JP2025500358A - Circular polyribonucleotides encoding antifusogenic polypeptides - Google Patents

Abstract

The present disclosure generally relates to compositions and methods for producing, purifying, and using circular RNAs encoding antifusogenic polypeptides.

Description

(Background Art)
The delivery of polynucleotides and proteins is important for a wide variety of therapeutic fields. However, current delivery modes are often ineffective. For example, the delivery of short polypeptides, such as polypeptides encoding antifusogenic polypeptides, often results in short half-life and rapid clearance of the polypeptide. Therefore, there is a need for improved compositions and methods for delivering antifusogenic polypeptides, for example, to treat or prevent viral infections.

The present disclosure provides compositions and methods for producing, purifying, and using circular RNAs encoding antifusogenic polypeptides.

In one aspect, the invention features a cyclic polyribonucleotide that includes a polyribonucleotide cargo that encodes an antifusogenic polypeptide. In some embodiments, the polyribonucleotide cargo includes an expression sequence that encodes an antifusogenic polypeptide.

In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo comprises an expressed 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 comprises an expressed 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.

In some embodiments, the circular polyribonucleotide comprises a splice junction joining the 5' exon fragment and the 3' exon fragment.

In some embodiments, the polyribonucleotide cargo comprises an IRES operably linked to an expression sequence encoding an antifusogenic polypeptide. The circular polyribonucleotide may further comprise 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. For example, 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. In some embodiments, the spacer region is 5-500 ribonucleotides in length. The spacer region may comprise a polyA, polyA-C, polyA-U, or polyA-G sequence. The spacer region may be a random sequence.

In some embodiments, the cyclic polyribonucleotide is at least 500 ribonucleotides in length. For example, the cyclic polyribonucleotide can 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. In some embodiments, the cyclic polyribonucleotide is 500-20,000 ribonucleotides in length.

In another aspect, the invention features a linear polyribonucleotide comprising, from 5' to 3', (A) a 3' 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' intron fragment.

In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding an antifusogenic polypeptide.

In some embodiments, the polyribonucleotide cargo comprises an IRES operably linked to an expression sequence encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1). The circular polyribonucleotide may further comprise a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The circular polyribonucleotide may further comprise a spacer region between one or more of (A), (B), (C), (D), (E), (F), and (G).

The spacer region can be at least 5 ribonucleotides in length. For example, the spacer region can 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. In some embodiments, the spacer region is 5-500 ribonucleotides in length. The spacer region can include poly A, poly A-C, poly A-U, or poly A-G sequences. The spacer region can be a random sequence.

In some embodiments, the circular polyribonucleotide lacks an IRES. In some embodiments, the circular polyribonucleotide lacks one or both of a 5' cap and a polyA sequence.

In some embodiments, the circular polyribonucleotide comprises a protein translation initiation site. In some embodiments, the protein translation initiation site comprises a Kozak sequence.

In some embodiments, the linear polyribonucleotide is at least 500 ribonucleotides in length. For example, the linear polyribonucleotide can 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. In some embodiments, the linear polyribonucleotide is 500-20,000 ribonucleotides in length.

Another aspect features a DNA vector encoding a polyribonucleotide (e.g., a linear or circular polyribonucleotide) described herein.

Another aspect features a method of expressing an antifusogenic polypeptide (e.g., a polypeptide of Table 1) in a cell. The method includes providing a circular polyribonucleotide, a linear polyribonucleotide, or a DNA vector described herein to the cell under conditions suitable for expressing the antifusogenic polypeptide.

Another aspect features a method of producing a circular polyribonucleotide from a linear polyribonucleotide described herein. The method includes providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to produce a circular polyribonucleotide.

In another aspect, the invention features a pharmaceutical composition that includes a circular polyribonucleotide, a linear polyribonucleotide, or a DNA vector of any of the above embodiments and a diluent, carrier, or excipient.

In another aspect, the invention features a method of expressing an antifusogenic polypeptide (e.g., a polypeptide of Table 1) in a subject. The method includes expressing an antifusogenic polypeptide in a subject 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). The method includes administering a first dose of the pharmaceutical composition in an amount sufficient to result in a serum concentration of an antifusogenic polypeptide (e.g., a polypeptide of Table 1) of 100ng/mL, 2,100ng/mL, 2,200ng/mL, 2,300ng/mL, 2,400ng/mL, 2,500ng/mL, 2,600ng/mL, 2,700ng/mL, 2,800ng/mL, 2,900ng/mL, 3,000ng/mL, or more.

In some embodiments, the method may further include administering a second dose of the pharmaceutical composition. The method may further include administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more doses of the pharmaceutical composition.

In some embodiments, the second dose is administered at least 1 hour (e.g., at least 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more) after the first dose of the pharmaceutical composition.

In some embodiments, the second dose is administered 1 hour to 1 year (e.g., 1 hour to 1 day, e.g., 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 1 day, e.g., 1 day to 1 week, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, e.g., 1 week to 1 month, e.g., 2 weeks, 3 weeks, or 1 month, e.g., 1 month to 1 year, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year) after the first dose of the pharmaceutical composition. In some embodiments, the second dose is administered within 1 day to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 6 days, days to 14 days, 6 to 7 days, 7 to 90 days, 7 to 45 days, 7 to 30 days, 7 to 14 days, 14 to 90 days, 14 to 45 days, 14 to 30 days, 21 to 90 days, 21 to 60 days, 21 to 45 days, 21 to 30 days, 30 to 90 days, 30 to 60 days, 30 to 45 days, 45 to 180 days, 45 to 120 days, 45 to 100 days, 45 to 90 days, 45 to 60 days, 60 to 180 days, 60 to 120 days, 60 to 100 days, 60 to 90 days, 90 to 100 days, 90 to 120 days, or 90 to 180 days).

In some embodiments, the third dose is administered at least 1 hour (e.g., at least 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more) after the second dose of the pharmaceutical composition.

In some embodiments, the third dose is administered 1 hour to 1 year (e.g., 1 hour to 1 day, e.g., 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 1 day, e.g., 1 day to 1 week, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, e.g., 1 week to 1 month, e.g., 2 weeks, 3 weeks, or 1 month, e.g., 1 month to 1 year, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year) after the second dose of the pharmaceutical composition. In some embodiments, the third dose is administered within 1 day to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 6 days, days to 14 days, 6 to 7 days, 7 to 90 days, 7 to 45 days, 7 to 30 days, 7 to 14 days, 14 to 90 days, 14 to 45 days, 14 to 30 days, 21 to 90 days, 21 to 60 days, 21 to 45 days, 21 to 30 days, 30 to 90 days, 30 to 60 days, 30 to 45 days, 45 to 180 days, 45 to 120 days, 45 to 100 days, 45 to 90 days, 45 to 60 days, 60 to 180 days, 60 to 120 days, 60 to 100 days, 60 to 90 days, 90 to 100 days, 90 to 120 days, or 90 to 180 days).

In some embodiments, the second dose is administered before the serum concentration of the antifusogenic polypeptide is less than about 500 ng/mL in the subject's serum.

In some embodiments, the method provides for the administration 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, ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL) for at least 1 hour (e.g., at least 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more).

In some embodiments, the method treats or prevents a viral infection in a subject. For example, the pharmaceutical composition may be administered to a subject in an amount and for a duration sufficient to treat or prevent a viral infection. The pharmaceutical composition may be administered to a subject to reduce the risk of a viral infection.

In some embodiments, the method treats or prevents human immunodeficiency virus (HIV) infection.

In some embodiments, the method treats or prevents a coronavirus infection (e.g., a betacoronavirus infection, e.g., a SARS-CoV-2 infection, such as a SARS-CoV-2 infection that results in symptoms of COVID-19 infection).

In some embodiments, the method treats or prevents Hepatitis C virus (HCV) infection.

In some embodiments, a circular polynucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1) is used to reduce viral entry.

Another aspect features a circular polyribonucleotide that includes a polyribonucleotide cargo encoding multiple antifusogenic polypeptides. The polyribonucleotide cargo can include expression sequences that encode antifusogenic polypeptides. In some embodiments, the antifusogenic polypeptides are directed against the same virus. Alternatively, the antifusogenic polypeptides can be directed against two or more viruses.

Definitions To facilitate understanding of the present disclosure, several terms are defined below. Terms defined herein have the meanings as commonly understood by those skilled in the art in the areas relevant to the present disclosure. Terms such as "a", "an" and "the" are not intended to refer to a singular entity only, but also include general classes, specific examples of which may be used for illustration. The term "or" is used to mean "and/or" unless expressly specified to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and/or". Although the terms in this specification are used to describe specific embodiments, their use should not be construed as limiting, except as outlined in the claims.

As used herein, any value provided in a range of values includes both the upper and lower limits, as well as any value that falls within the upper and lower limits.

As used herein, the term "about" refers to a value within ±10% of the recited value.

As used herein, the term "carrier" refers to a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., cyclic polyribonucleotide) to a cell, via a partially or fully encapsulated agent, or a combination thereof, by covalent modification of the cyclic polyribonucleotide. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles (e.g., nanoparticles encapsulated or covalently attached to cyclic polyribonucleotides), liposomes, fusosomes, exosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently attached to polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents).

As used herein, the terms "cyclic polyribonucleotide," "cyclic RNA," and "circRNA" are used interchangeably to refer to polyribonucleotide molecules having a structure with no free ends (i.e., free 3' and/or 5' ends), e.g., polyribonucleotide molecules that form a circular or endless structure through covalent or non-covalent bonds. A cyclic polyribonucleotide may be, for example, a covalently closed polyribonucleotide.

As used herein, the term "circularization efficiency" is a measure of the resulting circular polyribonucleotide relative to its non-circular starting material.

The term "diluent" refers to a vehicle comprising an inert solvent in which a composition described herein (e.g., a composition comprising a cyclic polyribonucleotide) may be diluted or dissolved. The diluent may be an RNA solubilizing agent, a buffer, an isotonicity agent, or a mixture thereof. The diluent may be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and 1,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, or powdered sugar.

As used herein, the terms "disease," "disorder," and "condition" each refer to a state of less than optimal health, e.g., a condition that is or would be typically diagnosed or treated by a medical professional.

As used herein, the term "expressed sequence" refers to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide (e.g., an antifusogenic polypeptide). An exemplary expressed sequence that encodes a peptide or polypeptide can include multiple nucleotide triplets, each of which can encode an amino acid, and are referred to as a "codon."

As used herein, the term "fragment" with respect to a polypeptide or nucleic acid sequence, e.g., an antifusogenic polypeptide or a nucleic acid sequence encoding an antifusogenic polypeptide, refers to a contiguous, less than full portion of the polypeptide or nucleic acid sequence. A fragment of a polypeptide or a nucleic acid sequence encoding a polypeptide refers to a contiguous, less than full fraction (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the total length) of a sequence, such as, for example, a sequence disclosed herein. It is understood that all of the present disclosure contemplates fragments of any antifusogenic polypeptide disclosed herein.

As used herein, the term "Fc domain" refers to a polypeptide chain comprising at least a hinge domain and a second and third antibody constant domain (C _H 2 and C _H 3) or a functional fragment thereof (e.g., a fragment capable of dimerizing and binding to an Fc receptor). The Fc domain may be of any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD (e.g., IgG). Further, the Fc domain may be of an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4) (e.g., IgG1). The 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). The Fc domain in the conjugates as described herein may contain one or more alterations from the 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 the Fc domain and the Fc receptor. Examples of suitable alterations are known in the art. Unless otherwise specified herein, the numbering of amino acid residues in an 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.

As used herein, the term "GC content" refers to the percentage of guanine (G) and cytosine (C) in a nucleic acid sequence. The formula for calculating GC content is (G+C)/(A+G+C+U) x 100% (for RNA) or (G+C)/(A+G+C+T) x 100% (for DNA). Similarly, the term "uridine content" refers to the percentage of uridine (U) in a nucleic acid sequence. The formula for calculating uridine content is U/(A+G+C+U) x 100%. Similarly, the term "thymidine content" refers to the percentage of thymidine (T) in a nucleic acid sequence. The formula for calculating thymidine content is T/(A+G+C+T) x 100%.

"Heterologous" means occurring under a situation other than the naturally occurring (natural) situation. A "heterologous" polynucleotide sequence indicates that the polynucleotide sequence is used in a manner other than that found in the sequence's native genome. For example, a "heterologous promoter" is used to drive transcription of a sequence that is not naturally transcribed by that promoter; therefore, a "heterologous promoter" sequence is often included in an expression construct using recombinant nucleic acid techniques. The term "heterologous" is also used to refer to a given sequence that is placed in a non-naturally occurring relationship with another sequence; for example, a heterologous coding or non-coding nucleotide sequence is commonly inserted into a genome by genome transformation techniques, resulting in a genetically modified or recombinant genome.

As used herein, 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. The intron fragment can be a 5' portion of an intron (e.g., the 5' portion of a catalytic intron) or a 3' portion of an intron (e.g., the 3' portion of a catalytic intron), such that a 5' intron fragment and a 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 that has been split into two portions. The term intron fragment is not meant to state, imply, or suggest that the two portions or halves are equal in length. The term intron fragment is used synonymously with the term split intron and may be used in place of the term "half intron."

As used herein, the term "linear counterpart" refers to a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence identity therebetween) as a circularized polyribonucleotide and has two free ends (i.e., a non-circularized version of a circularized polyribonucleotide (and fragments thereof)). In some embodiments, a linear counterpart (e.g., a pre-circularized version) is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence identity therebetween) as a circularized polyribonucleotide and the same or similar nucleic acid modifications and has two free ends (i.e., a non-circularized version of a circularized polyribonucleotide (and fragments thereof). In some embodiments, a linear counterpart is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage of sequence identity therebetween), and has a different or no nucleic acid modification, and has two free ends (i.e., a non-circularized version of a circularized polyribonucleotide (and fragments thereof). In some embodiments, a fragment of a polyribonucleotide molecule that is a linear counterpart is any portion of the linear counterpart polyribonucleotide molecule that is shorter than the linear counterpart polyribonucleotide molecule. In some embodiments, the linear counterpart further comprises a 5' cap. In some embodiments, the linear counterpart further comprises a polyadenosine tail. In some embodiments, the linear counterpart further comprises a 3' UTR. In some embodiments, the linear counterpart further comprises a 5' UTR.

As used herein, the terms "linear RNA," "linear polyribonucleotide," and "linear polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having 5' and 3' ends. One or both of the 5' and 3' ends may be free or may be attached to another moiety. Linear RNA includes RNA that has not been subjected to circularization (e.g., previously circularized) and can be used as starting material for circularization, for example, by splint ligation or chemical, enzymatic, ribozyme- or splicing-catalyzed circularization methods.

As used herein, the term "modified ribonucleotide" means a nucleotide having at least one modification to the sugar, nucleobase, or internucleoside linkage.

As used herein, the term "naked delivery" refers to a formulation for delivery to a cell without the aid of a carrier or covalent modifications to moieties that aid in delivery to the cell. Naked delivery formulations do not include transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation of a cyclic polyribonucleotide is a formulation that includes cyclic polyribonucleotide without covalent modifications and does not include a carrier.

As used herein, the terms "nicked RNA," "nicked linear polyribonucleotide," and "nicked linear polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having 5' and 3' ends that result from the nicking or degradation of circular RNA.

The term "pharmaceutical composition" is also intended to disclose that the cyclic or linear polyribonucleotides contained within the pharmaceutical composition may be used for the treatment of the human or animal body by therapy. It is therefore meant to be equivalent to "polyribonucleotides for use in therapy."

As used herein, the term "polynucleotide" refers to a molecule that includes 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 one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO ₃ ) groups. A nucleotide can include a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. A polyribonucleotide or ribonucleic acid, or RNA, can refer to a polymer that includes multiple ribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose. As used herein, a polyribonucleotide sequence that represents thymine (T) is understood to represent uracil (U).

As used herein, the term "polyribonucleotide cargo" includes any sequence comprising at least one polyribonucleotide. In embodiments, the polyribonucleotide cargo comprises one or more expressed sequences, each of which encodes a polypeptide. In embodiments, the polyribonucleotide cargo comprises one or more non-coding sequences, such as polyribonucleotides having regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo comprises a combination of expressed and non-coding sequences. In embodiments, the polyribonucleotide cargo comprises one or more polyribonucleotide sequences as described herein, e.g., one or more regulatory elements, internal ribosome entry site (IRES) elements, or spacer sequences.

As used interchangeably herein, the terms "polyA" and "polyA sequence" refer to an untranslated flanking region of a nucleic acid molecule that is at least 5 nucleotides in length and consists of adenosine residues. In some embodiments, the 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. In some embodiments, the polyA sequence is located 3' to (e.g., downstream of) an open reading frame (e.g., an open reading frame encoding a polypeptide), and the polyA sequence is 3' to a termination sequence (e.g., a stop codon) such that the polyA is not translated. In some embodiments, the polyA sequence is located 3' to the termination sequence and the 3' untranslated region.

