.
  • influenza virus e.g., the antifusogenic polypeptide inhibits viral entry of influenza.
  • influenza virus is influenza A virus (IAV).
  • influenza virus is influenza B virus (IBV).
  • influenza virus is an influenza virus expressing hemagglutinin (HA).
  • the virus is herpes simplex virus (HSV) (e.g., the antifusogenic polypeptide inhibits viral entry of HSV).
  • HSV herpes simplex virus
  • the HSV is HSV-1 .
  • the HSV is HSV-2.
  • the virus is human papilloma virus (HPV) (e.g., the antifusogenic polypeptide inhibits viral entry of HPV).
  • HPV human papilloma virus
  • the HPV is a high-risk HPV strain (e.g., HPV 16, HPV 18, HPV 31 , HPV 33, HPV 45, HPV 52, or HPV 58).
  • the HPV is a low-risk HPV strain (e.g., HPV 6, HPV 11 , HPV 42, HPV 43, HPV or 44).
  • the virus is any virus listed in Table 1 .
  • the GC content of a nucleic acid sequence encoding an antifusogenic polypeptide is at least 51% (e.g., at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%). In some embodiments, the GC content of a nucleic acid sequence encoding an antifusogenic polypeptide is at most 52%, 53%, 54%, 55%, 56%, 57%, 58% or 59%, or 60%. In some embodiments, the GC content of a nucleic acid sequence encoding an antifusogenic polypeptide is 51% to 60%, 52% to 60%, 53% to 60%, 54% to 60%, 55% to 60%, 52% to 58%, 53% to 58%.
  • the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding an antifusogenic polypeptide is more than 10% (e.g., more than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%).
  • the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding an antifusogenic polypeptide is at most 30% (e.g., at most 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%).
  • the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding an antifusogenic polypeptide is 20% to 28%, 21 % to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21 % to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21 % to 23%, or 22% to 23%.
  • the GC content of an expression sequence encoding the antifusogenic polypeptide refers to the GC content of the expression sequence that exclusively encodes the antifusogenic polypeptide with no other coding regions that encode polypeptides other than the antifusogenic polypeptide.
  • the uridine content or thymidine of an expression sequence encoding the antifusogenic polypeptide refers to the uridine content of the expression sequence that exclusively encodes the antifusogenic polypeptide with no other coding regions that encode polypeptides other than the antifusogenic polypeptide.
  • the calculation of the GC content or the uridine (or thymidine) content of the expression sequence encoding the antifusogenic polypeptide only takes into account the continuous nucleic acid sequence that starts in a 5’ to 3’ direction from the first nucleoside of the start codon of the open reading frame that encodes the antifusogenic polypeptide to the last nucleoside of the stop codon of the same open reading frame.
  • the calculation of the GC content or the uridine (or thymidine) content of the expression sequence encoding the antifusogenic polypeptide only takes into account the continuous nucleic acid sequence that starts in a 5’ to 3’ direction from the first nucleoside of the codon that encodes the N-terminal end amino acid residue of the antifusogenic polypeptide to the last nucleoside of the codon that encodes the C-terminal end amino acid residue of the antifusogenic polypeptide.
  • the nucleic acid sequence encoding the antifusogenic polypeptide has a uridine content of more than 20%. In some embodiments, the uridine content of a nucleic acid sequence encoding an antifusogenic polypeptide is more than 10% (e.g., more than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the uridine content of a nucleic acid sequence encoding an antifusogenic polypeptide is at most 30% (e.g., at most 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%).
  • the uridine content of a nucleic acid sequence encoding an antifusogenic polypeptide is 20% to 28%, 21% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21 % to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21% to 23%, or 22% to 23%.
  • the nucleic acid sequence encoding the antifusogenic polypeptide has a uridine content of 20% to 28%.
  • the circular polyribonucleotide encodes multiple expression sequences each encoding an antifusogenic polypeptide (e.g., two or more, such as 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, or 5 to 10 expression sequences).
  • an antifusogenic polypeptide e.g., two or more, such as 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, or 5 to 10 expression sequences.
  • the circular polyribonucleotide encodes two or more (e.g., 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, or 5 to 10) copies of the same antifusogenic polypeptide.
  • the circular polyribonucleotide encodes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different (e.g., sharing less than 100% sequence identity) antifusogenic polypeptides.
  • the two or more different antifusogenic polypeptides are each selected from an antifusogenic polypeptide of Table 1 .
  • the two or more different antifusogenic polypeptides each inhibit a different virus.
  • a circular polyribonucleotide may encode a first antifusogenic polypeptide that inhibits influenza and a second an antifusogenic polypeptide that inhibits RSV.
  • a circular polyribonucleotide may include a first antifusogenic polypeptide that inhibits influenza and a second antifusogenic polypeptide that inhibits SARS-CoV-2.
  • a circular polyribonucleotide may include a first antifusogenic polypeptide that inhibits HIV and a second antifusogenic polypeptide that inhibits SARS-CoV-2.
  • a circular polyribonucleotide may include a first antifusogenic polypeptide that inhibits HIV and a second antifusogenic polypeptide that inhibits HCV. Where either the first or second antifusogenic polypeptide inhibits a plurality of viruses, the first and second antifusogenic polypeptides may have different virus specificity.
  • a circular polyribonucleotide encodes two or more antifusogenic polypeptides
  • the antifusogenic polypeptides may be encoded in a single open reading frame or multiple open reading frames.
  • the disclosure provides a circular polyribonucleotide including an open reading frame (e.g., an open reading frame operably linked to an IRES) that includes two or more expression sequences, where each expression sequence encodes an antifusogenic polypeptide.
  • translation of the open reading frame produces a polypeptide fusion including the two or more antifusogenic polypeptides.
  • the antifusogenic polypeptides may be linked, e.g., by a linker described herein (e.g., a peptide linker encoded by the open reading frame, such as a glycine-serine linker described below with respect to peptide-Fc fusions).
  • the antifusogenic polypeptides may be separated by a cleavage domain (e.g., a stagger sequence), for example as described herein.
  • the disclosure provides a circular polyribonucleotide including a first open reading frame encoding a first antifusogenic polypeptide (e.g., operably linked to a first IRES) and a second open reading frame encoding a second antifusogenic polypeptide (e.g., operably linked to a second IRES).
  • a first antifusogenic polypeptide e.g., operably linked to a first IRES
  • a second open reading frame encoding a second antifusogenic polypeptide (e.g., operably linked to a second IRES).
  • the circular polyribonucleotide includes an expression sequence encoding a fusion protein including an antifusogenic polypeptide.
  • the fusion protein includes an antifusogenic polypeptide fused to an Fc domain (e.g., a single chain of an Fc domain) of an immunoglobulin.
  • the antifusogenic polypeptide is selected from Table 1 .
  • the circular polyribonucleotide includes an expression sequence encoding more than one fusion protein including an antifusogenic polypeptide.
  • the circular polyribonucleotide includes an expression sequence encoding more than one antifusogenic polypeptide.
  • the circular polyribonucleotide encoding more than one fusion protein reduces the chance for viral escape.
  • the Fc domain is an lgG4 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG 1 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an lgG2 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an lgG2a Fc domain or a fragment thereof. In some embodiments, the Fc domain is an lgG2b Fc domain or a fragment thereof. In some embodiments, the Fc domain is an lgG3 Fc domain or a fragment thereof.
  • the C-terminal amino acid residue of the antifusogenic polypeptide is fused to the N-terminal amino acid residue of the Fc domain, optionally via a peptide linker.
  • the N-terminal amino acid residue of the antifusogenic polypeptide is fused to the C- terminal amino acid residue of the Fc domain, optionally via a peptide linker.
  • the peptide linker between the antifusogenic polypeptide and the Fc domain includes at least two amino acid residues (e.g., 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 15, or at least 20 amino acid residues).
  • the peptide linker between the antifusogenic polypeptide and the Fc domain includes 2-200 amino acids residue (e.g., 2-200, 2-180, 2- 160, 2-140, 2-120, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 4-200, 5-200, 6-200, 7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200, 45-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, or 180-200 amino acids residues).
  • 2-200 amino acids residue e.g., 2-200, 2-180, 2- 160, 2-140, 2-120, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-45, 2-40, 2-3
  • the peptide linker consists of glycine (Gly) and serine (Ser) residues.
  • the peptide linker includes the amino acid sequence of any one of (GS)x, (GGS)x, (GGGGS)x, (GGSG)x, or (SGGG)x, wherein x is an integer from 1 to 50 (e.g., 1 -40, 1 -30, 1 -20, 1 -10, or 1 -5).
  • the peptide linker includes the amino acid sequence of any one of (GS)x, (GGS)x, (GGGGS)x, (GGSG)x, or (SGGG)x, wherein x is an integer from 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the peptide linker includes 6 to 36 amino acids. In some embodiments, the peptide linker includes 21 to 31 amino acids.
  • a polyribonucleotide cargo described herein includes any sequence including at least one polyribonucleotide.
  • the polyribonucleotide cargo includes an expression sequence, a non-coding sequence, or an expression sequence and a non-coding sequence.
  • the polyribonucleotide cargo includes an expression sequence encoding an antifusogenic polypeptide.
  • the polyribonucleotide cargo includes an IRES operably linked to an expression sequence encoding an antifusogenic polypeptide.
  • the polyribonucleotide cargo includes an expression sequence that encodes an antifusogenic polypeptide that has a biological effect on a subject.
  • a polyribonucleotide cargo may, for example, include at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1 ,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about
  • the polyribonucleotide cargo includes from 1 -20,000 nucleotides, 1 -10,000 nucleotides, 1 -5,000 nucleotides, 100-20,000 nucleotide, 100-10,000 nucleotides, 100-5,000 nucleotides, 500-20,000 nucleotides, 500-10,000 nucleotides, 500- 5,000 nucleotides, 1 ,000-20,000 nucleotides, 1 ,000-10,000 nucleotides, or 1 ,000-5,000 nucleotides.
  • the polyribonucleotide cargo includes one or multiple expression (or coding) sequences, wherein each expression (or coding) sequence encodes a polypeptide (e.g., an antifusogenic polypeptide).