As used herein, elements of nucleic acid are "operably connected" or "operably linked" when they are placed in a vector such that they can be transcribed to form linear RNA, which can then be circularized into circular RNA using the methods provided herein.

Polydeoxyribonucleotide, deoxyribonucleic acid, and DNA refer to a polymer comprising multiple deoxyribonucleotides polymerized through phosphodiester bonds. Nucleotides can be nucleoside monophosphates or nucleoside polyphosphates. Nucleotides refer to deoxyribonucleoside polyphosphates, such as deoxyribonucleoside triphosphates (dNTPs), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTPs, including detectable tags (e.g., fluorophores), such as luminescent tags or markers. Nucleotides can include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T, or U, or any other subunits that are specific to one or more complementary A, C, G, T, or U, or are complementary to a purine (i.e., A or G, or variants thereof) or pyrimidine (i.e., C, T, or U, or variants thereof). In some instances, the polynucleotide is a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a derivative or variant thereof. In some instances, the polynucleotide is a small interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a small hairpin RNA (shRNA), a small nuclear RNA (snRNA), a messenger RNA (mRNA), a pre-mRNA (pre-mRNA), an antisense RNA (asRNA), to name a few, and encompasses both the nucleotide sequence and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In some instances, the polynucleotide molecule is circular. The polynucleotide may have a variety of lengths. Nucleic acid molecules 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. Polynucleotides can be isolated from cells or tissues. Polynucleotide sequence embodiments can include isolated and purified DNA/RNA molecules, synthetic DNA/RNA molecules, and synthetic DNA/RNA analogs.

Polynucleotide embodiments, such as polyribonucleotides or polydeoxyribonucleotides, may include one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs, or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylketone, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, These include 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid, wybutoxocine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. In some cases, nucleotides can include modifications of their phosphate moieties, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications of the thiol moiety (e.g., α-thiotriphosphate and β-thiotriphosphate). In embodiments, the nucleic acid molecules are also modified at the base moiety, sugar moiety, or phosphate backbone (e.g., at one or more atoms typically available to form hydrogen bonds with a complementary nucleotide, or at one or more atoms typically not available to form hydrogen bonds with a complementary nucleotide). In embodiments, the nucleic acid molecules include amine-modifying groups, such as aminoallyl-dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP), to allow for covalent attachment of amine-reactive moieties, such as N-hydroxysuccinimide ester (NHS). Alternatives to standard DNA or RNA base pairs in the oligonucleotides of the present disclosure may provide higher bit density per cubic mm, greater safety (resistance to accidental or deliberate synthesis of natural toxins), easier identification in photoprogrammed polymerases, or subsecondary structure. Such alternative base pairs compatible with natural and mutant polymerases for de novo or amplicon synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diderichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul;8(7):612-4, incorporated herein by reference for all purposes.

As used herein, "polypeptide" refers to a polymer of amino acid residues (natural or non-natural) linked together, most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs thereof. Polypeptides can be single molecules or multi-molecular complexes such as dimers, trimers, or tetramers. Polypeptides can also include single chain or multi-chain polypeptides such as antibodies or insulin, which can be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

As used herein, the term "prevent" means reducing 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 reducing the severity or frequency of symptoms of a disease or disorder that subsequently develops. Therapeutic agents can be administered to subjects who are at increased risk of developing a viral infection compared to members of the general population to prevent the onset of a disease or condition or to reduce its severity. Therapeutic agents can be administered as a prophylactic, e.g., prior to the onset of any symptoms or manifestations of a viral infection.

As used herein, the term "regulatory element" refers to a portion of a nucleic acid sequence or the like that alters expression of an expression sequence within a circular or linear polyribonucleotide.

As used herein, "spacer" refers to any adjacent nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.

"Signal sequence" refers to a polypeptide sequence, e.g., 10-45 amino acids in length, present at the N-terminus of a polypeptide sequence of a nascent protein that targets the polypeptide sequence to the secretory pathway.

As used herein, the term "sequence identity" is determined by alignment of two peptide sequences or two nucleotide sequences using a global or local alignment algorithm. Sequences are said to be "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity when optimally aligned (aligned by a program 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 minimizing the number of gaps. Generally, the default parameters of GAP are used: gap creation penalty = 50 (nucleotides) / 8 (protein) and gap extension penalty = 3 (nucleotides) / 2 (protein). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and sequence identity percentage scores are determined using computer programs such as, for example, GCG Wisconsin Package, Version 10.3 or EmbossWin version 2.10.0 (using the program "Needle") available from Accelrys Inc., 9685 Scraton Road, San Diego, CA 92121-3752 USA. Alternatively, or in addition, percent identity is determined by searching a database using algorithms such as FASTA, BLAST, etc. Sequence identity refers to sequence identity over the entire length of the sequence.

As used herein, the term "subject" refers to an organism, such as an animal, a plant, or a microorganism. In embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In embodiments, the subject is a human. In embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a bison, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In embodiments, the subject is an avian, such as a member of an avian taxon such as Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate, such as an arthropod (e.g., insects, arachnids, crustaceans), nematodes, annelids, worms, or mollusks. In embodiments, the subject is an invertebrate that is an invertebrate agricultural pest or a parasite on an invertebrate or vertebrate host. In embodiments, the subject is a plant such as an angiosperm (which may be dicotyledonous or monocotyledonous) or gymnosperm (e.g., conifers, cycads, Gnetophytes, ginkgo), fern, horsetail, club moss, or bryophyte. In embodiments, the subject is a eukaryotic alga (unicellular or multicellular). In embodiments, the subject is an agriculturally or horticulturally important plant such as row crops, fruit-bearing plants and trees, vegetables, trees, and ornamentals, e.g., ornamental flowers, shrubs, trees, ground covers, and turf.

As used herein, the term "antifusogenic polypeptide" refers to a polypeptide, such as a 10-200 amino acid polypeptide, that inhibits a viral fusion-related event, such as viral entry or viral fusion. Antifusogenic polypeptides include, for example, the polypeptides in Table 1. Antifusogenic polypeptides include polypeptides as well as any biologically active fragments thereof (e.g., fragments of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids). Antifusogenic polypeptides include, for example, polypeptides that target HIV, SARS-CoV-2, HCV, or RSV. In some embodiments, the antifusogenic polypeptide comprises 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. Antifusogenic polypeptide also refers to a polynucleotide (e.g., a polyribonucleotide, e.g., a cyclic polyribonucleotide, encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1) or a biologically active fragment thereof.

As used herein, the terms "treat" and "treating" refer to 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 the severity of, curing, inhibiting progression of, reducing the likelihood of recurrence of, stabilizing (i.e., not worsening) the condition of the viral infection, or preventing the spread of the viral infection, as compared to the state or condition of the viral infection in the absence of therapeutic treatment.

As used herein, the term "termination element" refers to a portion of a nucleic acid sequence or the like that terminates translation of an expression sequence in a circular or linear polyribonucleotide.

As used herein, the term "translation efficiency" refers to the rate or amount of protein or peptide production from a ribonucleotide transcript. In certain embodiments, translation efficiency can be expressed as the amount of protein or peptide produced per a given amount of transcript encoding the protein or peptide, e.g., in a given translation system, e.g., a cell-free translation system such as rabbit reticulocyte lysate, for a given period of time.

As used herein, the term "translation initiation sequence" refers to a nucleic acid sequence that initiates translation of an expression sequence in a circular or linear polyribonucleotide.

As used herein, "vector" refers to a segment of DNA that is synthesized (e.g., using PCR) or taken from a virus, plasmid, or cell of a higher organism into which a foreign DNA segment can or has been inserted for cloning or expression purposes. In some embodiments, the vector can be stably maintained in the organism. The vector can include, for example, an origin of replication, a selectable marker or reporter gene, e.g., antibiotic resistance or GFP, or a multiple cloning site (MCS). The term includes linear DNA segments (e.g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like. In one embodiment, the vectors provided herein include a multiple cloning site (MCS). In another embodiment, the vectors provided herein do not include an MCS.

FIG. 1 is a schematic diagram showing the protein domains or regions of various coronavirus S proteins and the sequences of the HR1 and HR2 regions. FIG. 1 is a schematic diagram showing various antifusogenic polypeptides and sequences derived from the HR2 region of SARS CoV-2. FIG. 1 is a schematic diagram showing an exemplary embodiment of a multi-ORF antifusogenic polypeptide construct. 4A and 4B are graphs showing the inhibitory effect on fusion using Omicron and Delta pseudotyped viruses. Figure 4A shows the % inhibition of Delta and Omicron using either HR2 full length or HR2 full length with a HiBiT tag. Figure 4B shows the relative expression of antifusogenic polypeptides compared to mock controls. FIG. 1 is a graph showing in vivo expression of HR2 full-length antifusogenic polypeptide with and without a HiBiT tag at 6 hours and 24 hours. 6A and 6B are graphs showing in vitro neutralization of pseudotyped viruses of the Wuhan and Omicron strains of SARS CoV-2 using HR2A polypeptides. 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. 7A-7B are graphs showing the percent inhibition of SARS CoV-2 pseudotyped virus Omicron BA4 and BA.5 (FIG. 7A) or SARS CoV-1 pseudotyped virus (FIG. 7B) strains. 8A-8D are graphs showing the percent inhibition of SARS CoV-2 pseudotyped virus using full length HR2 (HR2 complete). Figure 8A shows inhibition of the Wuhan strain. Figure 8B shows inhibition of the Omicron BA.4 and BA.5 strains. Figure 8C shows inhibition of the Omicron BA.1 strain. Figure 8D shows inhibition of SARS CoV-1 pseudotyped virus. FIG. 1 is a schematic diagram showing the construct designs and sequences of various HIV antifusogenic polypeptides. 10A and 10B are a graph and a table showing the expression of various HIV antifusogenic polypeptides from circular RNA. 11A-B are graphs showing expression of various HIV antifusogenic polypeptides from circular RNA (FIG. 11A) or plasmid DNA (FIG. 11B). 1 is a table showing the expression of various HIV antifusogenic polypeptides from circular RNA.

The present invention features compositions containing cyclic polyribonucleotides (cyclic RNAs) encoding antifusogenic polypeptides and methods of using the same. The cyclic polyribonucleotides described herein are particularly useful for delivering polynucleotide cargo encoding antifusogenic polypeptides to target cells.

The cyclic polyribonucleotide may comprise a polyribonucleotide cargo encoding a polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo comprises an expressed 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 comprises an expressed 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.

Cyclic polyribonucleotides can be generated from precursors such as linear deoxyribonucleotides, cyclic deoxyribonucleotides, or cyclic polyribonucleotides.

The circular polyribonucleotide may, for example, include a splice junction connecting a 5' exon fragment and a 3' exon fragment.

The linear RNA molecules described herein are polyribonucleotides encoding antifusogenic polypeptides. In some embodiments, 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., a 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; (G) a 5' catalytic intron fragment. The catalytic intron fragment and the splice site can then allow the linear polyribonucleotide to self-splice, thus forming a circular polyribonucleotide encoding an antifusogenic polypeptide.

Also featured are methods of using the cyclic polyribonucleotides described herein. For example, the cyclic polyribonucleotides can be formulated as compositions (e.g., pharmaceutical compositions) for administration to a subject, e.g., a human subject. The pharmaceutical composition can be administered in one or more doses of the composition. The composition can be administered to a subject to treat or prevent a viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV).

Polynucleotides The present disclosure features cyclic polyribonucleotide compositions encoding antifusogenic polypeptides, their uses, and methods for making cyclic polyribonucleotides encoding antifusogenic polypeptides. In some embodiments, the cyclic polyribonucleotides are generated from linear polyribonucleotides (e.g., by self-splicing compatible ends of linear polyribonucleotides). In some embodiments, the linear polyribonucleotides are transcribed from deoxyribonucleotide templates (e.g., vectors, linearized vectors, or cDNAs). Thus, the present disclosure features deoxyribonucleotides, linear polyribonucleotides, and cyclic polyribonucleotides and compositions thereof that are useful for producing cyclic polyribonucleotides encoding antifusogenic polypeptides.

Template deoxyribonucleotides The present invention features template deoxyribonucleotides for making circular RNAs as described herein. In some embodiments, the deoxyribonucleotides include the following operably linked in a 5' to 3' direction: (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. In embodiments, the deoxyribonucleotides include additional elements, for example, outside or between any of elements (A), (B), (C), (D), (E), (F), or (G). In embodiments, any of elements (A), (B), (C), (D), (E), (F), or (G) are separated from each other by a spacer sequence as described herein.

In embodiments, the deoxyribonucleotides are, for example, a circular DNA vector, a linearized DNA vector, or linear DNA (e.g., generated from a DNA vector, e.g., cDNA).

In some embodiments, the deoxyribonucleotides further comprise an RNA polymerase promoter operably linked to the sequence encoding the linear RNA described herein. In embodiments, the RNA polymerase promoter is heterologous to the sequence encoding the linear RNA. In some embodiments, the RNA polymerase promoter is a T7 promoter, a T6 promoter, a T4 promoter, a T3 promoter, an SP6 viral promoter, or an SP3 promoter.

In some embodiments, the deoxyribonucleotides comprise a multiple cloning site (MCS).

In some embodiments, deoxyribonucleotides are used to generate circular RNAs having a size range of about 100 to about 20,000 nucleotides. In some embodiments, the circular RNAs are 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. In some embodiments, the circular RNA is less than or equal to 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 antifusogenic polypeptides. Linear polyribonucleotides can be used to generate circular polyribonucleotides, for example, by ligating or splicing (e.g., self-splicing) the linear polyribonucleotides to generate circular polyribonucleotides. In some embodiments, the linear polyribonucleotides include the following operably linked in a 5' to 3' direction: (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. In some embodiments, the linear polyribonucleotide includes additional elements, for example, additional elements outside 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) can be separated by a spacer sequence, as described herein.

In certain embodiments, provided herein is a method for generating a linear 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, a linearized vector, or a cDNA) encoding an antifusogenic polypeptide provided herein as a template (e.g., a vector, a linearized vector, or a cDNA provided herein in which an RNA polymerase promoter is located upstream of the region encoding the linear RNA).

In embodiments, the deoxyribonucleotide template is transcribed to produce a linear RNA containing the components described herein. Upon expression, the linear polyribonucleotide produces a splicing-compatible polyribonucleotide that can self-splice to produce a circular polyribonucleotide.

In some embodiments, the linear polyribonucleotide is 50-20,000, 100-20,000, 200-20,000, 300-20,000 (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, 00, 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. In embodiments, the linear polyribonucleotide is, for example, 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.

Circular polyribonucleotides In some embodiments, the present invention features a circular polyribonucleotide comprising an expression sequence encoding an antifusogenic polypeptide. In embodiments, the polyribonucleotide comprises an IRES operably linked to the expression sequence encoding the antifusogenic polypeptide. The circular polyribonucleotide may comprise a splice junction, for example joining a 5' exon fragment and a 3' exon fragment. The circular polyribonucleotide may comprise any one or more of the elements described herein. In some embodiments, the circular polyribonucleotide comprises any feature or any combination of features disclosed in WO 2019/118919, which is incorporated herein by reference in its entirety.

In embodiments, the circular polynucleotide further comprises a polyribonucleotide cargo. In embodiments, the polyribonucleotide cargo comprises an expressed (or coding) sequence, a non-coding sequence, or a combination of expressed (coding) and non-coding sequences. In embodiments, the polyribonucleotide cargo comprises an expressed (coding) sequence that encodes a polypeptide. In embodiments, the polyribonucleotide comprises an IRES operably linked to the expressed sequence that encodes the polypeptide. In some embodiments, the IRES is located upstream of the expressed sequence. In some embodiments, the IRES is located downstream of the expressed sequence. In some embodiments, the circular polyribonucleotide further comprises a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The spacer region may be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. The spacer region may be, for example, 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 some embodiments, the spacer region comprises a polyA sequence. In some embodiments, the spacer region comprises a polyA-C sequence. In some embodiments, the spacer region comprises a polyA-G sequence. In some embodiments, the spacer region comprises a polyA-T sequence. In some embodiments, the spacer region comprises a random sequence. In some embodiments, the first annealing region and the second annealing region are linked, thereby forming a circular polyribonucleotide.

In some embodiments, the circular RNA is generated by a deoxyribonucleotide template or a linear RNA as described herein. In some embodiments, the circular RNA is generated by any of the methods described herein.

In some embodiments, the cyclic 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, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

In some embodiments, the cyclic polyribonucleotide is 500-20,000 nucleotides, 1,000-20,000 nucleotides, 2,000-20,000 nucleotides, or 5,000-20,000 nucleotides. In some embodiments, the cyclic polyribonucleotide is 500-10,000 nucleotides, 1,000-10,000 nucleotides, 2,000-10,000 nucleotides, or 5,000-10,000 nucleotides.

As a result of its circularization, cyclic polyribonucleotides may contain certain characteristics that distinguish them from linear RNA. For example, cyclic polyribonucleotides are less susceptible to degradation by exonucleases compared to linear RNA. As such, cyclic polyribonucleotides are more stable than linear RNA, especially when incubated in the presence of exonucleases. The increased stability of cyclic polyribonucleotides compared to linear RNA makes them more useful as cell transformation reagents for producing polypeptides and can be stored more easily and for longer periods of time than linear RNA. The stability of exonuclease-treated cyclic polyribonucleotides can be tested using standard methods in the art to determine whether RNA degradation has occurred (e.g., by gel electrophoresis). Furthermore, unlike linear RNA, cyclic polyribonucleotides are less susceptible to dephosphorylation when cyclic polyribonucleotides are incubated with phosphatases, such as calf intestinal phosphatase.

The cyclic polyribonucleotides and compositions or pharmaceutical compositions thereof described herein may be used in therapeutic and veterinary methods of dosing to produce a level of cyclic polyribonucleotide, a level of binding to a target, or a level of protein in a plurality of cells after providing at least two doses of cyclic polyribonucleotide to the plurality of cells. In some embodiments, the cyclic polyribonucleotides are non-immunogenic in mammals, e.g., humans. In some embodiments, the cyclic polyribonucleotides can replicate or replicate in cells from aquaculture animals (such as fish, crabs, shrimp, oysters, etc.), mammalian cells, such as cells from pet or zoo animals (such as cats, dogs, lizards, birds, lions, tigers and bears), cells from farm or work animals (such as horses, cows, pigs, chickens, etc.), human cells, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, the invention includes cells comprising the cyclic polyribonucleotides described herein, the cells being cells from aquaculture animals (fish, crabs, shrimp, oysters, etc.), mammalian cells, such as cells from pet or zoo animals (cats, dogs, lizards, birds, lions, tigers, and bears, etc.), cells from farm or work animals (horses, cows, pigs, chickens, etc.), human cells, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, the cells are modified to comprise cyclic polyribonucleotides.

In some embodiments, the cyclic polyribonucleotide comprises a sequence for an expression product. In some embodiments, the cyclic polyribonucleotide comprises a binding site for binding to a target. In some embodiments, the cyclic polyribonucleotide is provided to the plurality of cells by any of the dosing regimens described herein. In some embodiments, the cyclic polyribonucleotide as described herein induces a response or level of response in the subject. In some embodiments, an expression product encoded by a sequence contained in the cyclic polyribonucleotide is expressed in one or more cells of the plurality of cells.

In some embodiments, the cyclic polyribonucleotide has a half-life at least that of its linear counterpart, e.g., the linear expression sequence or the linear polyribonucleotide. In some embodiments, the cyclic polyribonucleotide has a half-life that is increased over the half-life of its linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In some embodiments, the cyclic polyribonucleotide has a half-life or persistence in cells of 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 more. In some embodiments, the cyclic polyribonucleotide has a half-life or persistence in a cell of 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. In some embodiments, the cyclic polyribonucleotide has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the cyclic polyribonucleotide has a half-life or persistence in a cell after division. In certain embodiments, the cyclic polyribonucleotide has a half-life or persistence in dividing cells of 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 more. In certain embodiments, the cyclic polyribonucleotide has a half-life or persistence in dividing cells of 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.

In some embodiments, the cyclic polyribonucleotide modulates cellular function, e.g., transiently or chronically. In certain embodiments, the cellular function is stably altered, such as a modulation that lasts 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 more. In certain embodiments, the cellular function is stably altered, such as a modulation lasting for about 1 hour to about 60 days, e.g., about 1 hour to about 30 days, e.g., 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.

Polynucleotide Elements The polynucleotides described herein (e.g., circular polyribonucleotides) can include any one or more of the elements described herein and an expression sequence that encodes an antifusogenic polypeptide.

Anti-fusogenic polypeptides The present disclosure provides a circular polyribonucleotide encoding at least one expression sequence encoding an anti-fusogenic polypeptide. In some embodiments, the anti-fusogenic polypeptide inhibits viral entry. In some embodiments, the anti-fusogenic polypeptide inhibits viral fusion.

In some embodiments, the antifusogenic polypeptide is a polypeptide comprising an amino acid sequence selected from the sequences of Table 1 or a variant thereof. In some embodiments, the antifusogenic polypeptide is a polypeptide comprising 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 of Table 1. In some embodiments, the antifusogenic polypeptide is a polypeptide comprising 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 of Table 1. In some embodiments, the antifusogenic polypeptide is a polypeptide comprising a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence in Table 1. In some embodiments, the antifusogenic polypeptide is a variant of a sequence in Table 1 comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (e.g., substitutions, deletions, or insertions). In some embodiments, the cyclic polyribonucleotide comprises an expression sequence encoding two or more antifusogenic polypeptides. In some embodiments, the cyclic polyribonucleotide encoding two or more fusion proteins reduces the chance of viral escape.

In some embodiments, the antifusogenic polypeptide targets one or more (e.g., 2, 3, 4, or 5) viruses. Viruses that may be targeted by an antifusogenic polypeptide include, but are not limited to, all of the virus strains listed in Table 1. In some embodiments, the virus does not infect humans. In some embodiments, the virus infects humans.

In some embodiments, the virus is a human immunodeficiency virus (HIV) (e.g., the antifusogenic polypeptide inhibits viral entry of HIV). In some embodiments, the HIV is HIV-1. In some embodiments, the HIV is an HIV-1 strain (e.g., HIV-1 _HE , HIV-1 _IIIB , HIV-1 _MN , HIV-1 _NDK , HIV-1 _NL4-3 , HIV-1 _RF , or HIV-1 _SF2 ). In some embodiments, the HIV is HIV-2. In some embodiments, the HIV is an HIV-2 strain (e.g., HIV-2 _EHO or HIV-2 _ROD ). In some embodiments, the HIV is an HIV pseudovirion. In some embodiments, the antifusogenic polypeptide prevents HIV viral fusion by specifically binding to HIV glycoprotein 120 (gp120). In some embodiments, the antifusogenic polypeptide prevents gp120 binding to cluster of differentiation 4 (CD4) coreceptor. In some embodiments, the antifusogenic polypeptide reduces the affinity of gp120 for coreceptors (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 gp120 binding to coreceptors (e.g., CCR5 and CXCR4). In some embodiments, the antifusogenic polypeptide prevents HIV viral fusion by specifically binding to HIV glycoprotein 41 (gp41). In some embodiments, the antifusogenic polypeptide inhibits HIV viral core entry into cells. In some embodiments the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide (e.g. a polypeptide that inhibits HIV viral entry) 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. In some embodiments the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide having at least 85% (e.g. at least 90%, 95%, 97%, 99% or 100%) sequence identity to any one of the polypeptides in 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.

In some embodiments, the virus is a hepatitis virus (e.g., the antifusogenic polypeptide inhibits viral entry of a hepatitis virus). In some embodiments, the hepatitis virus is a hepatitis A virus (HAV). In some embodiments, the hepatitis virus is a hepatitis B virus (HBV). In some embodiments, the hepatitis virus is a hepatitis C virus (HCV). In some embodiments, the hepatitis virus is a hepatitis D virus (HDV). In some embodiments, the hepatitis virus is a hepatitis E virus (HEV). In some embodiments, the hepatitis virus is a duck hepatitis virus (DHV). In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide of any one of SEQ ID NOs: 104, 109, 112, 113, 141-145, 158-160, or 284 (e.g., a polypeptide that inhibits viral entry of a hepatitis virus, such as HCV). In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of the polypeptides in SEQ ID NOs: 104, 109, 112, 113, 141-145, 158-160, or 284.

In some embodiments, the virus is a coronavirus, e.g., a betacoronavirus (e.g., the antifusogenic polypeptide inhibits viral entry of a coronavirus). In some embodiments, the betacoronavirus is SARS-CoV type 1 (SARS-CoV-1). In some embodiments, the betacoronavirus is SARS-CoV type 2 (SARS-CoV-2). In some embodiments, the betacoronavirus is a pseudotyped virus that expresses a SARS-CoV-2 spike protein. In some embodiments, the polyribonucleotide cargo comprises an expression sequence that encodes a polypeptide of any one of SEQ ID NOs: 6-9, 119-123, 165-167, or 288-311 (e.g., a polypeptide that inhibits viral entry of a betacoronavirus, such as SARS-CoV-1 or SARS-CoV-2). In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of the polypeptides of SEQ ID NOs: 6-9, 119-123, 165-167, or 288-311.

In some embodiments, the virus is a respiratory syncytial virus (RSV) (e.g., the antifusogenic polypeptide inhibits viral entry of RSV). In some embodiments, the RSV is RSV subtype A (RSVA). In some embodiments, the RSV is RSV subtype B (RSVB). In some embodiments, the polyribonucleotide cargo comprises 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. In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of the polypeptides 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.

In some embodiments, the virus is influenza (e.g., seasonal influenza, pandemic influenza, influenza A, influenza H1N1, influenza B, influenza C, influenza D). In some embodiments, the influenza is influenza A. In some embodiments, the influenza is influenza B. In some embodiments, the influenza is influenza C. In some embodiments, the influenza is influenza D. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide (e.g., a polypeptide that inhibits influenza viral entry) of any one of SEQ ID NOs: 1, 4, 11, 245, 247, 253, 262, 269, 275, 277, 278, 279, 280, 281, or 283. In some embodiments, the polyribonucleotide cargo comprises an expressed 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, 4, 11, 245, 247, 253, 262, 269, 275, 277, 278, 279, 280, 281, or 283.

In some embodiments, the virus is an influenza virus (e.g., the antifusogenic polypeptide inhibits influenza viral entry). In some embodiments, the influenza virus is an influenza A virus (IAV). In some embodiments, the influenza virus is an influenza B virus (IBV). In some embodiments, the influenza virus is an influenza virus that expresses hemagglutinin (HA).

In some embodiments, the virus is a herpes simplex virus (HSV) (e.g., the antifusogenic polypeptide inhibits viral entry of HSV). In some embodiments, the HSV is HSV-1. In some embodiments, the HSV is HSV-2.

In some embodiments, the virus is a human papillomavirus (HPV) (e.g., the antifusogenic polypeptide inhibits viral entry of HPV). In some embodiments, the HPV is a high-risk HPV strain (e.g., HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, or HPV58). In some embodiments, the HPV is a low-risk HPV strain (e.g., HPV6, HPV11, HPV42, HPV43, HPV44).

In some embodiments, the virus is any virus listed in Table 1.

In some embodiments, the GC content of the nucleic acid sequence encoding the 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 the nucleic acid sequence encoding the antifusogenic polypeptide is at most 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 59%, or 60%. In some embodiments, the GC content of the nucleic acid sequence encoding the antifusogenic polypeptide is 51%-60%, 52%-60%, 53%-60%, 54%-60%, 55%-60%, 52%-58%, 53%-58%.

In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the antifusogenic polypeptide is greater than 10% (e.g., greater than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the antifusogenic polypeptide is at most 30% (e.g., at most 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%). In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the antifusogenic polypeptide is between 20% and 28%, between 21% and 26%, between 10% and 24%, between 15% and 24%, between 20% and 24%, between 21% and 24%, between 22% and 24%, between 23% and 24%, between 10% and 23%, between 15% and 23%, between 20% and 23%, between 21% and 23%, or between 22% and 23%.

The GC content of an expressed sequence encoding an anti-membrane fusion polypeptide refers to the GC content of the expressed sequence that exclusively encodes an anti-membrane fusion polypeptide and does not contain other coding regions that encode polypeptides other than the anti-membrane fusion polypeptide. Similarly, the uridine content or thymidine content of an expressed sequence encoding an anti-membrane fusion polypeptide refers to the uridine content of an expressed sequence that exclusively encodes an anti-membrane fusion polypeptide and does not contain other coding regions that encode polypeptides other than the anti-membrane fusion polypeptide. In some embodiments, the calculation of the GC content or uridine (or thymidine) content of an expressed sequence encoding an anti-membrane fusion polypeptide takes into account only the contiguous nucleic acid sequence that starts in the 5' to 3' direction from the first nucleoside of the start codon of the open reading frame encoding the anti-membrane fusion polypeptide to the last nucleoside of the stop codon of the same open reading frame. In other embodiments, the calculation of the GC content or uridine (or thymidine) content of an expressed sequence encoding an antifusogenic polypeptide takes into account only the contiguous nucleic acid sequence starting in the 5' to 3' direction from the first nucleoside of the codon encoding the N-terminal amino acid residue of the antifusogenic polypeptide to the last nucleoside of the codon encoding the C-terminal amino acid residue of the antifusogenic polypeptide.

In some embodiments, the nucleic acid sequence encoding the antifusogenic polypeptide has a uridine content of more than 20%. In some embodiments, the uridine content of the nucleic acid sequence encoding the 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 the nucleic acid sequence encoding the antifusogenic polypeptide is up to 30% (e.g., up to 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%). In some embodiments, the uridine content of the nucleic acid sequence encoding the antifusogenic polypeptide is 20%-28%, 21%-26%, 10%-24%, 15%-24%, 20%-24%, 21%-24%, 22%-24%, 23%-24%, 10%-23%, 15%-23%, 20%-23%, 21%-23%, or 22%-23%. In some embodiments, the nucleic acid sequence encoding the antifusogenic polypeptide has a uridine content of 20%-28%.

Multiple Antifusogenic Polypeptides In some embodiments, a circular polyribonucleotide encodes multiple expression sequences (e.g., two or more expression sequences, such as 2-100, 2-50, 2-20, 2-10, 5-100, 5-50, 5-20, or 5-10), each encoding an antifusogenic polypeptide.

In some embodiments, the circular polyribonucleotide encodes two or more (e.g., 2-100, 2-50, 2-20, 2-10, 5-100, 5-50, 5-20, or 5-10) copies of the same antifusogenic polypeptide.

In some embodiments, the cyclic 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) anti-fusogenic polypeptides. In some embodiments, each of the two or more different anti-fusogenic polypeptides is selected from the anti-fusogenic polypeptides of Table 1. In some embodiments, each of the two or more different anti-fusogenic polypeptides inhibits a different virus. For example, the cyclic polyribonucleotide may encode a first anti-fusogenic polypeptide that inhibits influenza and a second anti-fusogenic polypeptide that inhibits RSV. The cyclic polyribonucleotide may include a first anti-fusogenic polypeptide that inhibits influenza and a second anti-fusogenic polypeptide that inhibits SARS-CoV-2. The cyclic polyribonucleotide may include a first anti-fusogenic polypeptide that inhibits HIV and a second anti-fusogenic polypeptide that inhibits SARS-CoV-2. The circular polyribonucleotide may include a first antifusogenic polypeptide that inhibits HIV and a second antifusogenic polypeptide that inhibits HCV. If either the first or second antifusogenic polypeptide inhibits multiple viruses, the first and second antifusogenic polypeptides may have different virus specificities.

When a circular polyribonucleotide encodes two or more antifusogenic polypeptides, the antifusogenic polypeptides may be encoded in a single open reading frame or in multiple open reading frames.

In some embodiments, the disclosure provides a circular polyribonucleotide comprising an open reading frame (e.g., an open reading frame operably linked to an IRES) comprising two or more expression sequences, each of which encodes an antifusogenic polypeptide. In some embodiments, translation of the open reading frames produces a polypeptide fusion comprising two or more antifusogenic polypeptides. The antifusogenic polypeptides may be linked, for example, by a linker as described herein (e.g., a peptide linker encoded by the open reading frame, e.g., a glycine-serine linker as described below with respect to peptide-Fc fusions). In some embodiments, the antifusogenic polypeptides may be separated by a cleavage domain (e.g., a staggered sequence), for example, as described herein.

In some embodiments, the present disclosure provides a circular polyribonucleotide comprising 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).

Antifusogenic Polypeptide-Fc Fusions In some embodiments, the cyclic polyribonucleotide comprises an expression sequence encoding a fusion protein comprising an antifusogenic polypeptide. In some embodiments, the fusion protein comprises an antifusogenic polypeptide fused to an Fc domain (e.g., a single chain of an Fc domain) of an immunoglobulin. In some embodiments, the antifusogenic polypeptide is selected from Table 1. In some embodiments, the cyclic polyribonucleotide comprises an expression sequence encoding two or more fusion proteins comprising an antifusogenic polypeptide. In some embodiments, the cyclic polyribonucleotide comprises an expression sequence encoding two or more antifusogenic polypeptides. In some embodiments, the cyclic polyribonucleotide encoding two or more fusion proteins reduces the chance of viral escape.

In some embodiments, the Fc domain is an IgG4 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG1 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG2 Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG2a Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG2b Fc domain or a fragment thereof. In some embodiments, the Fc domain is an IgG3 Fc domain or a fragment thereof.