  • the polyribonucleotide cargo includes one or multiple noncoding sequences.
  • the polyribonucleotide cargo consists entirely of non-coding sequence(s).
  • the polyribonucleotide cargo includes a combination of expression (or coding) and noncoding sequences.
  • the polyribonucleotide includes any feature, or any combination of features as disclosed in International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
  • the polyribonucleotide described herein e.g., the polyribonucleotide cargo of the circular polyribonucleotide
  • the polyribonucleotide described herein includes one or more expression (or coding) sequences, wherein each expression sequence encodes an antifusogenic polypeptide.
  • the circular polyribonucleotide includes two, three, four, five, six, seven, eight, nine, ten or more expression (or coding) sequences.
  • Each encoded polypeptide may be linear or branched.
  • the polypeptide has a length from about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1 ,000 to about 2,500 amino acids, or any range therebetween.
  • the polypeptide has a length of less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1 ,500 amino acids, less than about 1 ,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less may be useful.
  • Polypeptides included herein may include naturally occurring polypeptides or non-naturally occurring polypeptides.
  • the polypeptide is or includes a functional fragment or variant of a reference polypeptide (e.g., a biologically active fragment or variant of an antifusogenic polypeptide).
  • the polypeptide may be a functionally active variant of any of the polypeptides described herein with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a polypeptide described herein or a naturally occurring polypeptide.
  • the polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to a protein of interest.
  • polypeptides include multiple polypeptides, e.g., multiple copies of one polypeptide sequence, or multiple different polypeptide sequences. In embodiments, multiple polypeptides are connected by linker amino acids or spacer amino acids.
  • the polynucleotide cargo includes a sequence encoding a signal peptide.
  • the Tat (Twin-arginine translocation) signal sequence is typically an N-terminal peptide sequence containing a consensus SRRxFLK “twin-arginine” motif, which serves to translocate a folded protein containing such a Tat signal peptide across a lipid bilayer.
  • Signal peptides are also useful for directing a protein to specific organelles; see, e.g., the experimentally determined and computationally predicted signal peptides disclosed in the Spdb signal peptide database, publicly available at proline. bic.nus.edu.sg/spdb.
  • the expression (or coding) sequence includes a poly-A sequence (e.g., at the 3’ end of an expression sequence).
  • the length of a poly-A sequence is greater than 10 nucleotides in length.
  • the poly-A sequence is greater than 15 nucleotides in length (e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, 1 ,100, 1 ,200, 1 ,300, 1 ,400, 1 ,500, 1 ,600, 1 ,700, 1 ,800, 1 ,900, 2,000, 2,500, and 3,000 nucleotides).
  • the poly- A sequence is designed according to the descriptions of the poly-A sequence in [0202]-[0204] of International Patent Publication No. WO2019/118919A1 , which is incorporated herein by reference in its entirety.
  • the expression sequence lacks a poly-A sequence (e.g., at the 3’ end of an expression sequence).
  • a circular polyribonucleotide includes a polyA, lacks a polyA, or has a modified polyA to modulate one or more characteristics of the circular polyribonucleotide.
  • the circular polyribonucleotide lacking a polyA or having modified polyA improves one or more functional characteristics, e.g., immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response), half-life, and/or expression efficiency.
  • a circular 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.
  • 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, and 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 (HRV-2), Homalodisca coagulata virus-1 , Human Immunodeficiency Virus type 1 , Homalodisca coagulata virus- 1 , Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 , Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis
  • the IRES is an IRES sequence of Coxsackievirus B3 (CVB3).
  • the IRES is an IRES sequence of Encephalomyocarditis virus.
  • the IRES is an IRES sequence of Theiler's encephalomyelitis virus.
  • the IRES sequence 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: TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGT ACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACA GTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGA CTGCTCACGCGGTTGAAGGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACAC CGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCA TTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCG GCTAAT
  • the IRES sequence is an Enterovirus 71 (EV17) IRES.
  • the terminal guanosine residue of the EV17 IRES sequence is modified to a cytosine residue.
  • the modified EV71 IRES may have the nucleic acid sequence of: ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCAT ATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGG GGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGG AAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCG ACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGT GCCACGTTGTGAGTTGGATAGTTGTGGA
  • 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 may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.
  • a polyribonucleotide described herein includes an IRES (e.g., an IRES operably linked to a coding region).
  • the polyribonucleotide may include any IRES as described in Chen et al. MOL. CELL 81 (20):4300-18, 2021 ; Jopling et al. ONCOGENE 20:2664-70, 2001 ; Baranick et al. PNAS 105(12):4733-38, 2008; Lang et al. MOLECULAR BIOLOGY OF THE CELL 13(5):1792- 1801 , 2002; Dorokhov et al. PNAS 99(8):5301 -06, 2002; Wang et al.
  • an antifusogenic 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 antifusogenic 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 antifusogenic polypeptide encoded by the circular polyribonucleotide does not include a secretion signal.
  • the signal sequence is selected from SecSP38 (MWWRLWWLLLLLLLLWPMVWA; SEQ ID NO: 327); SecD4 (MWWLLLLLLLLWPMVWA; SEQ ID NO: 328), gLuc (MGVKVLFALICIAVAEAK; SEQ ID NO: 329); INHC1 (MASRLTLLTLLLLLLAG DRASS; SEQ ID NO: 330); Epo (MGVHECPAWLWLLLSLLSLPLGLPVLG; SEQ ID NO: 331 ); and IL-2 (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 332).
  • a circular polyribonucleotide encodes multiple copies of the same antifusogenic polypeptide (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more).
  • at least one copy of the antifusogenic polypeptide includes a signal sequence and at least one copy of the antifusogenic polypeptide does not include a signal sequence.
  • a circular polyribonucleotide encodes plurality of an antifusogenic polypeptides (e.g., a plurality of different antifusogenic polypeptides or a plurality of an antifusogenic polypeptides having less than 100% sequence identity), where at least one of the plurality of an antifusogenic polypeptides includes a signal sequence and at least one copy of the plurality of an antifusogenic polypeptides does not include a signal sequence.
  • an antifusogenic polypeptides e.g., a plurality of different antifusogenic polypeptides or a plurality of an antifusogenic polypeptides having less than 100% sequence identity
  • the signal sequence is a wild-type signal sequence that is present on the N-terminus of the corresponding wild-type antifusogenic polypeptide, e.g., when expressed endogenously.
  • the signal sequence is heterologous to the antifusogenic polypeptide, e.g., is not present when the wild-type antifusogenic polypeptide is expressed endogenously.
  • a polyribonucleotide sequence encoding an antifusogenic 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 antifusogenic polypeptide to the secretory pathway.
  • the signal sequence may direct the antifusogenic polypeptide to reside in certain organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes).
  • the signal sequence directs the antifusogenic polypeptide to be secreted from the cell. For secreted proteins, the signal sequence may be cleaved after secretion, resulting in a mature protein.
  • the signal sequence may become embedded in the membrane of the cell or certain organelles, creating a transmembrane segment that anchors the protein to the membrane of the cell, endoplasmic reticulum, or Golgi apparatus.
  • the signal sequence of a transmembrane protein is a short sequence at the N-terminal of the polypeptide.
  • the first transmembrane domain acts as the first signal sequence, which targets the protein to the membrane.
  • the secretion signal is a human interleukin-2 (IL-2) secretion signal.
  • the IL-2 secretion signal has an amino acid sequence of at least 90% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 332).
  • the IL-2 secretion signal has an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 332.
  • the IL-2 secretion signal has an amino acid sequence of at least 99% sequence identity to SEQ ID NO: 332.
  • the IL-2 secretion signal has an amino acid sequence of 100% sequence identity to SEQ ID NO: 332.
  • the secretion signal is Gaussia luciferase secretion signal.
  • the Gaussia luciferase secretion signal has an amino acid sequence of at least 90% sequence identity of MGVKVLFALICIAVAEAK (SEQ ID NO: 329).
  • the Gaussia luciferase secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 329.
  • the Gaussia luciferase secretion signal has an amino acid sequence of at least 99% sequence identity of SEQ ID NO: 329.
  • the Gaussia luciferase secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 329.
  • the secretion signal is an EPO (e.g., a human EPO) secretion signal.
  • the EPO secretion signal has an amino acid sequence of at least 90% sequence identity of MGVHECPAWLWLLLSLLSLPLGLPVLGA (SEQ ID NO: 333).
  • the EPO secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 333.
  • the EPO secretion signal has an amino acid sequence of at least 99% sequence identity of SEQ ID NO: 333.
  • the EPO secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 333.
  • the secretion signal is a wildtype SARS-CoV-2 secretion signal.
  • the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of at least 90% sequence identity of MFVFLVLLPLVSS (SEQ ID NO: 334).
  • the wildtype SARS- CoV-2 secretion signal has an amino acid sequence of at least 95% sequence identity of SEQ ID NO: 334.
  • the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of at least 99% sequence identity of SEQ ID NO: 334.
  • the wildtype SARS-CoV-2 secretion signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 334.
  • an antifusogenic polypeptide encoded by a polyribonucleotide includes either a secretion signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence.
  • the polyribonucleotide described herein (e.g., the polyribonucleotide cargo 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 an amount 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.
  • 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: 336) (cis-acting hydrolase element) sequence.
  • the stagger element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP (SEQ ID NO: 337), where Xi is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid.
  • 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 des HDIETNPGP (SEQ ID NO: 351 ), HDVETNPGP (SEQ ID NO: 352), HDVEMNPGP (SEQ ID NO: 353), GDMESNPGP (SEQ ID NO: 354), GDVETNPGP (SEQ ID NO: 355), GDIEQNPGP (SEQ ID NO: 356), and DSEFNPGP (SEQ ID NO: 357).
  • 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. 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.
  • 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 intemucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage I to the phosphodiester backbone), and any combination thereof that can induce ribosomal pausing during translation.
  • Some of the exemplary modifications provided herein are described elsewhere herein.
  • 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.
  • 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.
  • a stagger element in some exemplary circular polyribonucleotides, 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 an antifusogenic polypeptides encoded by a circular ribonucleotide may be separated by an IRES between each antifusogenic polypeptide (e.g., each antifusogenic 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 antifusogenic polypeptides.
  • the IRES may be different between different antifusogenic polypeptides.
  • the plurality of an antifusogenic 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 antifusogenic polypeptide, a 2A, and a second antifusogenic polypeptide.
  • the plurality of an antifusogenic 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 antifusogenic polypeptide, a protease cleavage site (e.g., a furin cleavage site), and a second antifusogenic polypeptide.
  • the plurality of an antifusogenic polypeptides may be separated by a 2A self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage site).
  • a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first antifusogenic polypeptide, a 2A, a protease cleavage site (e.g., a furin cleavage site), and a second antifusogenic polypeptide.
  • a circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first antifusogenic polypeptide, a protease cleavage site (e.g., a furin cleavage site), a 2A, and a second antifusogenic 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).
  • an antifusogenic polypeptides encoded by the circular ribonucleotide may be separated by both IRES and 2A sequences.
  • an IRES may be between one antifusogenic polypeptide and a second antifusogenic polypeptide while a 2A peptide may be between the second antifusogenic polypeptide and the third antifusogenic polypeptide.
  • the selection of a particular IRES or 2A self-cleaving peptide may be used to control the expression level of an antifusogenic 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 between 2 and 10 cleavage sequences. In some embodiments, the circular polyribonucleotide includes between 2 and 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 antifusogenic polypeptides, e.g., where the two or more antifusogenic polypeptides are encoded by a single open-reading frame (ORF).
  • ORF open-reading frame
  • two or more antifusogenic 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 antifusogenic 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 antifusogenic polypeptides is cleaved upon expression, such that the two or more antifusogenic 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.
  • 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, IncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA.
  • 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, 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 sequence. Exemplary polyA sequences 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 sequence.
  • 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 sequence 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 sequence. 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. In some embodiments, the circular polyribonucleotide lacks a 5’ cap.
  • a circular polyribonucleotide includes one or more protein binding sites that allow a protein, e.g., a ribosome, to bind to an internal site in the RNA sequence.
  • a protein e.g., a ribosome
  • the circular polyribonucleotide may evade or have reduced detection by the host’s immune system, have modulated degradation, or modulated translation, by masking the circular polyribonucleotide from components of the host’s immune system.
  • a circular polyribonucleotide includes at least one immunoprotein binding site, for example to evade immune responses, e.g., CTL (cytotoxic T lymphocyte) responses.
  • the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and aids in masking the circular polyribonucleotide as exogenous.
  • the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and aids in hiding the circular polyribonucleotide as exogenous or foreign.
  • RNA binding to the capped 5' end of an RNA. From the 5' end, the ribosome migrates to an initiation codon, whereupon the first peptide bond is formed.
  • internal initiation i.e., cap-independent
  • a ribosome binds to a non-capped internal site, whereby the ribosome begins polypeptide elongation at an initiation codon.
  • the circular polyribonucleotide includes one or more RNA sequences including a ribosome binding site, e.g., an initiation codon.
  • Natural 5' UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ ID NO: 358), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5’ UTRs also have been known to form secondary structures which are involved in elongation factor binding.
  • a circular polyribonucleotide encodes a protein binding sequence that binds to a protein.
  • the protein binding sequence targets or localizes the circular polyribonucleotide to a specific target.
  • the protein binding sequence specifically binds an arginine-rich region of a protein.
  • the protein binding site includes, but is not limited to, a binding site to the protein such as ACIN1 , AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1 , CELF2, CPSF1 , CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21 , DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, ELAVL1 , ELAVL3, FAM120A, FBL, FIP1 L1 , FKBP4, FMR1 , FUS, FXR1 , FXR2, GNL3, GTF2F1 , HNRNPA1 , HNRNPA2B1 , HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1 , IGF2BP1 ,
  • the polyribonucleotide described herein includes one or more spacer sequences.
  • 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).
  • the polyribonucleotide further includes a first spacer region between the 5’ exon fragment of (C) and the polyribonucleotide cargo of (D).
  • the spacer may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length.
  • the polyribonucleotide further includes a second spacer region between the polyribonucleotide cargo of (D) and the 5’ exon fragment of (E).
  • 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. 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 sequence.
  • the first spacer region, the second spacer region, or the first spacer region and the second spacer region may include a 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 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-T 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.
  • Spacers may also be present within a nucleic acid region described herein.
  • a polynucleotide cargo region may include one or multiple spacers. Spacers may separate regions within the polynucleotide cargo.
  • 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 sequences, polyA-C sequences, polyC sequences, or poly- U sequences.
  • the spacer sequences can be polyA-T, polyA-C, polyA-G, or a random sequence.
  • the polyribonucleotide includes a 5’ spacer sequence (e.g., between the 5’ annealing region and the polyribonucleotide cargo).
  • the 5’ spacer sequence is at least 10 nucleotides in length.
  • the 5’ spacer sequence is at least 15 nucleotides in length.
  • the 5’ spacer sequence is at least 30 nucleotides in length.
  • the 5’ 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.
  • the 5’ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5’ spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the 5’ spacer sequence is
  • the 5’ spacer sequence is a polyA sequence. In another embodiment, the 5’ spacer sequence is a polyA-C sequence. In some embodiments, the 5’ spacer sequence includes a polyA-G sequence. In some embodiments, the 5’ spacer sequence includes a polyA-T sequence. In some embodiments, the 5’ spacer sequence includes a random sequence.
  • the polyribonucleotide includes a 3’ spacer sequence (e.g., between the 3’ annealing region and the polyribonucleotide cargo).
  • the 3’ spacer sequence is at least 10 nucleotides in length.
  • the 3’ spacer sequence is at least 15 nucleotides in length.
  • the 3’ spacer sequence is at least 30 nucleotides in length.
  • the 3’ 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.
  • the 3’ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 3’ spacer sequence is from 20 to 50 nucleotides in length. In certain embodiments, the 3’ spacer sequence is 10,
  • the 3’ spacer sequence is a polyA sequence.
  • the 5’ spacer sequence is a polyA-C sequence.
  • the 5’ spacer sequence includes a polyA-G sequence.
  • the 5’ spacer sequence includes a polyA-T sequence.
  • the 5’ spacer sequence includes a random sequence.
  • the polyribonucleotide includes a 5’ spacer sequence, but not a 3’ spacer sequence. In another embodiment, the polyribonucleotide includes a 3’ spacer sequence, but not a 5’ spacer sequence. In another embodiment, the polyribonucleotide includes neither a 5’ spacer sequence, nor a 3’ spacer sequence. In another embodiment, the polyribonucleotide does not include an IRES sequence. In a further embodiment, the polyribonucleotide does not include an IRES sequence, a 5’ spacer sequence or a 3’ spacer sequence.
  • the spacer sequence includes at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides, at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least 3 rib
  • 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 sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.).
  • the one or more post-transcriptional modifications can be any post- transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999).
  • the RNA Modification Database 1999 update.
  • the first isolated nucleic acid includes messenger RNA (mRNA).
  • the polyribonucleotide includes at least one nucleoside selected from the group such as those described in [0311] of International Patent Publication No. WO2019/118919A1 , which is incorporated herein by reference in its entirety.
  • a polyribonucleotide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage I to the phosphodiester backbone).
  • One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro).
  • modifications e.g., one or more modifications
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • a polyribonucleotide includes at least one N(6)methyladenosine (m6A) modification to increase translation efficiency.
  • the m6A modification can reduce immunogenicity (e.g., reduce the level of one or more marker of an immune or inflammatory response) of the 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'.
  • 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).
  • 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-(l- thiophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine (a- thio-cytidine), 5'-0-(l-thiophosphate)- guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0-(1 -thiophosphate)-pseudouridine).
  • alpha-thio-nucleoside e.g., 5'-0-(l- thiophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine (a- thio-cytidine), 5'-0-(l-thiophosphate)- guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0-(1 -thiophosphate)-pse
  • 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, l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5- fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5- fluoro-l-(tetrahydrofuran-2- yl)pyrimidine-2
  • Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinofuranosylcytosine, N4-octadecyl-1 -beta-D-arabinofuranosylcytosine, N4- palmitoyl-l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5'- elaidic acid ester).
  • a polyribonucleotide may or may not be uniformly modified along the entire length of the molecule.
  • one or more or all types of nucleotides e.g., naturally occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU
  • the 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.
  • inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.
  • nucleotides in a polyribonucleotide are modified.
  • the modification may include an m6A, which may augment expression; an inosine, which may attenuate an immune response; pseudouridine, which may increase RNA stability, or translational readthrough (stagger element), an m5C, which may increase stability; and a 2,2,7-trimethylguanosine, which aids subcellular translocation (e.g., nuclear localization).
  • nucleotide modifications may exist at various positions in a 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%
  • 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
  • a linear polyribonucleotide for circularization may be cyclized, or concatemerized. In some embodiments, the linear polyribonucleotide for circularization may be cyclized in vitro prior to formulation and/or delivery. In some embodiments, the circular polyribonucleotide may be in a mixture with linear polyribonucleotides. In some embodiments, the linear polyribonucleotides have the same nucleic acid sequence as the circular polyribonucleotides. In some embodiments, a linear polyribonucleotide for circularization is cyclized, or concatemerized using a chemical method to form a circular polyribonucleotide.
  • the 5'-end and the 3'-end of the nucleic acid includes chemically reactive groups that, when close together, may form a new covalent linkage between the 5'-end and the 3'-end of the molecule.
  • the 5'-end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino- terminated nucleotide on the 3'-end of a linear RNA molecule will undergo a nucleophilic attack on the 5'- NHS-ester moiety forming a new 5'73'-amide bond.
  • a DNA or RNA ligase is used to enzymatically link a 5'-phosphorylated nucleic acid molecule (e.g., a linear polyribonucleotide for circularization) to the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) forming a new phosphorodiester linkage.
  • a linear polyribonucleotide for circularization is incubated at 37°C for 1 hour with 1 -10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol.