In some embodiments, 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. In some embodiments, 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. In some embodiments, the peptide linker between the antifusogenic polypeptide and the Fc domain comprises 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). In some embodiments, the peptide linker between the antifusogenic polypeptide and the Fc domain is between 2 and 200 amino acid residues (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, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-20, 2-22, 2-24, 2-26, 2-28, 2-29, 2-30, 2-32, 2-36, 2-38, 2-39, 2-40, 2-42, 2-46, 2-48, 2-49, 2-50, 2-51, 2-52, 2-53, 2-54, 2-55, 2-56, 2-57, 2-58, 2-59, 2-60, 2-62, 2-64, 2-66, 2-68, 2-69, 2-70, 2-71, 2-72, 2-73, 2-74, 2-75, 2-76, 2-77, 2-78, 2-79, 2-80, In some embodiments, the peptide linker comprises glycine (Gly) and serine (Ser) residues. In some embodiments, the peptide linker comprises any one of the amino acid sequences: (GS) _x , (GGS) _x , (GGGGS) _x , (GGSG) _x , or (SGGG) _x , where x is an integer between 1 and 50 (e.g., 1-40, 1-30, 1-20, 1-10, or 1-5). In some embodiments, the peptide linker comprises any one of the amino acid sequences: (GS) _x , (GGS) _x , (GGGGS) _x , (GGSG) _x , or (SGGG) _x , where x is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the peptide linker comprises 6-36 amino acids. In some embodiments, the peptide linker comprises 21-31 amino acids.

Polyribonucleotide cargo The polyribonucleotide cargo described herein includes any sequence comprising at least one polyribonucleotide. In some embodiments, the polyribonucleotide cargo comprises an expressed sequence, a non-coding sequence, or an expressed sequence and a non-coding sequence. In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding an antifusogenic polypeptide. In some embodiments, the polyribonucleotide cargo comprises an IRES operably linked to an expressed sequence encoding an antifusogenic polypeptide. In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding an antifusogenic polypeptide having a biological effect on a subject.

The polyribonucleotide cargo may comprise, for example, 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 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments, the polyribonucleotide cargo comprises 1-20,000 nucleotides, 1-10,000 nucleotides, 1-5,000 nucleotides, 100-20,000 nucleotides, 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.

In embodiments, the polyribonucleotide cargo comprises one or more expressed (or coding) sequences, where each expressed (or coding) sequence encodes a polypeptide (e.g., an antifusogenic polypeptide). In embodiments, the polyribonucleotide cargo comprises one or more non-coding sequences. In embodiments, the polyribonucleotide cargo consists entirely of non-coding sequences. In embodiments, the polyribonucleotide cargo comprises a combination of expressed (or coding) sequences and non-coding sequences.

In some embodiments, the polyribonucleotide comprises any feature, or any combination of features, as disclosed in WO 2019/118919, the entirety of which is incorporated herein by reference.

Polypeptide Expression Sequences In some embodiments, the polyribonucleotides described herein (e.g., the polyribonucleotide cargo of a circular polyribonucleotide) comprise one or more expression (or coding) sequences, where each expression sequence encodes an antifusogenic polypeptide. In some embodiments, the circular polyribonucleotide comprises two, three, four, five, six, seven, eight, nine, ten or more expression (or coding) sequences.

Each encoded polypeptide may be linear or branched. In various embodiments, the polypeptide has a length of about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1,000 to about 2,500 amino acids, or any range therebetween. In some embodiments, the polypeptides may be useful having 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.

The polypeptides included herein may include naturally occurring or non-naturally occurring polypeptides. In some embodiments, 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). For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein that has 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 to a polypeptide described herein or a naturally occurring polypeptide sequence, e.g., over a specific region or over the entire sequence. In some cases, the polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identity to a protein of interest.

In embodiments, the polypeptide comprises multiple polypeptides, e.g., multiple copies of a single polypeptide sequence, or multiple different polypeptide sequences. In embodiments, the multiple polypeptides are connected by linker or spacer amino acids.

In embodiments, the polynucleotide cargo comprises a sequence encoding a signal peptide. Many signal peptide sequences have been described, for example, the Tat (twin arginine translocation) signal sequence is typically an N-terminal peptide sequence containing the consensus SRRxFLK "twin arginine" motif that serves to translocate folded proteins containing such Tat signal peptides through lipid bilayers. See also, for example, the publicly available signal peptide database at www[dot]signalpeptide[dot]de. Signal peptides are also useful for directing proteins to specific organelles; see, for example, the experimentally measured and computationally predicted signal peptides disclosed in the Spdb signal peptide database, publicly available at proline.bic.nus.edu.sg/spdb.

In some embodiments, the expressed (or coding) sequence includes a polyA sequence (e.g., at the 3' end of the expressed sequence). In some embodiments, the length of the polyA sequence is more than 10 nucleotides in length. In one embodiment, the polyA sequence is more 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). In some embodiments, the polyA sequence is designed according to the description of polyA sequences in paragraphs [0202] to [0204] of WO 2019/118919 A1, the entirety of which is incorporated herein by reference. In some embodiments, the expression sequence lacks a polyA sequence (e.g., at the 3' end of the expression sequence).

In some embodiments, the cyclic polyribonucleotide comprises a polyA, lacks a polyA, or has a modified polyA to modulate one or more characteristics of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide lacks a polyA or has a modified polyA to improve one or more functional properties, such as immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response), half-life, and/or expression efficiency.

Internal Ribosome Entry Site In some embodiments, the circular polyribonucleotides described herein contain one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences). In embodiments, the IRES is located between the heterologous promoter and the 5' end of the coding sequence.

IRES elements suitable for inclusion within a polyribonucleotide include RNA sequences capable of associating with eukaryotic ribosomes. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

In some embodiments, the IRES element is derived from DNA of organisms including, but not limited to, viruses, mammals, and Drosophila. Such viral DNA can be derived from, but is not limited to, encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA, as well as picornavirus complementary DNA (cDNA). In one embodiment, the Drosophila DNA from which the IRES element is derived includes, but is not limited to, the antennapedia gene from Drosophila melanogaster.

In some embodiments, the IRES sequence is selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invictavirus 1, Rhopalopsis aphid virus, Reticuloendotheliosis virus, Human poliovirus 1, German winged stink bug enteric 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 (Equine rhinitis virus), tea moth picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Crucifer tobamo virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen brood virus, aphid lethal paralysis virus, avian encephalomyelitis virus (AEV), acute bee paralysis virus, hibiscus chlorotic ringspot virus virus), classical swine fever virus, human FGF2, human SFTPA1, human AML1/RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n. myc, mouse Gtx, human p27kipl, human PDGF2/c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, dog Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Salivirus, Cosavirus, Parechovirus, Drosophila hairless, yeast (S.c erevisiae TFIID, yeast (S. cerevisiae) YAP1, human c-src, human FGF-1, picomavirus, Turnip crinkle virus, Aichi virus, Krohi virus, Echovirus 11, an aptamer for eIF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1/2) IRES sequence. In yet another embodiment, the IRES is a Coxsackievirus B3 (CVB3) IRES sequence. In a further embodiment, the IRES is an encephalomyocarditis virus IRES sequence. In a further embodiment, the IRES is a Theiler's encephalomyelitis virus IRES sequence.

The IRES sequence may have a modified sequence compared to the wild-type IRES sequence. In some embodiments, if 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 so that it is a cytosine residue. For example, the IRES sequence may be a CVB3 IRES sequence in which the terminal adenosine residue is modified to a cytosine residue. In some embodiments, the modified CVB3 IRES may have the following nucleic acid sequence:

In some embodiments, the IRES sequence is an Enterovirus 71 (EV17) IRES. In some embodiments, the terminal guanosine residue of the EV17 IRES sequence is modified to a cytosine residue. In some embodiments, the modified EV71 IRES may have the following nucleic acid sequence:

In some embodiments, the polyribonucleotide comprises at least one IRES flanking at least one (e.g., two, three, four, five or more) expression sequence. In some embodiments, the IRES flanks both sides of at least one (e.g., two, three, four, five or more) expression sequence. In some embodiments, the polyribonucleotide comprises one or more IRES sequences on one or both sides of each expression sequence, resulting in separation of the resulting peptides and/or polypeptides. For example, the polyribonucleotides described herein may comprise a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.

In some embodiments, the polyribonucleotides described herein include an IRES (e.g., an IRES operably linked to a coding region). For example, the polyribonucleotides 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. NUCLEIC ACIDS RESEARCH 33(7):2248-58,2005; Petz et al. NUCLEIC ACIDS RESEARCH 35(8):2473-82,2007; Chen et al. SCIENCE 268:415-417,1995; Fan et al. NATURE COMMUNICATION 13(1):3751-3765,2022, and any IRES as described in WO 2021/263124.

Signal sequence In some embodiments, the antifusogenic polypeptide expressed from the cyclic polyribonucleotide disclosed herein comprises a secretory protein, e.g., a protein that naturally contains a signal sequence, or a protein that does not normally encode a signal sequence but has been modified to contain one. In some embodiments, the antifusogenic polypeptide encoded by the cyclic polyribonucleotide comprises a secretory signal. For example, the secretory signal can be a secretory signal naturally encoded for the secretory protein. In another example, the secretory signal can be a modified secretory signal for the secretory protein. In other embodiments, the antifusogenic polypeptide encoded by the cyclic polyribonucleotide does not comprise a secretory signal.

In some embodiments, the signal sequence is selected from SecSP38 (MWWRLWWLLLLLLLLWPMVWA; SEQ ID NO: 327); SecD4 (MWWLLLLLLLLLWPMVWA; SEQ ID NO: 328); gLuc (MGVKVLFALICIAVAEAK; SEQ ID NO: 329); INHC1 (MASRLTLLTLLLLLLLAGDRASS; SEQ ID NO: 330); Epo (MGVHECPAWLWLLLSLLSLPLGLPVLG; SEQ ID NO: 331); and IL-2 (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 332).

In some embodiments, the cyclic polyribonucleotide encodes multiple copies of the same anti-fusion polypeptide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, at least one copy of the anti-fusion polypeptide includes a signal sequence and at least one copy of the anti-fusion polypeptide does not include a signal sequence. In some embodiments, the cyclic polyribonucleotide encodes multiple anti-fusion polypeptides (e.g., multiple different anti-fusion polypeptides or multiple anti-fusion polypeptides having less than 100% sequence identity), at least one of the multiple anti-fusion polypeptides includes a signal sequence and at least one copy of the multiple anti-fusion polypeptides does not include a signal sequence.

In some embodiments, the signal sequence is a wild-type signal sequence that is present at the N-terminus of the corresponding wild-type antifusogenic polypeptide, e.g., when endogenously expressed. In some embodiments, the signal sequence is heterologous to the antifusogenic polypeptide, e.g., is not present when the wild-type antifusogenic polypeptide is endogenously expressed. The polyribonucleotide sequence encoding the antifusogenic polypeptide may be modified to remove nucleotide sequences encoding the wild-type signal sequence and/or to add sequences encoding a heterologous signal sequence.

A polypeptide encoded by a polyribonucleotide (e.g., an antifusogenic polypeptide) may contain a signal sequence that directs the antifusogenic polypeptide to the secretory pathway. In some embodiments, the signal sequence may direct the antifusogenic polypeptide to be present in a specific organelle (e.g., the endoplasmic reticulum, the Golgi apparatus, or an endosome). In some embodiments, the signal sequence directs the antifusogenic polypeptide to be secreted from the cell. In the case of a secreted protein, the signal sequence may be cleaved after secretion to result in the mature protein. In other embodiments, the signal sequence may be embedded in the membrane of the cell or a specific organelle to form a transmembrane segment that anchors the protein to the membrane of the cell, the endoplasmic reticulum, or the Golgi apparatus. In certain embodiments, the signal sequence of a transmembrane protein is a short sequence at the N-terminus of the polypeptide. In other embodiments, the first transmembrane domain acts as a first signal sequence that targets the protein to the membrane.

In some embodiments, the secretion signal is a human interleukin-2 (IL-2) secretion signal. In some embodiments, the IL-2 secretion signal has an amino acid sequence with at least 90% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO:332). In some embodiments, the IL-2 secretion signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO:332. In some embodiments, the IL-2 secretion signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO:332. In some embodiments, the IL-2 secretion signal has an amino acid sequence with 100% sequence identity to SEQ ID NO:332.

In some embodiments, the secretion signal is a Gaussia luciferase secretion signal. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence of at least 90% sequence identity to MGVKVLFALICIAVAEAK (SEQ ID NO:329). In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence of at least 95% sequence identity to SEQ ID NO:329. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence of at least 99% sequence identity to SEQ ID NO:329. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence of 100% sequence identity to SEQ ID NO:329.

In some embodiments, the secretory signal is an EPO (e.g., human EPO) secretory signal. In some embodiments, the EPO secretory signal has an amino acid sequence with at least 90% sequence identity to MGVHECPAWLWLLLSLLSLPLGLPVLGA (SEQ ID NO: 333). In some embodiments, the EPO secretory signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 333. In some embodiments, the EPO secretory signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 333. In some embodiments, the EPO secretory signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 333.

In some embodiments, the secretion signal is a wild-type SARS-CoV-2 secretion signal. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 90% sequence identity to MFVFLVLLPLVSS (SEQ ID NO:334). In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO:334. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO:334. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with 100% sequence identity to SEQ ID NO:334.

In some embodiments, the antifusogenic polypeptide encoded by the polyribonucleotide includes either a secretory signal sequence, a transmembrane insertion signal sequence, or no signal sequence.

Regulatory Elements In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide cargo of a polyribonucleotide) described herein comprises one or more regulatory elements. In some embodiments, a polyribonucleotide comprises a regulatory element, e.g., a sequence that regulates expression of an expression sequence within the polyribonucleotide.

A regulatory element may include a sequence located adjacent to an expression sequence that encodes an expression product. The regulatory element may be operably linked to the adjacent sequence. A regulatory element may increase the amount of an expressed product compared to the amount of product expressed in the absence of the regulatory element. Furthermore, a single regulatory element may increase the amount of product expressed for multiple expression sequences linked side-by-side. Thus, a single regulatory element can promote expression of one or more expression sequences. Multiple regulatory elements are well known to those of skill in the art.

In some embodiments, the regulatory element is a translation modulator. The translation modulator may regulate translation of an expressed sequence in the polyribonucleotide. The translation modulator may be a translation enhancer or suppressor. In some embodiments, the polyribonucleotide comprises at least one translation modulator adjacent to at least one expressed sequence. In some embodiments, the polyribonucleotide comprises a translation modulator adjacent to each expressed sequence. In some embodiments, the translation modulator is on one or both sides of each expressed sequence, resulting in the separation of the expression products, e.g., peptides and/or polypeptides.

In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.

Further examples of regulatory elements are described, for example, in paragraphs [0154] to [0161] of WO 2019/118919, the entire contents of which are incorporated herein by reference.

Cleavage Domain Circular polyribonucleotides of the present disclosure can include a cleavage domain (e.g., a stagger element or cleavage sequence).

The term "stagger element" refers to a moiety, e.g., a nucleotide sequence, that induces ribosome pausing during translation. In some embodiments, the stagger element is a non-conserved sequence of amino acids with strong alpha-helical propensity followed by the consensus sequence -D(V/I)ExNPGP, where x=any amino acid (SEQ ID NO:335). In some embodiments, the stagger element can include a chemical moiety such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof.

In some embodiments, the cyclic polyribonucleotide comprises at least one stagger element flanking the expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element flanking each expressed sequence. In some embodiments, the stagger element is present on one or both sides of each expressed sequence, resulting in separation of the expression products, e.g., peptides and/or polypeptides. In some embodiments, the stagger element is part of one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide comprises one or more expressed sequences, each of which is separated from the subsequent expressed sequence by a stagger element on the cyclic polyribonucleotide. In some embodiments, the stagger element prevents the generation of a single polypeptide from (a) two translations of a single expressed sequence, or (b) one or more translations of two or more expressed sequences. In some embodiments, the stagger element is a separate sequence from one or more expressed sequences. In some embodiments, the stagger element comprises a portion of an expressed sequence of one or more expressed sequences.

In some embodiments, the circular polyribonucleotide comprises a stagger element. To avoid the production of continuous expression products, such as peptides or polypeptides, while maintaining rolling circle translation, a stagger element can be included to induce ribosome pausing during translation. In some embodiments, the stagger element is at the 3' end of at least one of the one or more expressed sequences. The stagger element can be configured to stall ribosomes during rolling circle translation of the circular polyribonucleotide. The stagger element can include, but is not limited to, 2A-like or CHYSEL (SEQ ID NO: 336) (cis-acting hydrolase element) sequences. In some embodiments, the stagger element encodes a sequence having a C-terminal consensus sequence that is X ₁ X ₂ X ₃ EX ₅ NPGP (SEQ ID NO: 337), where X ₁ is absent or G or H, X ₂ is absent or D or G, X ₃ is D or V or I or S or M, and X ₅ is any amino acid. In some embodiments, the sequence comprises a non-conserved sequence of amino acids with strong alpha-helical propensity followed by the consensus sequence -D(V/I)EXNPGP (SEQ ID NO:338), where x = any amino acid. Some non-limiting examples of stagger elements include GDVESNPGP (SEQ ID NO:339), GDIEENPGP (SEQ ID NO:340), VEPNPGP (SEQ ID NO:341), IETNPGP (SEQ ID NO:342), GDIESNPGP (SEQ ID NO:343), GDVELNPGP (SEQ ID NO:344), GDIETNPGP (SEQ ID NO:345), GDVENPGP (SEQ ID NO:346), GDVEENPGP (SEQ ID NO:347), GDV These include EQNPGP (SEQ ID NO:348), IESNPGP (SEQ ID NO:349), GDIELNPGP (SEQ ID NO:350), HDIETNNPGP (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).