  • the ligation reaction may occur in the presence of a linear nucleic acid capable of base-pairing with both the 5'- and 3'- region in juxtaposition to assist the enzymatic ligation reaction.
  • the ligation is splint ligation.
  • a splint ligase like SplintR® ligase, can be used for splint ligation, RNA ligase II, T4 RNA ligase, or T4 DNA ligase.
  • a single stranded polynucleotide like a single stranded 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 circular polyribonucleotide.
  • a DNA or RNA ligase is used in the synthesis of the circular polynucleotides.
  • either the 5'-or 3'-end of the linear polyribonucleotide for circularization can encode a ligase ribozyme sequence such that during in vitro transcription, the resultant linear polyribonucleotide for circularization includes an active ribozyme sequence capable of ligating the 5'-end of the linear polyribonucleotide for circularization to the 3'-end of the linear polyribonucleotide for circularization.
  • the ligase ribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
  • the ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.
  • a linear polyribonucleotide for circularization is cyclized or concatemerized 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 and/or near the 3' terminus of the linear polyribonucleotide for circularization in order to cyclize or concatemerized the linear polyribonucleotide for circularization.
  • the at least one non-nucleic acid moiety may be located in or linked to or near the 5' terminus and/or the 3' terminus of the linear polyribonucleotide for circularization.
  • the non-nucleic acid moieties contemplated may be homologous or heterologous.
  • the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage, and/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.
  • the linear polyribonucleotide for circularization is synthesized using IVT and an RNA polymerase, where the nucleotide mixture used for IVT may contain an excess of guanosine monophosphate relative to guanosine triphosphate to preferentially produce RNA with a 5’ monophosphate; the purified IVT product may be circularized using a splint DNA.
  • a linear polyribonucleotide for circularization is cyclized or concatemerized due to a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near or linked to the 5' and 3' ends of the linear polyribonucleotide for circularization.
  • one or more linear polyribonucleotides for circularization may be cyclized or concatemerized by intermolecular forces or intramolecular forces.
  • intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, Van der Waals forces, and London dispersion forces.
  • Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation and antibonding.
  • a linear polyribonucleotide for circularization may include a ribozyme RNA sequence near the 5' terminus and near the 3' terminus.
  • the ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme.
  • the peptides covalently linked to the ribozyme RNA sequence near the 5' terminus and the 3 'terminus may associate with each other causing a linear polyribonucleotide for circularization to cyclize or concatemerized.
  • 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 concatemerized 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 RNA of the present invention or a non-exhaustive listing of methods to incorporate and/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.
  • a linear polyribonucleotide for circularization may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase).
  • RppH RNA 5' pyrophosphohydrolase
  • apyrase an ATP diphosphohydrolase
  • the 5’ end of at least a portion of the linear polyribonucleotides includes a monophosphate moiety.
  • the population of polyribonucleotides including circular and linear polyribonucleotides is contacted with RppH prior to digesting at least a portion of the linear polyribonucleotides with a 5’ exonuclease and/or a 3’ exonuclease.
  • converting the 5' triphosphate of the linear polyribonucleotide for circularization into a 5' monophosphate may occur by a two-step reaction including: (a) contacting the 5' nucleotide of the linear polyribonucleotide for circularization with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphate.
  • a phosphatase e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase
  • a kinase e.g., Polynucleotide
  • circularization efficiency of the circularization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%.
  • the circularization method provided has a circularization efficiency of between about 10% and about 100%; for example, the circularization efficiency may be about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 99%.
  • the circularization efficiency is between about 20% and about 80%.
  • the circularization efficiency is between about 30% and about 60%.
  • the circularization efficiency is about 40%.
  • the circular polyribonucleotide includes an internal splicing element that when replicated the spliced ends are joined together.
  • Some examples may include miniature introns ( ⁇ 100 nt) with splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in (suptable4 enriched motifs) c/s-sequence elements proximal to back splice events such as sequences in the 200 bp preceding (upstream of) or following (downstream from) a back splice site with flanking exons.
  • the linear polyribonucleotide includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element.
  • the repetitive nucleotide sequence may include repeated sequences from the Alu family of introns.
  • a splicing-related ribosome binding protein can regulate circular polyribonucleotide biogenesis (e.g., the Muscle blind and Quaking (QKI) splicing factors).
  • the linear polyribonucleotide may include canonical splice sites that flank head-to-tail junctions of the circular polyribonucleotide.
  • the linear polyribonucleotide may include a bulge-helix-bulge motif, including a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage occurs at a site in the bulge region, generating characteristic fragments with terminal 5'-hydroxyl group and 2', 3'-cyclic phosphate. Circularization proceeds by nucleophilic attack of the 5'-OH group onto the 2', 3'-cyclic phosphate of the same molecule forming a 3', 5'-phosphodiester bridge.
  • the linear polyribonucleotide may include a multimeric repeating RNA sequence that harbors a HPR element.
  • the HPR includes a 2',3'-cyclic phosphate and 5'-OH termini.
  • the HPR element self-processes the 5'- and 3'-ends of the linear linear polyribonucleotide, thereby ligating the ends together.
  • the linear polyribonucleotide may include a sequence that mediates self-ligation.
  • the linear polyribonucleotide may include a HDV sequence, e.g., HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUG CUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (Beeharry et al 2004) (SEQ ID NO: 359) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUUUUCUGUAAAGAGGAGACUGCUGGACUCGC CGCC (SEQ ID NO: 360), to self-ligate.
  • a HDV sequence e.g., HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUUUUCUGUAAAGAGGAGACUGCUGGACUCGC CGCC (SEQ ID NO: 360), to self-ligate.
  • the linear polyribonucleotide may include loop E sequence (e.g., in PSTVd) to self-ligate.
  • the linear polyribonucleotide may include a self-circularizing intron, e.g., a 5' and 3’ slice junction, or a selfcircularizing catalytic intron such as a Group I, Group II or Group III Introns.
  • group I intron self-splicing sequences may include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena.
  • linear polyribonucleotides for circularization may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns. Repetitive nucleic acid sequence are sequences that occur within a segment of the linear polyribonucleotide.
  • the linear polyribonucleotide includes a repetitive nucleic acid sequence.
  • the repetitive nucleotide sequence includes poly CA or poly UG sequences.
  • the linear polyribonucleotide includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, the linear polyribonucleotide includes between 1 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, and 10) repetitive nucleic acid sequences that hybridize to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand.
  • the linear polyribonucleotide includes 2 repetitive nucleic acid sequences that hybridize to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand.
  • repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate linear polyribonucleotides hybridize to generate a single circularized polyribonucleotide, with the hybridized segments forming internal double strands.
  • the complementary sequences are found at the 5’ and 3’ ends of the linear polyribonucleotides for circularization.
  • the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.
  • 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.
  • enzymatic methods of circularization may be used to generate the circular polyribonucleotide.
  • a ligation enzyme e.g., DNA or RNA ligase, may be used to generate a template of the circular polyribonucleotide or complement, a complementary strand of the circular polyribonucleotide, or the circular polyribonucleotide.
  • Circularization of the linear polyribonucleotide may be accomplished by methods known in the art, for example, those described in “RNA circularization strategies in vivo and in vitro” by Petkovic and Muller from Nucleic Acids Res, 2015, 43(4): 2454-2465, and “In vitro circularization of RNA” by Muller and Appel, from RNA Biol, 2017, 14(8):1018-1027.
  • the circular polyribonucleotide may encode a sequence and/or motif useful for replication.
  • Exemplary replication elements are described in paragraphs [0280] - [0286] of International Patent Publication No. WO2019/118919, which is hereby incorporated by reference in its entirety.
  • linear polyribonucleotides may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns. Repetitive nucleic acid sequence are sequences that occur within a segment of the circular polyribonucleotide.
  • the linear polyribonucleotide includes a repetitive nucleic acid sequence.
  • the repetitive nucleotide sequence includes poly CA or poly UG sequences.
  • the linear polyribonucleotide includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand.
  • repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate linear polyribonucleotides hybridize to generate a single circularized polyribonucleotide, with the hybridized segments forming internal double strands.
  • the complementary sequences are found at the 5’ and 3’ ends of the linear polyribonucleotides.
  • the complementary sequences include about 3, 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.
  • 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.
  • RNA RNA sequence complementary metal-oxide-semiconductor
  • a deoxyribonucleotide template may be transcribed in a cell-free system (e.g., by in vitro transcription) to a produce a linear RNA.
  • the linear polyribonucleotide produces a splicing-compatible polyribonucleotide, which may be self-spliced to produce a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide (e.g., in a cell-free system) by providing a linear polyribonucleotide; and self-splicing linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide; thereby producing a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding the linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide, wherein the transcribing occurs in a solution under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.
  • the linear polyribonucleotide comprises a 5’ split-intron and a 3’ split-intron (e.g., a self-splicing construct for producing a circular polyribonucleotide). In some embodiments, the linear polyribonucleotide comprises a 5’ annealing region and a 3’ annealing region.
  • Suitable conditions for in vitro transcriptions and or self-splicing may include any conditions (e.g., a solution or a buffer, such as an aqueous buffer or solution) that mimic physiological conditions in one or more respects.
  • suitable conditions include between 0.1 -100mM Mg2+ ions or a salt thereof (e.g., 1 -1 OOmM, 1 -50mM, 1 -20mM, 5- 50mM, 5-20 mM, or 5-15mM).
  • suitable conditions include between 1 -1 OOOmM K+ ions or a salt thereof such as KCI (e.g., 1 -1 OOOmM, 1 - 500mM, 1 -200mM, 50- 500mM, 100-500mM, or 100-300mM).
  • suitable conditions include between 1 -1 OOOmM Cl- ions or a salt thereof such as KCI (e.g., 1 -1 OOOmM, 1 -500mM, 1 -200mM, 50- 500mM, 100-500mM, or 100-300mM).
  • suitable conditions include between 0.1 -1 OOmM Mn2+ ions or a salt thereof such as MnCI2 (e.g., 0.1 -1 OOmM, 0.1 -50mM, 0.1 -20mM, 0.1 - 10mM, 0.1 -5mM, 0.1 -2mM, 0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5-5mM, 0.5-2mM, or 0.1 -10mM).
  • MnCI2 e.g., 0.1 -1 OOmM, 0.1 -50mM, 0.1 -20mM, 0.1 - 10mM, 0.1 -5mM, 0.1 -2mM, 0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5-5mM, 0.5-2mM, or 0.1 -10mM.
  • suitable conditions include dithiothreitol (DTT) (e.g., 1 -1000 pM, 1 -500 pM, 1 -200pM, 50- 500pM, 100-500pM, 100-300pM, 0.1 -1 OOmM, 0.1 -50mM, 0.1 -20mM, 0.1 -1 OmM, 0.1 -5mM, 0.1 -2mM, 0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5-5mM, 0.5-2mM, or 0.1 -10mM).
  • DTT dithiothreitol
  • suitable conditions include between 0.1 mM and 100mM ribonucleoside triphosphate (NTP) (e.g., 0.1 -100 mM, 0.1 -50mM, 0.1 -1 OmM, 1 - 10OmM, 1 -50mM, or 1 -1 OmM).
  • NTP ribonucleoside triphosphate
  • suitable conditions include a pH of 4 to 10 (e.g., pH of 5 to 9, pH of 6 to 9, or pH of 6.5 to 8.5).
  • suitable conditions include a temperature of 4°C to 50°C (e.g., 10°C to 40°C, 15 °C to 40°C, 20°C to 40°C, or 30°C to 40°C),
  • the linear polyribonucleotide is produced from a deoxyribonucleic acid, e.g., a deoxyribonucleic acid described herein, such as a DNA vector, a linearized DNA vector, or a cDNA.
  • the linear polyribonucleotide is transcribed from the deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).
  • the disclosure also provides methods of producing a circular RNA in a cell, e.g., a prokaryotic cell or a eukaryotic cell.
  • an exogenous polyribonucleotide is provided to a cell (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding for the transcription of a linear polyribonucleotide described here).
  • the linear polyribonucleotides may be transcribed in the cell from an exogenous DNA molecule provided to the cell.
  • the linear polyribonucleotide may be transcribed in the cell from an exogenous recombinant DNA molecule transiently provided to the cell.
  • the exogenous DNA molecule does not integrate into the cell’s genome.
  • the linear polyribonucleotide is transcribed in the cell from a recombinant DNA molecule that is incorporated into the cell’s genome.
  • the cell is a prokaryotic cell.
  • the prokaryotic cell including the polyribonucleotides described herein may be a bacterial cell or an archaeal cell.
  • the prokaryotic cell including the polyribonucleotides described herein may be E coli, halophilic archaea (e.g., Haloferax volcaniii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira) spp., and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp.
  • the prokaryotic cells may be grown in a culture medium.
  • the prokaryotic cells may be contained in a bioreactor.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell including the polyribonucleotides described herein is a unicellular eukaryotic cell.
  • the unicellular eukaryotic is a unicellular fungal cell such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp, and Pichia spp.).
  • the unicellular eukaryotic cell is a unicellular animal cell.
  • a unicellular animal cell may be a cell isolated from a multicellular animal and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular animal cell may be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. A unicellular plant cell may be a cell isolated from a multicellular plant and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular plant cell may be dedifferentiated. In some embodiments, the unicellular plant cell is from a plant callus. In embodiments, the unicellular cell is a plant cell protoplast.
  • the unicellular eukaryotic cell is a unicellular eukaryotic algal cell, such as a unicellular green alga, a diatom, a euglenid, or a dinoflagellate.
  • Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris spp., Protosiphon botryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas spp.
  • the unicellular eukaryotic cell is a protist cell.
  • the unicellular eukaryotic cell is a protozoan cell.
  • the eukaryotic cell is a cell of a multicellular eukaryote.
  • the multicellular eukaryote may be selected from the group consisting of a vertebrate animal, an invertebrate animal, a multicellular fungus, a multicellular alga, and a multicellular plant.
  • the eukaryotic organism is a human.
  • the eukaryotic organism is a non-human vertebrate animal.
  • the eukaryotic organism is an invertebrate animal.
  • the eukaryotic organism is a multicellular fungus.
  • the eukaryotic organism is a multicellular plant.
  • the eukaryotic cell is a cell of a human or a cell of a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., bovids including cattle, buffalo, bison, sheep, goat, and musk ox; pig; camelids including camel, llama, and alpaca; deer, antelope; and equids including horse and donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare).
  • a non-human primate e.g., monkeys, apes
  • ungulate e.g., bovids including cattle, buffalo, bison, sheep, goat, and musk ox
  • pig camelids including camel, llama, and alpaca
  • the eukaryotic cell is a cell of a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).
  • avian taxa Galliformes e.g., chickens, turkeys, pheasants, quail
  • Anseriformes e.g., ducks, geese
  • Paleaognathae e.g., ostriches, emus
  • Columbiformes e.g., pigeons, doves
  • the eukaryotic cell is a cell of an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc.
  • the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte.
  • the eukaryotic cell is a cell of a eukaryotic multicellular alga.
  • the eukaryotic cells may be grown in a culture medium.
  • the eukaryotic cells may be contained in a bioreactor.
  • the linear polyribonucleotide is substantively enriched or pure (e.g., purified) prior to self-splicing the linear polyribonucleotide.
  • the linear polyribonucleotide is not purified prior to self-splicing the linear polyribonucleotide.
  • the resulting circular RNA is purified.
  • Purification may include separating or enriching the desired reaction product from one or more undesired components, such as any unreacted stating material, byproducts, enzymes, or other reaction components.
  • purification of linear polyribonucleotide following transcription in a cell-free system may include separation or enrichment from the DNA template prior to self-splicing the linear polyribonucleotide.
  • Purification of the circular RNA product following splicing may be used to separate or enrich the circular RNA from its corresponding linear RNA. Methods of purification of RNA are known to those of skill in the art and include enzymatic purification or by chromatography.
  • the methods of purification result in a circular polyribonucleotide that has less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) linear polyribonucleotides.
  • any method of producing a circular polyribonucleotide described herein may be performed in a bioreactor.
  • a bioreactor refers to any vessel in which a chemical or biological process is carried out which involves organisms or biochemically active substances derived from such organisms. Bioreactors may be compatible with the cell-free methods for production of circular RNA described herein.
  • a vessel for a bioreactor may include a culture flask, a dish, or a bag that may be single use (disposable), autoclavable, or sterilizable.
  • a bioreactor may be made of glass, or it may be polymer-based, or it may be made of other materials.
  • bioreactors include, without limitation, stirred tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibromixers, fluidized bed reactors, and membrane bioreactors.
  • the mode of operating the bioreactor may be a batch or continuous processes.
  • a bioreactor is continuous when the reagent and product streams are continuously being fed and withdrawn from the system.
  • a batch bioreactor may have a continuous recirculating flow, but no continuous feeding of reagents or product harvest.
  • Some methods of the present disclosure are directed to large-scale production of circular polyribonucleotides.
  • the method may be performed in a volume of 1 liter (L) to 50 L, or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more).
  • the method may be performed in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15
  • a bioreactor may produce at least 1g of circular RNA. In some embodiments, a bioreactor may produce 1 -200g of circular RNA (e.g., 1 -10g, 1 -20g, 1 -50g, 10-50g, 10-
  • the amount produced is measured per liter (e.g., 1 -200g per liter), per batch or reaction (e.g., 1 -200g per batch or reaction), or per unit time (e.g., 1 -200g per hour or per day).
  • more than one bioreactor may be utilized in series to increase the production capacity (e.g., one, two, three, four, five, six, seven, eight, or nine bioreactors may be used in series).
  • a circular polyribonucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1 ) is used for the treatment or prevention of a viral infection (e.g., HIV, SARS-CoV- 2, HCV, influenza, or RSV).
  • a viral infection e.g., HIV, SARS-CoV- 2, HCV, influenza, or RSV.
  • a circular polynucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1 ) is used for reducing viral entry.
  • a circular polynucleotide encoding an antifusogenic polypeptide may be administered to a subject to reduce the risk of a viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV).
  • a viral infection e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV.
  • a circular polyribonucleotide as described herein may be administered to a subject (e.g., in a pharmaceutical composition).
  • 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 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 viral infection 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 viral infection in a subject, e.g., in need thereof.
  • the method may be used to treat or prevent HIV.
  • the circular polyribonucleotide encodes an antifusogenic polypeptide that targets HIV, and the composition may be used to treat or prevent HIV.
  • the method may be used to treat or prevent SARS-CoV-2.
  • the circular polyribonucleotide encodes an antifusogenic polypeptide that targets SARS-CoV-2, and the composition may be used to treat or prevent SARS-CoV-2.
  • the method may be used to treat or prevent HCV.
  • the circular polyribonucleotide encodes an antifusogenic polypeptide that targets HCV, and the composition may be used to treat or prevent HCV.
  • the method may be used to treat or prevent RSV.
  • the circular polyribonucleotide encodes an antifusogenic polypeptide that targets RSV, and the composition may be used to treat or prevent RSV.
  • a method of dosing to produce a level of circular polyribonucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1 ) or express a level of an antifusogenic polypeptide (e.g., a polypeptide of Table 1 ) 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 or express a level of an antifusogenic polypeptide e.g., a polypeptide of Table 1 , e.g., a polypeptide of any one of Tables 2-4
  • a subject e.g., a mammal, e.g., a human
  • the composition includes a circular polyribonucleotide encoding an antifusogenic polypeptide 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.
  • the composition containing a circular polyribonucleotide further includes a pharmaceutically acceptable carrier or excipient.
  • the circular polyribonucleotide encodes an antifusogenic polypeptide, which can be expressed in a cell, e.g., following administration.
  • 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,
  • 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.
  • 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/
  • multiple doses are provided to produce a level of the composition or express a level of the antifusogenic 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 antifusogenic 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 antifusogenic 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, 3,000 ng/mL, or more) of an antifus
  • a method of administering multiple doses of a composition of a nucleic acid molecule described herein includes providing two or more compositions over a period of time, to a cell, tissue or subject (e.g., a mammal).