In some embodiments, the stagger elements described herein cleave the expression product, such as between the G and P of the consensus sequences described herein. As one non-limiting example, the cyclic polyribonucleotide includes at least one stagger element for cleaving the expression product. In some embodiments, the cyclic polyribonucleotide includes a stagger element adjacent to at least one expressed sequence. In some embodiments, the cyclic polyribonucleotide includes a stagger element after each expressed sequence. In some embodiments, the cyclic polyribonucleotide includes a stagger element present on one or both sides of each expressed sequence, resulting in translation of individual peptides and/or polypeptides from each expressed sequence.

In some embodiments, the stagger element comprises one or more modified or non-natural nucleotides that induce ribosome pausing during translation. Non-natural nucleotides can include peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Examples such as these are distinguished from naturally occurring DNA or RNA by changes in the backbone of the molecule. Exemplary modifications can include any modification to the sugar, nucleobase, internucleoside bond (e.g., to the linking phosphate/to the phosphodiester bond/to the phosphodiester backbone), and any combination thereof that can induce ribosome pausing during translation. Some of the exemplary modifications provided herein are described elsewhere herein.

In some embodiments, the stagger element is present in the circular polyribonucleotide in other forms. For example, in some exemplary circular polyribonucleotides, the 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 expression following the first expression sequence. In some examples, the first stagger element of the first expression sequence is upstream (5') of the first translation initiation sequence of expression following the first expression sequence in the circular polyribonucleotide. In some cases, the first expression sequence and the expression sequence following 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 may allow for continuous translation of the first expression sequence and the subsequent expression sequence.

In some embodiments, the first stagger element comprises a termination element, which separates the expression product of a first expression sequence from the expression product of the subsequent expression sequence, thereby creating discrete expression products. In some cases, a circular polyribonucleotide comprising a first stagger element upstream of a first translation initiation sequence of a subsequent sequence in the circular polyribonucleotide is translated continuously, and a corresponding circular polyribonucleotide comprising a stagger element of a second expression sequence upstream of a second translation initiation sequence of a subsequent expression sequence of a second expression sequence is not translated continuously. In some cases, there is only one expression sequence in the circular polyribonucleotide, and the first expression sequence and its subsequent expression sequence are the same expression sequence. In some exemplary circular polyribonucleotides, the stagger element comprises a first termination element of a first expression sequence in the circular polyribonucleotide and a nucleotide spacer sequence that separates the termination element from a downstream translation initiation sequence. In some such examples, the first stagger element is upstream (5') of the first translation initiation sequence of a first expression sequence in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence allows for continuous translation of the first expressed sequence and any subsequent expressed sequences.

In some embodiments, the first stagger element separates the expression products of one round of the first expression sequence from the expression products of the next round of the first expression sequence, thereby creating discrete expression products. In some cases, a circular polyribonucleotide that includes a first stagger element upstream of a first translation initiation sequence of a first expression sequence in a circular polyribonucleotide is translated continuously, and a corresponding circular polyribonucleotide that includes a stagger element upstream of a second translation initiation sequence of a second expression sequence in a corresponding circular polyribonucleotide is not translated continuously. In some cases, the distance between the second stagger element and the second translation initiation sequence is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater in the corresponding circular polyribonucleotide than the distance between the first stagger element and the first translation initiation in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation start 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 more. In some embodiments, the distance between the second stagger element and the second translation start 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 start. In some embodiments, the circular polyribonucleotide comprises two or more expressed sequences.

Examples of stagger elements are described in paragraphs [0172] to [0175] of WO 2019/118919, the entire contents of which are incorporated herein by reference.

In some embodiments, the multiple antifusogenic polypeptides encoded by the cyclic ribonucleotide may be separated by an IRES between each antifusogenic polypeptide (e.g., each antifusogenic polypeptide is operably linked to a separate IRES). For example, the cyclic 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 the antifusogenic polypeptides. The IRES may be different between the different antifusogenic polypeptides.

In some embodiments, the antifusogenic polypeptides can be separated by a 2A self-cleaving peptide. For example, a circular polyribonucleotide can encode a first antifusogenic polypeptide, 2A, and an IRES operably linked to an open reading frame encoding a second antifusogenic polypeptide.

In some embodiments, the antifusogenic polypeptides may be separated by a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first antifusogenic polypeptide, a protease cleavage site (e.g., a furin cleavage site), and a second antifusogenic polypeptide.

In some embodiments, the multiple antifusogenic polypeptides may be separated by a 2A autocleavage peptide and a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode a first antifusogenic polypeptide, 2A, a protease cleavage site (e.g., a furin cleavage site), and an IRES operably linked to an open reading frame encoding a second antifusogenic polypeptide. A circular polyribonucleotide may also encode a first antifusogenic polypeptide, a protease cleavage site (e.g., a furin cleavage site), 2A, and an IRES operably linked to an open reading frame encoding a second antifusogenic polypeptide. The tandem 2A and furin cleavage site may be referred to as furin 2A (including furin-2A or 2A-furin, arranged in either orientation).

Furthermore, multiple antifusogenic polypeptides encoded by cyclic ribonucleotides may be separated by both an IRES and a 2A sequence. For example, 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 a third antifusogenic polypeptide. Selection of a particular IRES or 2A self-cleaving peptide may be used to control the expression level of the antifusogenic polypeptide under the control of the IRES or 2A sequence. For example, depending on the IRES and/or 2A peptide selected, expression on the polypeptide may be higher or lower.

In some embodiments, the cyclic polyribonucleotide comprises 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, the cleavage sequence is included in an expression sequence. In some embodiments, the cyclic polyribonucleotide comprises 2-10 cleavage sequences. In some embodiments, the cyclic polyribonucleotide comprises 2-5 cleavage sequences. In some embodiments, multiple cleavage sequences are between multiple expression sequences, for example, the cyclic polyribonucleotide may comprise three expression sequences, two cleavage sequences, such that there is a cleavage sequence between each expression sequence. In some embodiments, the cyclic polyribonucleotide comprises a cleavage sequence, such as in a sacrificial circRNA or a cleavable circRNA or a self-cleaving circRNA. In some embodiments, the cyclic polyribonucleotide comprises two or more cleavage sequences that result in the separation of the cyclic polyribonucleotide into multiple products, such as miRNAs, linear RNAs, smaller cyclic polyribonucleotides, etc.

In some embodiments, the cleavage sequence comprises a ribozyme RNA sequence. Ribozymes (from ribonucleic acid enzymes, also called RNA enzymes or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many naturally occurring ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds or the hydrolysis of bonds in other RNAs, but have also been found to catalyze the aminotransferase activity of ribosomes. Catalytic RNAs can be "evolved" by in vitro methods. Similar to the riboswitch activity described above, ribozymes and their reaction products can regulate gene expression. In some embodiments, catalytic RNAs or ribozymes can be placed within a larger non-coding RNA such that the ribozyme is present in many copies within the cell for the purpose of chemical transformation of molecules from bulk volume. In some embodiments, the aptamer and ribozyme can both be encoded in the same non-coding RNA.

In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, the polyribonucleotide may encode two or more antifusogenic polypeptides, e.g., two or more antifusogenic polypeptides are encoded by a single open reading frame (ORF). For example, two or more antifusogenic polypeptides may be encoded by a single open reading frame, the expression of which is controlled by an IRES. In some embodiments, the ORF further encodes a polypeptide linker, e.g., such that the expression product of the ORF encodes two or more antifusogenic polypeptides, each separated by a sequence encoding a polypeptide linker (e.g., a linker of 5-200, 5-100, 5-50, 5-20, 50-100, or 50-200 amino acids). The polypeptide linker may include a cleavage site, e.g., a cleavage site that is recognized and cleaved by a protease (e.g., an endogenous protease in the subject following administration of the polyribonucleotide to the subject). In such embodiments, a single expression product containing the amino acid sequences of two or more antifusogenic polypeptides is cleaved during expression, resulting in the separation of two or more antifusogenic polypeptides after expression. Exemplary protease cleavage sites, such as amino acid sequences that act as protease cleavage sites recognized by metalloproteinases (e.g., matrix metalloproteinases (MMPs), e.g., any one or more of MMP1-28), disintegrins and metalloproteinases (ADAMs, e.g., any one or more of ADAM2, 7-12, 15, 17-23, 28-30, and 33), serine proteases (e.g., furin), urokinase-type plasminogen activator, matriptase, cysteine proteases, aspartic acid proteases, or cathepsin proteases, are known to those of skill in the art. In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is matriptase.

In some embodiments, the cyclic polyribonucleotide described herein is a sacrificial cyclic polyribonucleotide, a cleavable cyclic polyribonucleotide, or a self-cleaving cyclic polyribonucleotide. The cyclic polyribonucleotide can deliver a cellular component, including, for example, RNA, lncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA. In some embodiments, the cyclic polyribonucleotide comprises an miRNA separated by (i) a self-cleaving element; (ii) a cleavage recruitment site; (iii) a degradable linker; (iv) a chemical linker; and/or (v) a spacer sequence. In some embodiments, the circRNA comprises an siRNA separated by (i) a self-cleaving element; (ii) a cleavage recruitment site (e.g., ADAR); (iii) a degradable linker (e.g., glycerol); (iv) a chemical linker; and/or (v) a spacer sequence. Non-limiting examples of self-cleaving elements include hammerheads, splicing elements, hairpins, Hepatitis Delta Virus (HDV), Varkud satellite (VS), and glmS ribozymes.

Translation initiation sequence In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide cargo of a polyribonucleotide) described herein comprises at least one translation initiation sequence. In some embodiments, the polyribonucleotide comprises a translation initiation sequence that is operably linked to an expression sequence.

In some embodiments, the polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence, e.g., a Kozak sequence, flanking an expressed sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, a translation initiation sequence, e.g., a Kozak sequence, is present on one or both sides of each expressed sequence, resulting in separation of the expression products. In some embodiments, the polyribonucleotide includes at least one translation initiation sequence flanking an expressed sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single-stranded region of the circular polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs [0163] to [0165] of WO 2019/118919, which is incorporated herein by reference in its entirety.

A polyribonucleotide may include two or more start codons, 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 begin at the first start codon or may begin downstream of the first start codon.

In some embodiments, the polyribonucleotide may initiate at the first start codon, e.g., a codon that is not AUG. Translation of the polyribonucleotide may initiate at an alternative translation start sequence, e.g., but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG. In some embodiments, translation initiates at an alternative translation start sequence under selective conditions, e.g., stress-inducing conditions. As a non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, e.g., ACG. As another non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, CTG/CUG. As another non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, GTG/GUG. As another non-limiting example, polyribonucleotides can initiate translation at alternative translation initiation sequences, including repeat-associated non-AUG (RAN) sequences, e.g., short stretches of repetitive RNA, e.g., CGG, GGGGCC, CAG, CTG.

Termination Sequence In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide cargo of a polyribonucleotide) described herein comprises at least one termination sequence. In some embodiments, the polyribonucleotide comprises a termination sequence that is operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination sequence.

In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence. In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence, such that the polyribonucleotide is continuously translated. Exclusion of the termination sequence may result in rolling circle translation or continuous expression of the expression product.

In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence, such that the circular polyribonucleotide is translated continuously. The omission of the termination sequence may result in rolling circle translation or continuous expression of expression products, such as peptides or polypeptides, due to lack of ribosome stalling or shedding. In such embodiments, the rolling circle translation expresses continuous expression products through each expression sequence. In some other embodiments, the termination sequence of the expression sequence may be part of a stagger element. In some embodiments, one or more expression sequences in the circular polyribonucleotide include a termination sequence. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the circular polyribonucleotide is performed. In such cases, the expression product may fall off the ribosome when the ribosome encounters a termination sequence, e.g., a stop codon, terminating translation. In some embodiments, translation is terminated while the ribosome, e.g., at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.

In some embodiments, the circular polyribonucleotide includes a termination sequence at the end of one or more expressed sequences. In some embodiments, the one or more expressed sequences include two or more subsequent termination sequences. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome is completely separated from the circular polyribonucleotide. In some such embodiments, the generation of the subsequent (e.g., second, third, fourth, fifth, etc.) expressed sequence in the circular polyribonucleotide may require the ribosome to reassociate with the circular polyribonucleotide before the start of translation. Generally, the termination sequence includes an in-frame nucleotide triplet, e.g., UAA, UGA, UAG, that signals the termination of translation. In some embodiments, one or more termination sequences in the circular polyribonucleotide are frame-shifted termination sequences, such as, but not limited to, off-frame or -1 and +1 shifted reading frames (e.g., cryptic stops) that may terminate translation. Frameshifted termination sequences include the triplet nucleotides TAA, TAG, and TGA that occur in the second and third reading frames of the expressed sequence. Frameshifted termination sequences can be important to prevent misreading of the mRNA, which is often harmful to the cell. In some embodiments, the termination sequence is a stop codon.

Further examples of termination sequences are described in paragraphs [0169] to [0170] of WO 2019/118919, the entire contents of which are incorporated herein by reference.

Untranslated Region In some embodiments, the circular polyribonucleotide comprises an untranslated region (UTR). The UTR of a genomic region that includes a gene may be transcribed but not translated. In some embodiments, the UTR may be included upstream of the translation initiation sequence of an expression sequence described herein. In some embodiments, the UTR may be included downstream of an expression sequence described herein. In some cases, one UTR for a first expression sequence is the same as, contiguous with, or overlaps with another UTR for a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, such as ZKSCAN1.

Exemplary untranslated regions are described in paragraphs [0197] to [201] of WO 2019/118919, the entire contents of which are incorporated herein by reference.

In some embodiments, the cyclic polyribonucleotide comprises a polyA sequence. Exemplary polyA sequences are described in paragraphs [0202]-[0205] of WO 2019/118919, which is incorporated by reference in its entirety. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence.

In some embodiments, the cyclic polyribonucleotide comprises a UTR that contains one or more stretches of adenosines and uridines. These AU-rich signatures may increase the turnover rate of the expression product.

Introduction, removal, or modification of AU-rich elements (AREs) in UTRs can be useful for modulating the stability or immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response) of a cyclic polyribonucleotide. When modifying a particular cyclic polyribonucleotide, one or more copies of an ARE may be introduced into the cyclic polyribonucleotide, which copies of the ARE may modulate the translation and/or production of the expression product. Similarly, AREs can be identified and removed or modified into a cyclic polyribonucleotide to modulate the intracellular stability and thus affect the translation and production of the resulting protein.

It should be understood that any UTR from any gene may be incorporated into each flanking region of the circular polyribonucleotide.

In some embodiments, the cyclic polyribonucleotide lacks a 5'UTR and is capable of protein expression from its one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a 3'UTR and is capable of protein expression from its one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence and is capable of protein expression from its one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a termination sequence and is capable of protein expression from its one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site and is capable of protein expression from its one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a cap and is capable of protein expression from its one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a 5'UTR, a 3'UTR, and an IRES and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide comprises one or more of the following sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding an exogenous gene, a sequence encoding a therapeutic, a regulatory element (e.g., a translation modulator, e.g., a translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids (e.g., siRNA, lncRNA, shRNA) targeting an endogenous gene, and a sequence encoding a therapeutic mRNA or protein.

In some embodiments, the cyclic polyribonucleotide lacks a 5'UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3'UTR. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence. In some embodiments, the cyclic polyribonucleotide lacks a termination sequence. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks susceptibility to degradation by an exonuclease. In some embodiments, the fact that the cyclic polyribonucleotide lacks susceptibility to degradation can mean that the cyclic polyribonucleotide is not degraded by an exonuclease or is degraded to a limited extent in the presence of an exonuclease, e.g., is equivalent or similar in the absence of an exonuclease. In some embodiments, the cyclic polyribonucleotide is not degraded by an exonuclease. In some embodiments, the cyclic polyribonucleotide has reduced degradation when exposed to an exonuclease. In some embodiments, the cyclic polyribonucleotide lacks a linkage to a cap-binding protein. In some embodiments, the cyclic polyribonucleotide lacks a 5' cap.

Protein Binding Sequences In some embodiments, the cyclic polyribonucleotide comprises one or more protein binding sites that allow a protein, e.g., a ribosome, to bind to an internal site within the RNA sequence. By engineering a protein binding site, e.g., a ribosome binding site, into the cyclic polyribonucleotide, the cyclic polyribonucleotide can evade or reduce detection by the host's immune system by masking the cyclic polyribonucleotide from components of the host's immune system, modulating degradation, or modulating translation.