  • 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, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical or veterinary composition).
  • a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • 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).
  • 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 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.
  • 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). 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.
  • 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. 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.
  • 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.
  • the nucleic acid molecule e.g., circular polyribonucleotide
  • 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.
  • the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding an antifusogenic 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 an antifusogenic 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 antifusogenic polypeptide.
  • the different compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding different antifusogenic 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 antifusogenic polypeptide e.g., a plasma antifusogenic polypeptide
  • a level of the antifusogenic 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 antifusogenic polypeptide e.g., a plasma antifusogenic polypeptide level
  • a level of the antifusogenic 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 antifusogenic 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 antifusogenic polypeptide e.g., a plasma antifusogenic polypeptide level
  • a level of the antifusogenic 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 antifusogenic polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).
  • a circular polyribonucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1 ) described herein may be included in pharmaceutical compositions with a carrier or without a carrier.
  • compositions described herein may be formulated for example including a carrier, such as a pharmaceutical carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to a subject in need thereof (e.g., a human or non-human agricultural or domestic animal, e.g., cattle, dog, cat, horse, poultry).
  • a carrier such as a pharmaceutical carrier and/or a polymeric carrier, e.g., a liposome
  • transfection e.g., lipid- mediated, cationic polymers, calcium phosphate, dendrimers
  • electroporation or other methods of membrane disruption e.g., nucleofection
  • viral delivery e.g., lentivirus, retrovirus, adenovirus, AAV
  • microinjection microprojectile bombardment (“gene gun”)
  • fugene direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.
  • circular polyribonucleotides may be delivered in a “naked” delivery formulation.
  • a naked delivery formulation delivers a circular polyribonucleotide to a cell without the aid of a carrier and without covalent modification of the circular polyribonucleotide or partial or complete encapsulation of the circular polyribonucleotide.
  • 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 and the circular polyribonucleotide is not partially or completely encapsulated.
  • a circular polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may be a polyribonucleotide that is not covalently bound to a moiety, such as a protein, small molecule, a particle, a polymer, or a biopolymer that aids in delivery to a cell.
  • circular polyribonucleotides may be delivered in a delivery formulation with protamine or a protamine salt (e.g., protamine sulfate).
  • a polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may not contain a modified phosphate group.
  • a polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may not contain phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.
  • a naked delivery formulation may be free of any or all of: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers.
  • a naked delivery formulation may be 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),
  • DOTAP
  • a naked delivery formulation may include a non-carrier excipient.
  • a noncarrier excipient may include an inactive ingredient that does not exhibit an active cell-penetrating effect.
  • a non-carrier excipient may include a buffer, for example PBS.
  • a non-carrier excipient may be 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 may include a diluent, such as a parenterally acceptable diluent.
  • a diluent e.g., a parenterally acceptable diluent
  • a diluent may be 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.
  • Examples of an isotonic agent include glycerin, mannitol, polyethylene glycol, prop
  • the formulation includes a cell-penetrating agent.
  • the formulation is a topical formulation and includes a cell-penetrating agent.
  • the cell-penetrating agent can include organic compounds such as alcohols having one or more hydroxyl function groups.
  • the cell-penetrating agent includes an alcohol such as, but not limited to, monohydric alcohols, polyhydric alcohols, unsaturated aliphatic alcohols, and alicyclic alcohols.
  • the cell-penetrating agent can include one or more of methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzyl alcohol, specially denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol, menthol, polyethylene glycols (PEG)-400, ethoxylated fatty acids, or hydroxyethylcellulose.
  • the cellpenetrating agent includes ethanol.
  • the cell-penetrating agents can include any cell-penetrating agent in any amount or in any formulation as described in WO 2020/180751 or WO 2020/180752, which are hereby incorporated by reference in their entirety.
  • the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein is in parenteral nucleic acid delivery system.
  • the parental nucleic acid delivery system may include the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein, and a parenterally acceptable diluent.
  • the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein in the parenteral nucleic acid delivery system is free of any carrier.
  • the disclosure is further directed to a host or host cell including the circular polyribonucleotide described herein.
  • the host or host cell is a vertebrate, mammal (e.g., human), or other organism or cell.
  • the circular polyribonucleotide has a decreased, or fails to produce a, undesired response by the host’s immune system as compared to the response triggered by a reference compound, e.g., a linear polynucleotide corresponding to the described circular polyribonucleotide.
  • a reference compound e.g., a linear polynucleotide corresponding to the described circular polyribonucleotide.
  • the circular polyribonucleotide is non-immunogenic in the host.
  • Some immune responses include, but are not limited to, humoral immune responses (e.g., production of immunogen-specific antibodies) and cell-mediated immune responses (e.g., lymphocyte proliferation).
  • a host or a host cell is contacted with (e.g., delivered to or administered to) the circular polyribonucleotide.
  • the host is a mammal, such as a human.
  • the amount of the circular polyribonucleotide or linear, expression product, or both in the host can be measured at any time after administration. In certain embodiments, a time course of host growth in a culture is determined. If the growth is increased or reduced in the presence of the circular polyribonucleotide or linear, the circular polyribonucleotide or expression product or both is identified as being effective in increasing or reducing the growth of the host.
  • a method of delivering a circular polyribonucleotide molecule as described herein to a cell, tissue, or subject includes administering the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein to the cell, tissue, or subject.
  • the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an ungulate cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is an immune cell. In some embodiments, the tissue is a connective tissue, a muscle tissue, a nervous tissue, or an epithelial tissue. In some embodiments, the tissue is an organ (e.g., liver, lung, spleen, kidney, etc.).
  • the method of delivering is an in vivo method.
  • a method of delivery of a circular polyribonucleotide as described herein includes parenterally administering to a subject in need thereof, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein to the subject in need thereof.
  • a method of delivering a circular polyribonucleotide to a cell or tissue of a subject includes administering parenterally to the cell or tissue the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein.
  • the circular polyribonucleotide is in an amount effective to elicit a biological response in the subject.
  • the circular polyribonucleotide is an amount effective to have a biological effect on the cell or tissue in the subject.
  • the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein includes a carrier.
  • the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein includes a diluent and is free of any carrier.
  • the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered parenterally.
  • the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, intralymphaticly, subcutaneously, or intramuscularly.
  • parenteral administration is intravenously, intramuscularly, ophthalmically, subcutaneously, intradermally or topically.
  • the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered intramuscularly. In some embodiments, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered subcutaneously. In some embodiments, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered topically. In some embodiments, the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered intratracheally.
  • the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product is administered by injection.
  • the administration can be systemic administration or local administration.
  • any of the methods of delivery as described herein are performed with a carrier. In some embodiments, any methods of delivery as described herein are performed without the aid of a carrier or cell penetrating agent.
  • the circular polyribonucleotide or a product translated from the circular polyribonucleotide is detected in the cell, tissue, or subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days after the administering step.
  • the presence of the circular polyribonucleotide or a product translated from the circular polyribonucleotide is evaluated in the cell, tissue, or subject before the administering step.
  • the presence of the circular polyribonucleotide or a product translated from the circular polyribonucleotide is evaluated in the cell, tissue, or subject after the administering step.
  • 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 gg/ ml, 10 gg/ml, 50 gg/ml, 100 gg/ml, 200 g/ml, 300 gg/ml, 400 gg/ml, 500 gg/ml, 600 gg/ml, 700 gg/ml, 800 gg/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.
  • protein contamination e.g., cell protein such as a host cell protein or protein process impurities
  • endotoxin e.g., mononucleotide molecules
  • a process-related impurity e.g., cell protein such as a host cell protein or protein process impurities
  • 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).
  • 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.
  • 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.
  • 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 between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and 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-1 ,2-ethanediyl) groups, e.g., octoxynol-9 (Triton X-100, or t- octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (le
  • the one or more detergents and/or surfactants can be present only at trace amounts.
  • the composition can include less than 1 mg/ml of each of octoxynol-10 and polysorbate 80.
  • Non-ionic surfactants can be used herein.
  • Surfactants can be classified by their “HLB” (hydrophile/lipophile balance). In some cases, surfactants have a HLB of at least 10, at least 15, and/or at least 16.
  • the polyribonucleotide can be present in either linear or circular form.
  • a composition 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.
  • 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 phtoglycogen 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
  • a naked delivery formulation includes a non-carrier excipient.
  • a non-carrier excipient includes an inactive ingredient that does not exhibit a cellpenetrating 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. 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
  • 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.
  • 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
  • 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).
  • 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.
  • 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.
  • 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.
  • the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs.
  • the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs.
  • 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 include phtoglycogen 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, aminoglycosidepolyamine, 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), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-di
  • 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.
  • Exosomes can also be used as drug delivery vehicles for a linear polyribonucleotide composition or preparation described herein.
  • 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.
  • 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 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 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.
  • 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., US71 15583; 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): 1 153-1 166; Rychak, J.J. et al., Adv Drug Deliv Rev. 2014 Jun; 72: 82-93.
  • 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.
  • 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.
  • the particle size may be optimized for depositions of the circular polyribonucleotide into draining lymph node cells.
  • compositions, methods, and delivery systems provided by the present disclosure may employ any suitable carrier or delivery modality described herein, including, in certain embodiments, lipid nanoparticles (LNPs).
  • Lipid nanoparticles include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of 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
  • 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
  • DAG P
  • sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009/127060 or US2010/0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.
  • the lipid particle includes an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol.
  • the amounts of these components can be varied independently and to achieve desired properties.
  • the lipid nanoparticle includes an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids.
  • the ratio of total lipid to nucleic acid can be varied as desired.
  • the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1 .
  • the lipid to nucleic acid ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1 :1 to about 25:1 , from about 10:1 to about 14:1 , from about 3:1 to about 15:1 , from about 4:1 to about 10:1 , from about 5:1 to about 9:1 , or about 6:1 to about 9:1 .
  • the amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher.