In some embodiments, the cyclic polyribonucleotide comprises at least one immunity protein binding site, e.g., to evade an immune response, e.g., a CTL (cytotoxic T lymphocyte) response. In some embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and aids in masking the cyclic polyribonucleotide as exogenous. In some embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and aids in masking the cyclic polyribonucleotide as exogenous or foreign.

The traditional mechanism of ribosome association to linear RNA involves ribosome binding to the capped 5' end of the RNA. The first peptide bond is formed as soon as the ribosome moves from the 5' end to the start codon. According to the present disclosure, internal initiation of translation of a circular polyribonucleotide (i.e., cap-independent) does not require a free or capped end. Rather, the ribosome binds to an uncapped internal site, whereby the ribosome initiates polypeptide elongation at the start codon. In some embodiments, the circular polyribonucleotide comprises one or more RNA sequences that include a ribosome binding site, e.g., a start codon.

Natural 5'UTRs have characteristics that play a role in translation initiation. They contain signatures such as the Kozak sequence, which is commonly known to be involved in the process by which the ribosome initiates the translation of many genes. The Kozak sequence has 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), followed by another "G". 5'UTRs are also known to form secondary structures involved in elongation factor binding.

In some embodiments, the cyclic polyribonucleotide encodes a protein binding sequence that binds to a protein. In some embodiments, the protein binding sequence targets or localizes the cyclic polyribonucleotide to a specific target. In some embodiments, the protein binding sequence specifically binds to an arginine-rich region of a protein.

In some embodiments, the protein binding sites include, but are not limited to, ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, EL AVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, LIN28A, LIN28B , m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2, POLR2A, PRPF8, PTBP1, RBFOX 2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF 1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1, TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF1, WDR33, XRN2, YBX1, YTHDC1, YTHDF1, YTHDF2, YWHAG, ZC3H7B, PDK1, AKT1, and any other protein that binds to RNA.

Spacer Sequence In some embodiments, the polyribonucleotides described herein comprise one or more spacer sequences. A spacer refers to any adjacent nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. A spacer may be present between any of the nucleic acid elements described herein. A spacer may also be present within the nucleic acid elements described herein.

For example, the nucleic acid includes two or more of any 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' Group I 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, a spacer may be present 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).

In some embodiments, the polyribonucleotide further comprises a first spacer region between the 5' exon fragment of (C) and the polyribonucleotide cargo of (D). The spacer may be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, the polyribonucleotide further comprises a second spacer region between the polyribonucleotide cargo of (D) and the 5' exon fragment of (E).

Spacer sequences can be used to separate the IRES from adjacent structural elements to modulate the structure and function of the IRES or adjacent elements. Spacers can be specifically engineered depending on the IRES. In some embodiments, RNA folding computer software such as RNAFold can be used to guide the design of various elements of the vector, including spacers.

The spacer may be, for example, 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, for example, 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 include a poly A-G sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region include a poly A-T sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region include a random sequence.

Spacers may also be present within the nucleic acid regions described herein. For example, a polynucleotide cargo region may include one or more spacers. Spacers may separate regions within a polynucleotide cargo.

In some embodiments, the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 30 nucleotides in length. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the spacer sequence is 20-50 nucleotides in length. In certain embodiments, the spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

The spacer sequence can be a polyA sequence, a polyA-C sequence, a polyC sequence, or a polyU sequence.

In some embodiments, the spacer sequence can be poly A-T, poly A-C, poly A-G, or a random sequence.

Exemplary spacer sequences are described in paragraphs [0293] to [0302] of WO 2019/118919, which is incorporated herein by reference in its entirety.

In some embodiments, the polyribonucleotide comprises a 5' spacer sequence (e.g., between the 5' annealing region and the polyribonucleotide cargo). In some embodiments, the 5' spacer sequence is at least 10 nucleotides in length. In another embodiment, the 5' spacer sequence is at least 15 nucleotides in length. In further embodiments, the 5' spacer sequence is at least 30 nucleotides in length. In some embodiments, 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. In some embodiments, 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 20-50 nucleotides in length. In certain embodiments, the 5' 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. In one embodiment, 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 comprises a polyA-G sequence. In some embodiments, the 5' spacer sequence comprises a polyA-T sequence. In some embodiments, the 5' spacer sequence comprises a random sequence.

In some embodiments, the polyribonucleotide comprises a 3' spacer sequence (e.g., between the 3' annealing region and the polyribonucleotide cargo). In some embodiments, the 3' spacer sequence is at least 10 nucleotides in length. In another embodiment, the 3' spacer sequence is at least 15 nucleotides in length. In further embodiments, the 3' spacer sequence is at least 30 nucleotides in length. In some embodiments, 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. In some embodiments, 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 20-50 nucleotides in length. In certain embodiments, the 3' 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. In one embodiment, the 3' 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 comprises a polyA-G sequence. In some embodiments, the 5' spacer sequence comprises a polyA-T sequence. In some embodiments, the 5' spacer sequence comprises a random sequence.

In one embodiment, the polyribonucleotide comprises a 5' spacer sequence but does not comprise a 3' spacer sequence. In another embodiment, the polyribonucleotide comprises a 3' spacer sequence but does not comprise a 5' spacer sequence. In another embodiment, the polyribonucleotide does not comprise a 5' or 3' spacer sequence. In another embodiment, the polyribonucleotide does not comprise an IRES sequence. In a further embodiment, the polyribonucleotide does not comprise an IRES sequence, a 5' spacer sequence, or a 3' spacer sequence.

In some embodiments, the spacer sequence is 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. nucleotides, at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least about 200 ribonucleotides, at least about 250 ribonucleotides, at least about 300 ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 600 ribonucleotides, at least about 700 ribonucleotides, at least about 800 ribonucleotides, at least about 900 ribonucleotides, or at least about 100 ribonucleotides.

Modifications The polyribonucleotides described herein (e.g., circular polyribonucleotides) may contain one or more substitutions, insertions and/or additions, deletions, and covalent modifications relative to a reference sequence, particularly a parent polyribonucleotide, and are included within the scope of the present disclosure.

In some embodiments, the circular polyribonucleotide comprises one or more post-transcriptional modifications (e.g., capping, truncation, polyadenylation, splicing, polyA sequences, 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 over 100 different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27:196-197). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the polyribonucleotide comprises at least one nucleoside selected from the group, such as those described in paragraph [0311] of WO 2019/118919, the entire contents of which are incorporated herein by reference.

Polyribonucleotides may include any useful modification to the sugar, nucleobase, or internucleoside linkage (e.g., to the linking phosphate/to the phosphodiester linkage/to the phosphodiester backbone), etc. One or more atoms of the pyrimidine nucleobase may be replaced or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl) or a halo (e.g., chloro or fluoro). In certain embodiments, a modification (e.g., one or more modifications) is present in each of the sugar and the internucleoside linkage. The modification may be from ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Further modifications are described herein.

In some embodiments, the polyribonucleotide comprises at least one N(6) methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the m6A modification can reduce the immunogenicity of the cyclic polyribonucleotide (e.g., reduce the level of one or more markers of an immune or inflammatory response).

In some embodiments, the modifications may include chemical or cell-induced modifications. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions" in Nat Reviews Mol Cell Biol, 2017, 18:202-210.

In some embodiments, chemical modifications to the ribonucleotides of the cyclic polyribonucleotides may enhance immune evasion. The cyclic polyribonucleotides may be modified according to 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 incorporated herein by reference. Modifications include, for example, end modifications, such as 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, reverse linkage, etc.), 3' end modifications (conjugation, DNA nucleotides, reverse linkage, 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. Modified ribonucleotide bases can also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications can modulate the expression, immune response, stability, subcellular localization of cyclic polyribonucleotides, to name a few functional effects. In some embodiments, modifications include biorthogonal nucleotides, such as unnatural bases. See, for example, Kimoto et al., Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039/c7cc06661a, which is incorporated herein by reference.

In some embodiments, the sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions of one or more ribonucleotides of a cyclic polyribonucleotide may include modifications or substitutions of phosphodiester linkages, as well as backbone modifications. Specific examples of cyclic polyribonucleotides include, but are not limited to, cyclic polyribonucleotides that include modified backbones or do not include natural internucleoside linkages, such as internucleoside modifications that include modifications or substitutions of phosphodiester linkages. Cyclic polyribonucleotides with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For purposes of this application, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In certain embodiments, a cyclic polyribonucleotide will include ribonucleotides that have a phosphorus atom in their internucleoside backbone.

Modified polyribonucleotide backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with reverse polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments, the cyclic polyribonucleotides may be negatively or positively charged.

Modified nucleotides that may be incorporated into polyribonucleotides may be modified at the internucleoside linkage (e.g., the phosphate backbone). In the context of polynucleotide backbones, the terms "phosphate" and "phosphodiester" are used interchangeably herein. The backbone phosphate group may be modified by replacing one or more of the oxygen atoms with different substituents. Additionally, modified nucleosides and nucleotides may include bulk replacement of the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linked oxygens replaced with sulfur. Phosphate linkers can also be modified by replacing the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates).

The α-thio substituted phosphate moieties are provided to impart stability to RNA and DNA polymers via unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently longer half-lives in the cellular environment. Phosphorothioates attached to cyclic polyribonucleotides are predicted to reduce innate immune responses via weaker binding/activation of cellular innate immune molecules.

In certain embodiments, the modified nucleoside comprises an alpha-thionucleoside (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).

Other internucleoside linkages that may be used in accordance with the present disclosure are described herein, including internucleoside linkages that do not contain a phosphorus atom.

In some embodiments, the cyclic polyribonucleotide may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into the cyclic polyribonucleotide, such as a bifunctional modification. Cytotoxic nucleosides may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), and 6-mercaptopurine. Further examples include fludarabine phosphate, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidate).

Polyribonucleotides may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purines or pyrimidines, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in a cyclic polyribonucleotide or a given predetermined sequence region thereof. In some embodiments, the cyclic polyribonucleotide includes pseudouridine. In some embodiments, the cyclic polyribonucleotide includes inosine, which may assist the immune system in characterizing cyclic polyribonucleotides as endogenous RNA versus viral RNA. Incorporation of inosine may also mediate improved RNA stability/reduced degradation. See, e.g., Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as "self". Cell Res. 25, 1283-1284 (incorporated herein by reference in its entirety).

In some embodiments, all nucleotides in a polyribonucleotide (or a given sequence region thereof) are modified. In some embodiments, modifications may include translational read-through (stagger elements), such as m6A, which may enhance expression; inosine, which may attenuate immune responses; pseudouridine, which may increase RNA stability, or m5C, which may increase stability; and 2,2,7-trimethylguanosine, which aids in cellular internalization (e.g., nuclear localization).

Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) can be present at various positions of the cyclic polyribonucleotide. One of skill in the art will appreciate that nucleotide analogs or other modifications can be placed at any position of the cyclic polyribonucleotide such that the function of the cyclic polyribonucleotide is not substantially diminished. Modifications can also be non-coding region modifications. Circular polyribonucleotides can contain from about 1% to about 100% modified nucleotides (either in relation to the overall nucleotide content or in relation to any one or more types of nucleotides, i.e., A, G, U, or C), or any intervening percentage (e.g., 1% to less than 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 1% to 10 ... 0%-90%, 10%-95%, 10%-100%, 20%-25%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20-95%, 20%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%).

Methods of Circularization The present disclosure provides methods for generating circular polyribonucleotides encoding antifusogenic polypeptides (e.g., polypeptides of Table 1), including, for example, recombinant techniques or chemical synthesis. For example, the DNA molecules used to generate the RNA circle can include DNA sequences of the original nucleic acid sequence occurring in nature, modified versions thereof, or DNA sequences encoding synthetic polypeptides 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, classical mutagenesis techniques and recombinant techniques, such as site-specific mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of a nucleic acid sequence, synthesis of mixtures of oligonucleotides and ligation of mixtures into a mixture of nucleic acid molecules "assembly," and combinations thereof.

In some embodiments, the linear polyribonucleotide for circularization may be circularized or concatemerized. In some embodiments, the linear polyribonucleotide for circularization may be circularized in vitro prior to formulation and/or delivery. In some embodiments, the circular polyribonucleotide may be a mixture with linear polyribonucleotides. In some embodiments, the linear polyribonucleotide has the same nucleic acid sequence as the circular polyribonucleotide.

In some embodiments, a linear polyribonucleotide for circularization is circularized or concatemerized using chemical methods to form a circular polyribonucleotide. In some chemical methods, the 5' and 3' ends of a nucleic acid (e.g., a linear polyribonucleotide for circularization) contain chemically reactive groups that, when in close proximity to one another, can form a new covalent bond between the 5' and 3' ends of the molecule. The 5' end can contain an NHS-ester reactive group and the 3' end can contain a 3' amino terminal nucleotide, such that in an organic solvent, the 3' amino terminal nucleotide on the 3' end of the linear RNA molecule undergoes nucleophilic attack on the 5'-NHS-ester moiety to form a new 5'/3' amide bond.

In some embodiments, a DNA or RNA ligase is used to enzymatically ligate 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 bond. In an exemplary reaction, a linear polyribonucleotide for circularization is incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) for 1 hour at 37°C according to the manufacturer's protocol. The ligation reaction can be performed in the presence of a linear nucleic acid that can base pair with both the 5' and 3' regions in juxtaposition to aid in the enzymatic ligation reaction. In some embodiments, the ligation is a splint ligation. For example, a splint ligase such as SplintR® ligase can be used for splint ligation, RNA ligase II, T4 RNA ligase, or T4 DNA ligase. For splint ligation, a single-stranded polynucleotide (splint), such as a single-stranded RNA, can be designed to hybridize with both ends of a linear polyribonucleotide, such that the two ends can be juxtaposed upon hybridization with the single-stranded splint. Thus, a splint ligase can catalyze the ligation of the two juxtaposed ends of a linear polyribonucleotide to generate a circular polyribonucleotide.

In some embodiments, DNA ligase or RNA ligase is used in the synthesis of the circular polynucleotide. In some embodiments, either the 5' or 3' end of the linear polyribonucleotide for circularization can encode a ligase ribozyme sequence, such that the linear polyribonucleotide for circularization can contain an active ribozyme sequence that can ligate the 5' end of the linear polyribonucleotide for circularization to the 3' end of the linear polyribonucleotide for circularization during in vitro transcription. The ligase ribozyme can be derived from group I introns, hepatitis delta virus, hairpin ribozymes, or selected by SELEX (Systematic Evolution of Ligands by Exponential Enrichment). The ribozyme ligase reaction can take 1-24 hours at temperatures between 0-37°C.

In some embodiments, the linear polyribonucleotide for circularization is circularized or concatemerized by using at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety may react with a region or feature near the 5' end and/or near the 3' end of the linear polyribonucleotide for circularization to circularize or concatemerize the linear polyribonucleotide for circularization. In another aspect, the at least one non-nucleic acid moiety may be located within or linked near the 5' end and/or 3' end of the linear polyribonucleotide for circularization. The contemplated non-nucleic acid moieties may be homogeneous or heterogeneous. As a non-limiting example, the non-nucleic acid moiety may be a bond such as a hydrophobic bond, an ionic bond, a biodegradable bond, and/or a cleavable bond. As another non-limiting example, the non-nucleic acid moiety is a ligation moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.

In some embodiments, linear polyribonucleotides for circularization are synthesized using IVT and an RNA polymerase, where the nucleotide mixture used for IVT may contain an excess of guanosine monophosphate compared to guanosine triphosphate to preferentially produce RNA with a 5' monophosphate, and the purified IVT product may be circularized using splinted DNA.

In some embodiments, the linear polyribonucleotide for circularization is circularized or concatemerized due to non-nucleic acid moieties that cause attractive forces between atoms, molecular surfaces at or near the 5' and 3' ends of the linear polyribonucleotide for circularization. As a non-limiting example, one or more linear polyribonucleotides for circularization may be circularized or concatemerized due to intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, invisible bonds, dipolar bonds, conjugation, hyperconjugation, and antibonds.

In some embodiments, the linear polyribonucleotide for circularization may include a ribozyme RNA sequence near the 5' end and near the 3' end. The ribozyme RNA sequence may be covalently attached to a peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, the peptides covalently attached to the ribozyme RNA sequence near the 5' end and near the 3' end may associate with each other to circularize or concatemerize the linear polyribonucleotide for circularization. In another aspect, the peptides covalently attached to the ribozyme RNA near the 5' end and near the 3' end may be subjected to ligation using various methods known in the art, such as, but not limited to, protein ligation, to circularize or concatemerize the linear primary construct or linear mRNA. A non-limiting list of examples of ribozymes or methods for incorporating and/or covalently linking peptides for use in the linear primary constructs or linear RNAs of the present invention is described in U.S. Patent Application Publication No. 20030082768, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, linear polyribonucleotides for circularization may include 5' triphosphates of nucleic acids that have been converted to 5' monophosphates, for example, by contacting the 5' triphosphates with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase (apyrase). In some embodiments, the 5' ends of at least some of the linear polyribonucleotides include monophosphate moieties. In some embodiments, a population of polyribonucleotides including circular and linear polyribonucleotides is contacted with RppH prior to digesting at least some of the linear polyribonucleotides with a 5' exonuclease and/or a 3' exonuclease. Alternatively, converting the 5' triphosphate of a linear polyribonucleotide for circularization to a 5' monophosphate can occur by a two-step reaction that includes: (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.

In some embodiments, the cyclization efficiency of the cyclization 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 cyclization efficiency of the cyclization methods provided herein is at least about 40%. In some embodiments, the provided cyclization methods have a cyclization efficiency of about 10% to about 100%, for example, the cyclization efficiency can 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%. In some embodiments, the cyclization efficiency is about 20% to about 80%. In some embodiments, the cyclization efficiency is about 30% to about 60%. In some embodiments, the cyclization efficiency is about 40%.

In some embodiments, the circular polyribonucleotide contains an internal splicing element where the spliced ends are joined together when duplicated. Some examples may include motifs found in cis sequence elements proximal to a back splice event (suptable4 enriched motifs), such as miniature introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences of adjacent introns, Alu elements of adjacent introns, and sequences within 200 bp before (upstream) or after (downstream) a back splice site with an adjacent exon. In some embodiments, the linear polyribonucleotide contains at least one repeated nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repeated nucleotide sequence may include a repeated sequence from the Alu family of introns. In some embodiments, the splicing-related ribosome-binding protein can regulate circular polyribonucleotide biosynthesis (e.g., Muscle blind and Quaking (QKI) splicing factor).

In some embodiments, the linear polyribonucleotide may include a canonical splice site adjacent to the head-to-tail junction of the circular polyribonucleotide.

In some embodiments, a linear polyribonucleotide may contain a bulge-helix-bulge motif, which comprises a four base pair stem flanked by two three nucleotide bulges. Cleavage occurs at the site of the bulge region, generating characteristic fragments with terminal 5'-hydroxyl groups and 2',3'-cyclic phosphates. Circularization proceeds by nucleophilic attack of the 5'-OH group on the 2',3'-cyclic phosphate of the same molecule forming a 3',5'-phosphodiester bridge.

In some embodiments, the linear polyribonucleotide may comprise a multimeric repeat RNA sequence having an HPR element. The HPR comprises a 2',3'-cyclic phosphate and a 5'-OH terminus. The HPR element self-processes the 5' and 3' ends of the linear polyribonucleotide, thereby joining the ends together.

In some embodiments, the linear polyribonucleotide may include a sequence that mediates self-ligation. In one embodiment, the linear polyribonucleotide may include a HDV sequence for self-ligation, e.g., the HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (Beeharry et al 2004) (SEQ ID NO: 359) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCGAGCC (SEQ ID NO: 360). In one embodiment, the linear polyribonucleotide may include a loop E sequence for self-ligation (e.g., PSTVd). In another embodiment, the linear polyribonucleotide may include a self-circularizing intron, such as a 5' and 3' slice junction, or a self-circularizing catalytic intron, such as a group I, group II, or group III intron. Non-limiting examples of group I intron self-splicing sequences may include the self-splicing permuted intron-exon sequence from the T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena.

In some embodiments, linear polyribonucleotides for circularization may contain complementary sequences that include either repeat or non-repetitive nucleic acid sequences within individual introns or across adjacent introns. Repeat nucleic acid sequences are sequences that are present within a segment of a linear polyribonucleotide. In some embodiments, the linear polyribonucleotide comprises a repeat nucleic acid sequence. In some embodiments, the repeat nucleotide sequence comprises a polyCA or polyUG sequence. In some embodiments, the linear polyribonucleotide comprises at least one repeat nucleic acid sequence that hybridizes to a complementary repeat nucleic acid sequence in another segment of the linear polyribonucleotide, the hybridized segment forming an internal duplex. In some embodiments, the linear polyribonucleotide comprises 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, and 10) repeat nucleic acid sequences that hybridize to a complementary repeat nucleic acid sequence in another segment of the linear polyribonucleotide, the hybridized segment forming an internal duplex. In some embodiments, the linear polyribonucleotide comprises two repeating nucleic acid sequences that hybridize to complementary repeating nucleic acid sequences in another segment of the linear polyribonucleotide, and the hybridized segments form an internal duplex. In some embodiments, the repeating nucleic acid sequences and complementary repeating nucleic acid sequences from two separate linear polyribonucleotides hybridize to generate a single circularized polyribonucleotide, and the hybridized segments form an internal duplex. In some embodiments, the complementary sequences are found at the 5' and 3' ends of the linear polyribonucleotide for circularization. In some embodiments, the complementary sequence comprises 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.

In some embodiments, chemical methods of circularization may be used to generate circular polyribonucleotides. 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, hemiamine limine crosslinking, base modification, and any combination thereof.

In some embodiments, enzymatic methods of circularization can be used to generate circular polyribonucleotides. In some embodiments, a ligation enzyme, such as a DNA or RNA ligase, can be used to generate a circular polyribonucleotide or a complement, a complementary strand of a circular polyribonucleotide, or a template for a circular polyribonucleotide.

Circularization of linear polyribonucleotides can be achieved by methods known in the art, for example, as described in "RNA circularization strategies in vivo and in vitro" by Petkovic and Muller in Nucleic Acids Res, 2015, 43(4):2454-2465 and "In vitro circularization of RNA" by Muller and Appel in RNA Biol, 2017, 14(8):1018-1027.

Circular polyribonucleotides may encode sequences and/or motifs useful for replication. Exemplary replication elements are described in paragraphs [0280]-[0286] of WO 2019/118919, which is incorporated by reference in its entirety.

In some embodiments, a linear polyribonucleotide may contain complementary sequences that include either repeat or non-repetitive nucleic acid sequences within individual introns or across adjacent introns. The repeat nucleic acid sequence is a sequence that is present within a segment of the circular polyribonucleotide. In some embodiments, the linear polyribonucleotide comprises a repeat nucleic acid sequence. In some embodiments, the repeat nucleotide sequence comprises a polyCA or polyUG sequence. In some embodiments, the linear polyribonucleotide comprises at least one repeat nucleic acid sequence that hybridizes to a complementary repeat nucleic acid sequence in another segment of the linear polyribonucleotide, and the hybridized segment forms an internal duplex. In some embodiments, the repeat nucleic acid sequence and the complementary repeat nucleic acid sequence from two separate linear polyribonucleotides hybridize to generate a single circularized polyribonucleotide, and the hybridized segment forms an internal duplex. In some embodiments, the complementary sequences are found at the 5' and 3' ends of the linear polyribonucleotide. In some embodiments, the complementary sequence comprises 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.

In some embodiments, chemical methods of circularization may be used to generate circular polyribonucleotides. 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, hemiamine limine crosslinking, base modification, and any combination thereof.

The method for producing the cyclic polyribonucleotides described herein is described, for example, in Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); in Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); Muller and Appel (from RNA Biol, 2017, 14(8):1018-1027); and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, 1999, 14(8):1018-1027. Acids, (First Edition), Wiley-VCH (2012). Other methods for making circular polyribonucleotides are described, for example, in WO 2022/247943, U.S. Pat. No. 11000547, WO 2018/191722, WO 2019/236673, WO 2020/023595, WO 2022/204460, WO 2022/204464, and WO 2022/204466.

Various methods for synthesizing cyclic polyribonucleotides have also been described elsewhere (see, e.g., U.S. Pat. No. 6,210,931, U.S. Pat. No. 5,773,244, U.S. Pat. No. 5,766,903, U.S. Pat. No. 5,712,128, U.S. Pat. No. 5,426,180, U.S. Patent Publication No. 20100137407, WO 1992001813, WO 2010084371, and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015), the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the circular polyribonucleotides are purified, e.g., free ribonucleic acid, linear or nicked RNA, DNA, proteins, etc. In some embodiments, the circular polyribonucleotides can be purified by any known method commonly used in the art. Non-limiting examples of purification methods include column chromatography, gel excision, size exclusion, etc.

Production method Production method in cell-free system The present disclosure also provides a method for producing circular RNA.For example, deoxyribonucleotide template can be transcribed in cell-free system (e.g., by in vitro transcription) to produce linear RNA.Linear polyribonucleotide produces splicing-compatible polyribonucleotide, which can self-splice to produce circular polyribonucleotide.

In some embodiments, the present disclosure provides a method of producing a circular polyribonucleotide (e.g., in a cell-free system) by providing a linear polyribonucleotide; and a 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.

In some embodiments, the disclosure provides a method of producing a circular polyribonucleotide by providing deoxyribonucleotides encoding a linear polyribonucleotide; transcribing the deoxyribonucleotides in a cell-free system to produce a 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.

In some embodiments, the disclosure provides a method of producing a circular polyribonucleotide by providing deoxyribonucleotides encoding a linear polyribonucleotide; transcribing the deoxyribonucleotides in a cell-free system to produce a linear polyribonucleotide, where the transcription is in solution under conditions suitable for splicing of the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide. In some embodiments, the linear polyribonucleotide comprises a 5' split-intron and a 3' split-intron (e.g., a self-splicing construct to produce a circular polyribonucleotide). In some embodiments, the linear polyribonucleotide comprises a 5' annealing region and a 3' annealing region.

Suitable conditions for in vitro transcription and self-splicing can include any conditions (e.g., a solution or buffer, such as an aqueous buffer or solution) that recapitulate physiological conditions in one or more respects. In some embodiments, suitable conditions include 0.1-100 mM Mg2+ ions or a salt thereof (e.g., 1-100 mM, 1-50 mM, 1-20 mM, 5-50 mM, 5-20 mM, or 5-15 mM). In some embodiments, suitable conditions include 1-1000 mM K+ ions or a salt thereof, such as KCl (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50-500 mM, 100-500 mM, or 100-300 mM). In some embodiments, suitable conditions include 1-1000 mM Cl- ions or a salt thereof such as KCl (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50-500 mM, 100-500 mM, or 100-300 mM). In some embodiments, suitable conditions include 0.1-100 mM Mn2+ ions or a salt thereof such as MnCl2 (e.g., 0.1-100 mM, 0.1-50 mM, 0.1-20 mM, 0.1-10 mM, 0.1-5 mM, 0.1-2 mM, 0.5-50 mM, 0.5-20 mM, 0.5-15 mM, 0.5-5 mM, 0.5-2 mM, or 0.1-10 mM). In some embodiments, suitable conditions include dithiothreitol (DTT) (e.g., 1-1000 μM, 1-500 μM, 1-200 μM, 50-500 μM, 100-500 μM, 100-300 μM, 0.1-100 mM, 0.1-50 mM, 0.1-20 mM, 0.1-10 mM, 0.1-5 mM, 0.1-2 mM, 0.5-50 mM, 0.5-20 mM, 0.5-15 mM, 0.5-5 mM, 0.5-2 mM, or 0.1-10 mM). In some embodiments, suitable conditions include 0.1 mM and 100 mM ribonucleoside triphosphates (NTPs) (e.g., 0.1-100 mM, 0.1-50 mM, 0.1-10 mM, 1-100 mM, 1-50 mM, or 1-10 mM). In some embodiments, suitable conditions include a pH of 4-10 (e.g., a pH of 5-9, a pH of 6-9, or a pH of 6.5-8.5). In some embodiments, 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).

In some embodiments, the linear polyribonucleotide is generated from a deoxyribonucleic acid, such as a deoxyribonucleic acid described herein, such as a DNA vector, a linearized DNA vector, or a cDNA. In some embodiments, the linear polyribonucleotide is transcribed from the deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).

Methods of production in a cell The present disclosure also provides a method of producing a circular RNA in a cell, for example, a prokaryotic or eukaryotic cell. In some embodiments, an exogenous polyribonucleotide is provided to the cell (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding the transcription of a linear polyribonucleotide described herein). The linear polyribonucleotide 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 that is transiently provided to the cell. In some embodiments, the exogenous DNA molecule is not integrated into the genome of the cell. In some embodiments, the linear polyribonucleotide is transcribed in the cell from a recombinant DNA molecule that is integrated into the genome of the cell.

In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell comprising the polyribonucleotides described herein may be a bacterial cell or an archaeal cell. For example, the prokaryotic cell comprising the polyribonucleotides described herein may be a bacterial cell or an archaeal cell. For example, the prokaryotic cell comprising the polyribonucleotides described herein may be a bacterial cell or an archaeal cell. elongatus), Spirulina (Arthrospira spp., and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp. (e.g., Bacillus subtilis, Bacillus anthracis, Bacillus cereus, cereus), betaproteobacteria (e.g., Burkholderia), alphaproteobacteria (e.g., Agrobacterium), Pseudomonas (e.g., Pseudomonas putida), and enterobacteria. The prokaryotic cells may be grown in a culture medium. The prokaryotic cells may be contained within a bioreactor.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell comprising the polyribonucleotides described herein is a unicellular eukaryotic cell. In some embodiments, the unicellular eukaryotic organism is a unicellular fungal cell, such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces species), Brettanomyces species, Schizosaccharomyces species, Torulaspora species, and Pichia species. In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. The unicellular animal cell may be a cell isolated from a multicellular animal and grown in culture, or a daughter cell thereof. In some embodiments, the unicellular animal cell may be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. The unicellular plant cell may be a cell isolated from a multicellular animal and grown in culture, or a daughter cell thereof. In some embodiments, the unicellular plant cell may be dedifferentiated. In some embodiments, the unicellular plant cell is derived from a plant callus. In embodiments, the unicellular cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algae cell, such as a unicellular green algae, diatom, euglenid, or dinoflagellate. Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris species, Protosiphon botryoides, Botryococcus braunii, and the like. braunii), Cryptococcus spp., Chlamydomonas reinhardtii, and other Chlamydomonas spp. In some embodiments, the unicellular eukaryotic cell is a protist cell. In some embodiments, the unicellular eukaryotic cell is a protozoan cell.

In some embodiments, the eukaryotic cell is a cell of a multicellular eukaryotic organism. For example, the multicellular eukaryotic organism may be selected from the group consisting of a vertebrate, an invertebrate, a multicellular fungus, a multicellular alga, and a multicellular plant. In some embodiments, the eukaryotic organism is a human. In some embodiments, the eukaryotic organism is a non-human vertebrate. In some embodiments, the eukaryotic organism is an invertebrate. In some embodiments, the eukaryotic organism is a multicellular fungus. In some embodiments, the eukaryotic organism is a multicellular plant. In embodiments, the eukaryotic cell is a human cell or a cell of a non-human mammal, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., bovids including cows, buffalo, bison, sheep, goats, and muskoxen; pigs; camelids including camels, llamas, and alpacas; deer, antelope; and equines including horses and donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare). In embodiments, the eukaryotic cell is an avian, e.g., a member of the avian taxonomic group Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, geese), Paleaognathae (e.g., ostrich, emu), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrot) cell. In embodiments, the eukaryotic cell is an arthropod (e.g., insect, arachnid, crustacean), nematode, annelid, worm, or mollusc cell. In embodiments, the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm (which may be dicotyledonous or monocotyledonous) or gymnosperm (e.g., conifer, cycad, gnetophyte, ginkgo), fern, horsetail, club moss, or bryophyte. In embodiments, 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 within a bioreactor.

Purification Methods One or more purification steps may be included in the methods described herein. For example, in some embodiments, the linear polyribonucleotide is substantially concentrated or pure (e.g., purified) prior to self-splicing the linear polyribonucleotide. In other embodiments, the linear polyribonucleotide is not purified prior to self-splicing the linear polyribonucleotide. In some embodiments, the resulting circular RNA is purified.

Purification may include separating or concentrating the desired reaction product from one or more undesired components, such as any unreacted starting materials, by-products, enzymes, or other reaction components. For example, purification of linear polyribonucleotides after transcription in a cell-free system (e.g., in vitro transcription) may include separation or concentration from the DNA template prior to self-splicing of the linear polyribonucleotide. Purification of the circular RNA product after splicing can be used to separate or concentrate the circular RNA from its corresponding linear RNA. Methods for purifying RNA are known to those of skill in the art and include enzymatic purification or chromatographic purification.

In some embodiments, the purification method results in cyclic polyribonucleotides having less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) linear polyribonucleotides.

Bioreactor In some embodiments, any method for producing circular polyribonucleotides described herein may be carried out in a bioreactor. A bioreactor refers to any container in which a chemical or biological process involving an organism or a biochemically active substance derived from such an organism is carried out. The bioreactor may be adapted for the cell-free method for producing circular RNA described herein. The container for the bioreactor may include a culture flask, dish, or bag, which may be single-use (disposable), autoclavable, or sterilizable. The bioreactor may be made of glass, or may be polymer-based, or may be manufactured from other materials.

Examples of bioreactors include, but are not limited to, stirred tank (e.g., well-mixed) and flat plate (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibratory mixers, fluidized bed reactors, and membrane bioreactors. The mode of operation of the bioreactor may be a batch or continuous process. A bioreactor is continuous when the flow of reagents and products is continuously provided and removed from the system. A batch bioreactor may have a continuous recirculation flow but may not have a continuous supply of reagents or product recovery.