  • the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg/ml to about 30 mg/mL.
  • lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein includes,
  • an LNP including Formula (i) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (ii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (iii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • an LNP including Formula (v) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (vi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (viii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (ix) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • X 1 is O, NR 1 , or a direct bond
  • X 2 is C2-5 alkylene
  • R 1 is H or Me
  • R 3 is C1 -3 alkyl
  • R 2 is C1 -3 alkyl
  • X 1 is NR 1 , R 1 and R 2 taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R 2 taken together with R 3 and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring
  • Y 1 is C2-12 alkylene
  • Y 2 is selected from
  • R 4 is C1 -15 alkyl
  • Z 1 is C1 -6 alkylene or a direct bond
  • R 5 is C5-9 alkyl or C6-10 alkoxy
  • R 6 is C5-9 alkyl or C6-10 alkoxy
  • W is methylene or a direct bond
  • R 4 is linear C5 alkyl
  • Z 1 is C2 alkylene
  • Z 2 is absent
  • W is methylene
  • R 7 is H
  • R 5 and R 6 are not Cx alkoxy.
  • an LNP including Formula (xii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including Formula (xi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP includes a compound of Formula (xiii) and a compound of Formula
  • an LNP including Formula (xv) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • an LNP including a formulation of Formula (xvi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a lipid compound used to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein is made by one of the following reactions:
  • an LNP including Formula (xxi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • the LNP of Formula (xxi) is an LNP described by WO2021 1 13777 (e.g., a lipid of Formula (1 ) such as a lipid of Table 1 of WO2021 1 13777). wherein each n is independently an integer from 2-15; Li 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, alkyloxy
  • R2 is selected from a group consisting of:
  • an LNP including Formula (xxii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • the LNP of Formula (xxii) is an LNP described by WO2021 1 13777 (e.g., a lipid of Formula (2) such as a lipid of Table 2 of WO2021 1 13777). wherein each n is independently an integer from 1 -15;
  • R3 is selected from a group consisting of:
  • an LNP including Formula (xxiii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.
  • a polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • the LNP of Formula (xxiii) is an LNP described by WO2021 1 13777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of WO2021 1 13777).
  • X is selected from -O-, -S-, or -OC(O)-*, wherein * indicates the attachment point to R1 ;
  • R1 is selected from a group consisting of: and
  • R2 is selected from a group consisting of:
  • a composition described herein e.g., a nucleic acid (e.g., a circular polyribonucleotide, a linear polyribonucleotide) or a protein
  • an LNP that includes an ionizable lipid.
  • the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of 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, 1 1 , 12, 13, 14, 15, 16, 17-tetradecahydro-IH- cyclopenta[a]phenanthren-3-yl 3-(1 H-imidazol-4-yl)propanoate, e.g., Structure (I) from W02020/106946 (incorporated by reference herein in its entirety).
  • ICE Imidazole cholesterol ester
  • an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine- containing lipid that can be readily protonated.
  • the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.
  • Exemplary cationic lipids include one or more amine group(s) which bear the positive charge.
  • the lipid particle includes a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids.
  • the cationic lipid may be an ionizable cationic lipid.
  • An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0.
  • a lipid nanoparticle may include a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid.
  • a lipid nanoparticle may include between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA (e.g., a circular polyribonucleotide, a linear polyribonucleotide)) described herein, encapsulated within or associated with the lipid nanoparticle.
  • a nucleic acid e.g., RNA (e.g., a circular polyribonucleotide, a linear polyribonucleotide)
  • 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 US2016/031 1759; I of US201503761 15 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or HA of US20170210967; l-c of US20150140070; A of US2013/0178541 ; I of US2013/0303587 or US2013/0123338; I of US2015/0141678; II, III, IV, or V of US2015/0239926; I of US2017/01 19904; I or II of WO2017/1 17528; A of US2012/0149894; A of US2015/0057373; A of WO2013/1 16126; A of US2013/0090372; A of US2013
  • the ionizable lipid is MC3 (6Z,9Z,28Z,3 IZ)-heptatriaconta- 6,9,28,3 I- 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-13, 16-dien-l-amine (Compound 32), e.g., as described in Example 1 1 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), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DO
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, 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).
  • 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.
  • the noncationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , or 8:1).
  • the lipid nanoparticles do not include any phospholipids.
  • the lipid nanoparticle can further include a component, such as a sterol, to provide membrane integrity.
  • a component such as a sterol
  • a sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof.
  • cholesterol derivatives include polar analogues such as 5a-cholestanol, 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)-butyl ether.
  • exemplary cholesterol derivatives are described in PCT publication W02009/127060 and US patent publication US2010/0130588, each of which is incorporated herein by reference in its entirety.
  • the component providing membrane integrity such as a sterol
  • such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
  • the lipid nanoparticle can include a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization.
  • PEG polyethylene glycol
  • exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof.
  • the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)- conjugated lipid.
  • PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 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- methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero
  • PEG-lipid conjugates are described, for example, in US5,885,613, US6,287,591 , US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, US2018/0028664, and WO2017/099823, the contents of all of which are incorporated herein by reference in their entirety.
  • a PEG-lipid is a compound of Formula III, lll-a-l, lll-a-2, lll-b-1 , lll-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety.
  • a PEG-lipid is of Formula II of US20150376115 or US2016/0376224, the content of both of which is incorporated herein by reference in its entirety.
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG- distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG- dipalmitoylglycerol, PEG- disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1 -[8'-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG- DMB (3,4- Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1 ,2- dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glyco
  • the PEG-lipid includes PEG-DMG, 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid includes a structure selected from:
  • lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid.
  • PEG-lipid conjugates 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.
  • POZ polyoxazoline
  • GPL cationic-polymer 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. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar ratios of the ionizable lipid, non- cationic-lipid, sterol, and PEG-conjugated lipid can be varied as needed.
  • the lipid particle can include 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non-cationic lipid by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition.
  • the composition includes 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10- 20% non-cationic-lipid by mole or by total weight of the composition.
  • the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic lipid, by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition.
  • the composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% 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
  • the lipid particle formulation includes ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5: 1 .5.
  • the lipid particle includes ionizable lipid, non-cationic lipid (e.g., phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non-cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.
  • non-cationic lipid e.g., phospholipid
  • a sterol e.g., cholesterol
  • PEG-ylated lipid e.g., PEG-ylated lipid
  • the lipid particle includes ionizable lipid I non-cationic- lipid / sterol I conjugated lipid at a molar ratio of 50:10:38.5:1 .5.
  • the disclosure provides a lipid nanoparticle formulation including phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.
  • 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.
  • 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.
  • 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- 9,12-dienoate) or another ionizable lipid.
  • lipids of WO2019/067992, WO/2017/173054, WO2015/095340, and WO2014/136086 as well as references provided therein.
  • the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.
  • the average LNP diameter of the LNP formulation may be between 10s of nm and 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.
  • DLS dynamic light scattering
  • 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.
  • 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 I 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 polydispersity index of a LNP may be from about 0.10 to about 0.20.
  • the zeta potential of an LNP may be used to indicate the electrokinetic potential of the composition.
  • 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.
  • 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.
  • lipids, formulations, methods, and characterization of LNPs are taught by W02020/061457 and WO2021/113777, 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 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 GenVoyJLM 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.
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane
  • DLin-MC3-DMA or MC3 dilinoleylmethyl-4-dimethylaminobutyrate
  • 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.
  • 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 e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • a portion e.g., an antigenic portion of a protein or polypeptide described herein
  • the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid
  • the LNPs have a mean particle size of between 80 nm and 160 nm
  • the polyribonucleotide e.g., a circular polyribonucleotide, a linear polyribonucleotide
  • the polyribonucelotide e.g., circular polyribonucleotide, linear polyribonucleotide
  • formulated in an LNP is a vaccine.
  • Exemplary dosing of polyribonucleotide (e.g., a circular polyribonucleotide, a linear 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 (e.g., a circular polyribonucleotide, a linear polyribonucleotide) antigenic composition described herein is between 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg.
  • the disclosure provides a kit.
  • the kit includes (a) a circular polyribonucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1 ) 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.
  • 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.
  • the informational material relates to methods for administering a dosage form of the pharmaceutical composition.
  • the informational material relates to methods for administering a dosage form of the circular polyribonucleotide.
  • 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.
  • 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.
  • a pharmaceutical composition or nucleic acid molecule e.g., a circular polyribonucleotide
  • 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.
  • inert conditions e.g., under Nitrogen or another inert gas such as Argon.
  • anhydrous conditions e.g., with a desiccant
  • 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.
  • 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
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the kit may include a scored tablet to allow the user to administered divided doses, as needed.
  • Example 1 Expression of antifusogenic polypeptides from RNA in mammalian cells
  • This example demonstrates expression of one or more open reading frames (ORFs) encoding one or more OC43-HR2P and EK1 peptides in Huh-7 cells.
  • ORFs open reading frames
  • Circular RNAs encoding one OC43-HR2P peptide (SEQ ID NO: 289), one EK1 peptide (SEQ ID NO: 288), multiple OC43-HR2P peptides, multiple EK1 peptides, and a combination of OC43-HR2P peptides, peptide analogs, and EK1 peptides are designed.
  • Circular RNAs are designed to include an IRES, a secretion signal, a furin site, one or more OC43-HR2P peptides, analogs, and/or EK1 sequences, and a spacer element.
  • the circular RNAs are transfected into Huh-7 cells using Lipofectamine MessengerMax (Invitrogen LMRNA001 ) according to the manufacturer’s instructions. In one study, peptide expression is monitored in vitro over a time course.
  • Example 2 Inhibition of MERS-CoV S protein-mediated cell-cell fusion
  • MERS-CoV S protein- mediated cell-cell fusion assay uses 293T cells that are transfected with the plasmid pAAV-IRES-EGFP encoding the EGFP (293T/EGFP) or pAAV-IRES-MERS-EGFP encoding the MERS-CoV S protein and EGFP (293T/MERS/EGFP) and cultured in DMEM containing 10% FBS at 37 °C for 48 h.
  • Huh-7 cells (5 x 10 4 ) expressing the MERS-CoV receptor DPP4, prepared according to Example 1 , are incubated in 96-well plates at 37 °C for 5 h, followed by the addition of 1 x 10 4 293T/EGFP or 293T/MERS/EGFP cells, respectively.
  • the 293T/MERS/EGFP cells (293T/EGFP cells are used as the negative control) fused or unfused with Huh-7 cells are counted under an inverted fluorescence microscope (Nikon Eclipse Ti-S).
  • the fused cell is seen as one that is 2-fold or more larger than the unfused cell, and the differences of intensity of fluorescence in the fused cell is compared to that of the unfused cell.
  • the percent inhibition of cell-cell fusion can be calculated using the following formula: (1 -(E-A/)/(P-A/)) x 100. ‘E represents the % cell-cell fusion in the experimental group.
  • ‘P represents the % cell-cell fusion in the positive control group, to which no circRNA was added. ‘/V is the % cell-cell fusion in negative control group, in which 293T/MERS/EGFP cells are replaced by 293T/EGFP cells.
  • the concentration for 50% inhibition (IC50) can be calculated using the CalcuSyn software.
  • Co-culture can continue at 37 °C for 48 h and measurements may be taken, for example of syncytium formation.
  • In-cell S protein ELISA can be adapted to measure antiviral activities two days following viral challenge.
  • Example 3 Inhibition of pseudotyped SARS-CoV-2 and MERS-CoV infection
  • SARS or MERS pseudovirus bearing SARS-CoV-2 or MERS-CoV S protein, respectively, and a defective HIV-1 genome that expresses luciferase as reporter are prepared by co-transfecting 293T cells with the plasmid pNL4-3.luc.RE (encoding Env-defective, luciferase-expressing HIV-1 ) and pcDNA3.1 - MERS-CoV-S plasmid.
  • ACE2-transfected 293T (293T/ACE2) cells and Huh-7 cells (10 4 per well in 96-well plates) that have and have not been transfected with circRNAs of the present invention are respectively infected with SARS or MERS-CoV pseudovirus.
  • the culture is re-fed with fresh medium 12 h post-infection and incubated for an additional 72 h.
  • Cells are washed with PBS and lysed using lysis reagent included in a luciferase kit (Promega).
  • This example demonstrates expression of SARS-CoV-2 antifusogenic polypeptides from circular RNAs.
  • FIG. 1 Several SARS-CoV-2 antifusogenic polypeptides were designed (FIGS. 2 and 3) based on the HR2 region show in FIG. 1.
  • Circular RNAs were designed to include an IRES and a nucleotide sequence encoding a SARS-CoV-2 antifusogenic polypeptide.
  • DNA constructs were designed to include a spacer element and a polynucleotide cargo.
  • the constructs were designed to include a combination of an IRES and an ORF as the polynucleotide cargo.
  • the ORF was designed to include an IL-2 secretion signal sequence, a nucleotide sequence encoding a SARS-CoV-2 antifusogenic polypeptide, and a nucleotide sequence encoding a HiBiT tag with a GGGGS peptide linker.
  • the IRES was EMCV.
  • LFKKIS (SEQ ID NO: 383) HR2B ATGTATAGAATGCAGCTTCTGTCTTGTATTGCTCTGTCTCTTGCTCTGGTTACTAATTCTaggctgagaag agttgttattggtattgttaataatactgtttacgatcctcttcagcctgaacttgattctttttaaggaagaactggataagtattttaaaatcacacttctcct gatggaggaggaggagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 384)
  • Circular RNAs were generated by self-splicing using a method described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from a DNA template including the motifs listed above in the presence of 7.5 mM of NTP. Template DNA was removed by treating with DNase. Synthesized linear RNA was purified with an RNA clean up kit (New England Biolabs, T2050). Self-splicing occurred during transcription. Circular RNAs encoding an antifusogenic peptide were purified by urea polyacrylamide gel electrophoresis (Urea-PAGE) or by reversed phase column chromatography.
  • Urea-PAGE polyacrylamide gel electrophoresis
  • This example demonstrates inhibition of pseudotyped SARS-CoV2- infection by antifusogenic polypeptides expressed from circular RNAs.
  • Circular RNAs were designed to include an internal ribosome entry site (IRES) and a nucleotide sequence encoding an anitfusogenic polypeptide of SARS-CoV-2.
  • DNA constructs were designed to include a spacer element and a combination of an EMCV IRES and an ORF as the polynucleotide cargo.
  • the ORF was designed to include an IL-2 secretion signal sequence and a nucleotide sequence encoding an HR2 full length antifusogenic polypeptide, and a nucleotide sequence encoding a HiBiT peptide tag.
  • An ORF was also designed to include an IL-2 secretion signal sequence and a nucleotide sequence encoding an HR2 full length antifusogenic polypeptide without a nucleotide sequence encoding a HiBiT peptide tag.
  • Circular RNAs were generated by self-splicing using a method described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from a DNA template including the motifs listed above in the presence of 7.5 mM of NTP. Template DNA was removed by treating with DNase. Synthesized linear RNA was purified with an RNA clean up kit (New England Biolabs, T2050). Self-splicing occurred during transcription. Circular RNAs encoding the HR2 full length antifusogenic polypeptide were purified by urea polyacrylamide gel electrophoresis (Urea-PAGE) or by reversed phase column chromatography.
  • Urea-PAGE polyacrylamide gel electrophoresis
  • ACE2-transfected 293T (293T/ACE2) cells (10 4 per well in 96-well plates) that have and have not been transfected with circular RNAs were respectively infected with SARS-CoV-2 pseudovirus.
  • Transfection reagent alone (with no circular RNA) was used as a control (“Mock”). Following infection, the culture was re-fed with fresh medium 12 hours post-infection and incubated for an additional 72 hours. Cells were washed with PBS and lysed using lysis reagent included in a luciferase kit (Promega).
  • RNAs formulated in lipid nanoparticles were delivered to mice via intravenous injection. Expression was measured by Nano- Glo® HiBiT Extracellular Detection System (#N3030, Promega) 10% Serum. The antifusogenic polypeptide was highly expressed at 6 hours and significantly decreased by 24 hours as shown in Table 3 and FIG. 5.
  • This example demonstrates pseudotyped SARS-CoV2- infection using antifusogenic polypeptides.
  • Antifusogenic polypeptides of SARS-CoV-2 were created based on the HR2, HR2A, HR2B, and HR2C regions of SARS-CoV-2 Spike polypeptide and the EK1 polypeptide (FIG. 2).
  • a functional assay was performed to measured pseudoviral neutralization by EK-1 and HR2A polypeptides in vitro.
  • HR2A polypeptides showed efficacy against Wuhan and Omicron strains (FIGS. 6A and 6B).
  • HR2 full length polypeptide (“HR2Complete”) was shown to successfully inhibit the fusion of Omicron BA.4/BA.5, SARS CoV-1 , and Wuhan strains (FIGS. 7A, 7B, and 8A-8D).
  • This example demonstrates expression of HIV antifusogenic polypeptides from circular RNAs.
  • Circular RNAs were designed to include an IRES and a nucleotide sequence encoding an HIV antifusogenic polypeptide.
  • DNA constructs were designed to include a spacer element and a polynucleotide cargo (FIG. 9). The constructs were designed to include a combination of an IRES and an ORF as the polynucleotide cargo.
  • the ORF was designed to include an IL-2 secretion signal sequence (SEQ ID NO: 332), a nucleotide sequence encoding an HIV antifusogenic polypeptide, and a nucleotide sequence encoding a HiBiT tag (having sequence VSGWRLFKKIS (SEQ ID NO: 362) with a GGS or GGGGS peptide linker.
  • the IRES was either EMCV or a modified CVB3. Circular RNAs were generated by self-splicing using a method described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from a DNA template including the motifs listed above in the presence of 7.5 mM of NTP. Template DNA was removed by treating with DNase.
  • RNA clean up kit New England Biolabs, T2050. Self-splicing occurred during transcription. Circular RNAs encoding an antifusogenic peptide were purified by urea polyacrylamide gel electrophoresis (Urea-PAGE) or by reversed phase column chromatography.
  • RNA was delivered to HEK293 cells using lipofectamine. Expression was measured after 48 hours. As shown in FIGS. 10A and 10B, the polypeptides were expressed in HEK293 cells. As shown in FIGS. 11 A and 11 B, expression was comparable between circular RNA and DNA plasmid. Total expression (ng/mL and nM) is shown in FIG. 12 for the various polypeptides.
  • WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 318) tggcaggagtgggaacagaagatcactgctctgctggaacaggctcagattcagcaggaaaagaacgaatacgaactgcagaagctggataa gtgggcttctctgtgggagtggttc (with EMCV IRES) (SEQ ID NO: 365)
  • TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALREL actacttgggaagcttgggatagagctatcgctgaatacgctgctagaattgaagctctgctgagagctctgcaggaacagcaggaaaagaacga agctgctctgagagaactg (with EMCV IRES) (SEQ ID NO: 367)
  • TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALRELDKWASLWNWF (SEQ ID NO: 317) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagcagcaagagaaga acgaggccgcctgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 369)
  • TTWQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 319) accacctggcaggagtgggagcagaagatcaccgccctgctggagcaggcccagatccagcaagagaagaacgagtacgagctgcagaag ctggacaagtgggccagcctgtgggagtggttc (with modified CVB3 IRES) (SEQ ID NO: 372)
  • TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELREL (SEQ ID NO: 323) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagccaggagcagcaagagaaga acgaggccgagctgcgggagctg (with modified CVB3 IRES) (SEQ ID NO: 373)
  • TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELRELDKWASLWNWF (SEQ ID NO: 324) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagccaggagcagcaagagaaga acgaggccgagctgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 374)
  • TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL (SEQ ID NO: 320) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaaga acgaggccgcactgcgggagctg (with modified CVB3 IRES) (SEQ ID NO: 375)
  • TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF (SEQ ID NO: 321 ) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaaga acgaggccgcactgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 376) 2635_3.0

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