Some methods of the present disclosure are directed to large-scale production of circular polyribonucleotides. For large-scale production methods, the methods can be performed in volumes 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). In some embodiments, the method can be performed with a volume of 5L to 10L, 5L to 15L, 5L to 20L, 5L to 25L, 5L to 30L, 5L to 35L, 5L to 40L, 5L to 45L, 10L to 15L, 10L to 20L, 10L to 25L, 20L to 30L, 10L to 35L, 10L to 40L, 10L to 45L, 10L to 50L, 15L to 20L, 15L to 25L, 15L to 30L, 15L to 35L, 15L to 40L, 15L to 45L, or 15 to 50L.

In some embodiments, the bioreactor may produce at least 1 g of circular RNA. In some embodiments, the bioreactor may produce 1-200 g of circular RNA (e.g., 1-10 g, 1-20 g, 1-50 g, 10-50 g, 10-100 g, 50-100 g, 50-200 g of circular RNA). In some embodiments, the amount produced is measured per liter (e.g., 1-200 g per liter), per batch or reaction (e.g., 1-200 g per batch or reaction), or per unit of time (e.g., 1-200 g per hour or day).

In some embodiments, two or more bioreactors can be utilized in series to increase production capacity (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 bioreactors can be used in series).

Methods of Use In some embodiments, circular polyribonucleotides encoding antifusogenic polypeptides (e.g., polypeptides of Table 1) are used to treat or prevent viral infections (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV).

In some embodiments, a circular polynucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1) is used to reduce viral entry.

In some embodiments, a circular polynucleotide encoding an antifusogenic polypeptide (e.g., a polypeptide in Table 1) can be administered to a subject to reduce the risk of viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV).

For example, a cyclic polyribonucleotide produced by the methods described herein may be administered to a subject (e.g., in a pharmaceutical, veterinary, or agricultural composition). In some embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., monkey, ape), an ungulate (e.g., cow, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horse, donkey), a carnivore (e.g., dog, cat), a rodent (e.g., rat, mouse), or a lagomorph (e.g., rabbit). In embodiments, the subject is an avian, such as a member of an avian taxon such as Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate, such as an arthropod (e.g., insects, arachnids, crustaceans), nematodes, annelids, worms, or mollusks.

In some embodiments, the disclosure provides a method of modifying a subject by providing a composition or preparation described herein to the subject. In some embodiments, the composition or preparation is or comprises a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or preparation is or comprises a eukaryotic or prokaryotic cell that contains a nucleic acid described herein.

In some embodiments, the disclosure provides a method of treating a viral infection in a subject in need thereof by providing a composition or preparation described herein to the subject. In some embodiments, the composition or preparation is or comprises a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and a polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or preparation is or comprises a eukaryotic or prokaryotic cell that comprises a nucleic acid described herein. In some embodiments, the polyribonucleotide is provided, for example, in an amount and for a duration sufficient to treat a viral infection in a subject in need of such treatment.

In some embodiments, the method may be used to treat or prevent HIV. For example, in some embodiments, the circular polyribonucleotide encodes an antifusogenic polypeptide that targets HIV, and the composition may be used to treat or prevent HIV.

In some embodiments, the methods can be used to treat or prevent SARS-CoV-2. For example, in some embodiments, the cyclic polyribonucleotide encodes an antifusogenic polypeptide that targets SARS-CoV-2, and the composition can be used to treat or prevent SARS-CoV-2.

In some embodiments, the method may be used to treat or prevent HCV. For example, in some embodiments, the cyclic polyribonucleotide encodes an antifusogenic polypeptide that targets HCV, and the composition may be used to treat or prevent HCV.

In some embodiments, the methods can be used to treat or prevent RSV. For example, in some embodiments, the cyclic polyribonucleotide encodes an antifusogenic polypeptide that targets RSV, and the composition can be used to treat or prevent RSV.

Methods of Administration Disclosed herein are dosing methods for producing a level of cyclic polyribonucleotides encoding an antifusogenic polypeptide (e.g., a polypeptide of Table 1) or expressing 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 cyclic polyribonucleotides. Disclosed herein are administration methods for producing a level of cyclic polyribonucleotides or expressing a level of an antifusogenic polypeptide (e.g., a polypeptide of Table 1, e.g., a polypeptide of any one of Tables 2-4) in a subject after providing (e.g., administering) at least two doses or compositions of cyclic polyribonucleotides to a subject (e.g., a mammal, e.g., a human). The composition comprises a cyclic polyribonucleotide encoding an antifusogenic polypeptide as described herein. The administration method can include administering two or more doses of a cyclic polyribonucleotide composition, e.g., over a short or long period of time. In some embodiments, the composition containing cyclic polyribonucleotide further comprises a pharma- ceutically acceptable carrier or excipient. The cyclic polyribonucleotide encodes an antifusogenic polypeptide that can be expressed in the cell, e.g., after administration.

The methods described herein can be used to determine whether a subject has 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, The method may include administering a first dose of the pharmaceutical composition in an amount sufficient to result in a serum concentration of the antifusogenic polypeptide of 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.

In some embodiments, the method may further include administering a second dose of the pharmaceutical composition. The method may further include administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more doses of the pharmaceutical composition. In some embodiments, subsequent doses provide a total serum IL-10 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, 200 ng/mL, 2100 ng/mL, 2200 ng/mL, 2300 ng/mL, 2400 ng/mL, 2500 ng/mL, 2600 ng/mL, 2700 ng/mL, 2800 ng/mL, 2900 ng/mL, 3000 ng/mL, 3100 ng/mL, 3200 ng/mL, 3300 ng/mL, 3400 ng/mL, 3500 ng/mL, 3600 ng/mL, 3700 ng/mL, 3800 ng/mL, 3900 ng/mL, 4000 ng/mL, 4100 ng/mL, 4200 ng/mL, 4300 ng/mL, 4400 ng/mL, 4500 ng/mL, 4600 ng/mL, 47 In some embodiments, the subsequent doses are administered before the serum concentration falls below 500 ng/mL of the antifusogenic polypeptide in the subject.

In some embodiments, multiple doses are provided to produce or maintain a level of the composition or to 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, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 21, or 24 months, or at least 1, 2, 3, 4, or 5 years.

In some embodiments, the second dose is administered at least 1 hour (e.g., at least 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more) after the first dose of the pharmaceutical composition.

In some embodiments, the second dose is administered 1 hour to 1 year (e.g., 1 hour to 1 day, e.g., 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 1 day, e.g., 1 day to 1 week, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, e.g., 1 week to 1 month, e.g., 2 weeks, 3 weeks, or 1 month, e.g., 1 month to 1 year, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year) after the first dose of the pharmaceutical composition. In some embodiments, the second dose is administered within 1 day to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 6 days, days to 14 days, 6 to 7 days, 7 to 90 days, 7 to 45 days, 7 to 30 days, 7 to 14 days, 14 to 90 days, 14 to 45 days, 14 to 30 days, 21 to 90 days, 21 to 60 days, 21 to 45 days, 21 to 30 days, 30 to 90 days, 30 to 60 days, 30 to 45 days, 45 to 180 days, 45 to 120 days, 45 to 100 days, 45 to 90 days, 45 to 60 days, 60 to 180 days, 60 to 120 days, 60 to 100 days, 60 to 90 days, 90 to 100 days, 90 to 120 days, or 90 to 180 days).

In some embodiments, the third dose is administered at least 1 hour (e.g., at least 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more) after the second dose of the pharmaceutical composition.

In some embodiments, the third dose is administered 1 hour to 1 year (e.g., 1 hour to 1 day, e.g., 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 1 day, e.g., 1 day to 1 week, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, e.g., 1 week to 1 month, e.g., 2 weeks, 3 weeks, or 1 month, e.g., 1 month to 1 year, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year) after the second dose of the pharmaceutical composition. In some embodiments, the third dose is administered within 1 day to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 6 days, days to 14 days, 6 to 7 days, 7 to 90 days, 7 to 45 days, 7 to 30 days, 7 to 14 days, 14 to 90 days, 14 to 45 days, 14 to 30 days, 21 to 90 days, 21 to 60 days, 21 to 45 days, 21 to 30 days, 30 to 90 days, 30 to 60 days, 30 to 45 days, 45 to 180 days, 45 to 120 days, 45 to 100 days, 45 to 90 days, 45 to 60 days, 60 to 180 days, 60 to 120 days, 60 to 100 days, 60 to 90 days, 90 to 100 days, 90 to 120 days, or 90 to 180 days).

In some embodiments, the second dose is administered before the serum concentration of the antifusogenic polypeptide is less than about 500 ng/mL in the subject's serum.

In some embodiments, the method provides for the administration 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 antifusogenic polypeptide, for example, at least 1 hour (e.g., at least 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, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more).

A method of administering multiple doses of a nucleic acid molecule (e.g., cyclic polyribonucleotide) composition described herein comprises providing two or more compositions to a cell, tissue or subject (e.g., a mammal) over a period of time. According to certain embodiments, multiple doses of a nucleic acid molecule composition described herein can be administered to a subject over a defined time course. A method according to this aspect of the invention comprises administering a nucleic acid molecule composition described herein (e.g., cyclic polyribonucleotide, linear polyribonucleotide, cyclic polydeoxyribonucleotide, linear polydeoxyribonucleotide) (e.g., in a pharmaceutical or veterinary composition) multiple times to a subject. As used herein, "administering sequentially" means that each dose of a nucleic acid molecule composition described herein is administered to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). In some embodiments, the invention provides a method comprising sequentially administering to a subject a single initial dose of a nucleic acid molecule composition described herein, followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition.

The terms "initial dose", "secondary dose" and "tertiary dose" refer to the temporal order of administration of the nucleic acid molecule compositions described herein. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen, a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary and tertiary doses may all contain the same amount of the nucleic acid molecule compositions described herein, and in certain embodiments may differ from each other in terms of frequency of administration. In certain embodiments, the amount of the nucleic acid molecule compositions described herein contained in the initial, secondary and/or tertiary doses differ from each other during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, one or more (e.g., 2, 3, 4 or 5) doses are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

In certain embodiments, each secondary and/or tertiary dose is administered after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" refers to a dose of a composition of a nucleic acid molecule described herein that is administered to a subject in a multiple dose sequence prior to administration of the very next dose in the sequence without any intervening doses. In certain embodiments, each secondary and/or tertiary dose is administered every day, every 2nd day, every 3rd day, every 4th day, every 5th day, every 6th day, or every 7th day after the immediately preceding dose. In certain embodiments, each secondary and/or tertiary dose is administered every 0.5 weeks, every week, every 2 weeks, every 3 weeks, or every 4 weeks after the immediately preceding dose.

The method according to this aspect of the invention may include administering to the subject any number of secondary and/or tertiary doses of a composition of nucleic acid molecules described herein. For example, in certain embodiments, only a single secondary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the subject. Similarly, in certain embodiments, only a single tertiary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the subject.

In certain embodiments, the frequency at which secondary and/or tertiary doses are administered to a subject may vary over the course of a treatment regimen. The frequency of administration may also be adjusted during the course of treatment.

In some embodiments, the method includes providing (e.g., administering) at least a first composition and a second composition to a cell, 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, a fourth composition, a fifth composition, a sixth composition, a seventh composition, an eighth composition, a ninth composition, a tenth composition, or more compositions. In some embodiments, the additional compositions are provided over the lifespan of the cell. In some embodiments, the additional compositions are provided (e.g., administered) while the cell, tissue, or subject is benefiting from the compositions.

In some embodiments, the first composition in the multiple dose regimen comprises a first amount of a nucleic acid molecule (e.g., a cyclic polyribonucleotide) disclosed herein. In some embodiments, the second composition in the multiple dose regimen comprises a second amount of a nucleic acid molecule (e.g., a cyclic polyribonucleotide) disclosed herein. In some embodiments, the third composition, the fourth composition, the fifth composition, the sixth composition, the seventh composition, the eighth composition, the ninth composition, the tenth composition or more compositions in the multiple dose regimen comprise a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amounts of a nucleic acid molecule (e.g., a cyclic polyribonucleotide) disclosed herein. In some embodiments, the second amount of a nucleic acid molecule (e.g., a cyclic polyribonucleotide) is the same as the first amount of a nucleic acid molecule (e.g., a cyclic polyribonucleotide). In some embodiments, the third amount of nucleic acid molecules (e.g., circular polyribonucleotides) is the same as the first amount of nucleic acid molecules (e.g., circular polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of nucleic acid molecules (e.g., circular polyribonucleotides) is the same as the first amount of nucleic acid molecules (e.g., circular polyribonucleotides). In some embodiments, the second amount of nucleic acid molecules (e.g., circular polyribonucleotides) is less than the first amount of nucleic acid molecules (e.g., circular polyribonucleotides). In some embodiments, the third amount of nucleic acid molecules (e.g., circular polyribonucleotides) is less than the first amount of nucleic acid molecules (e.g., circular polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of nucleic acid molecules (e.g., circular polyribonucleotides) is less than the first amount of nucleic acid molecules (e.g., circular polyribonucleotides). In some embodiments, the second amount of nucleic acid molecules (e.g., circular polyribonucleotides) is greater than the first amount of nucleic acid molecules (e.g., circular polyribonucleotides). In some embodiments, the third amount of nucleic acid molecules (e.g., circular polyribonucleotides) is greater than the first amount of nucleic acid molecules (e.g., circular polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of nucleic acid molecules (e.g., circular polyribonucleotides) is greater than the first amount of nucleic acid molecules (e.g., circular polyribonucleotides). In some embodiments, the amount of nucleic acid molecules (e.g., circular polyribonucleotides) of the second composition varies by no more than 1%, 5%, 10%, 15%, 20%, or 25% of the amount of nucleic acid molecules (e.g., circular polyribonucleotides) of the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the second composition is no more than 1%, 5%, 10%, 15%, 20%, or 25% less than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the second composition is 0.1-1000 times more than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the second composition is 0.1-, 1-, 5-, 10-, 100-, or 1000-fold more than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the subsequent composition (e.g., a composition administered after the first composition) is 0.1, 1, 5, 10, 100, or 1000 times greater than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the second composition is 0.1-1000 times lower than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the second composition is 0.1, 1, 5, 10, 100, or 1000 times lower than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the first composition. In some embodiments, the amount of nucleic acid molecule (e.g., cyclic polyribonucleotide) in the subsequent composition (e.g., a composition administered after a first composition) is 0.1, 1, 5, 10, 100, or 1000 times lower than the amount of nucleic acid molecule (e.g., cyclic polyribonucleotide) in the first composition. In some embodiments, the amount of nucleic acid molecule (e.g., cyclic polyribonucleotide) in the subsequent composition (e.g., after an amount of nucleic acid molecule (e.g., cyclic polyribonucleotide) in the first composition) is 0.1 to 1000 times higher or lower than the amount of nucleic acid molecule (e.g., cyclic polyribonucleotide) in the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in a subsequent composition (e.g., after a first composition with an amount of nucleic acid molecules (e.g., cyclic polyribonucleotides)) is 0.1 times, 1 times, 5 times, 10 times, 100 times, or 1000 times higher or lower than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the first composition. For example, the first composition contains 1 times the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) compared to the first composition, the second composition contains 5 times the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) compared to the first composition, and the third composition contains 0.2 times the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) compared to the first composition. In some embodiments, the second composition contains at least 5 times the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) compared to the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) in the first composition.

In some embodiments, the first composition comprises a greater amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) than the second composition. In some embodiments, the first composition comprises a greater amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth compositions.

In some embodiments, the multiple (e.g., two or more) compositions of nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding antifusogenic polypeptides administered in a multiple dosing regimen as described herein are the same composition. In some embodiments, the multiple (e.g., two or more) compositions of nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding antifusogenic polypeptides administered in a multiple dosing regimen as described herein are different compositions. In some embodiments, the same composition comprises nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding the same antifusogenic polypeptide. In some embodiments, the different compositions comprise nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding different antifusogenic polypeptides or combinations thereof.

In some embodiments, in a multiple dose regimen, the methods of administering a nucleic acid molecule (e.g., a cyclic polyribonucleotide) provided herein include administering the nucleic acid molecule to a subject in need thereof 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, for 1 to 56 days, e.g., about 49 days, 42 days, 35 days, 28 days, 21 days, 14 days, or 7 days. In some embodiments, in a multiple dose regimen, the methods provided herein include administering the nucleic acid molecule to a subject in need thereof at least three times, spaced apart by about 7 days. In some embodiments, in subjects receiving multiple doses of a nucleic acid molecule provided herein (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses), levels of antifusogenic polypeptides (e.g., plasma antifusogenic polypeptides) are maintained at levels with less than 50%, 40%, 30%, 20%, or 10% variation for a period of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or 20 weeks after the last dose. In some embodiments, in a subject receiving multiple doses of a nucleic acid molecule provided herein (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses), the level of an antifusogenic polypeptide (e.g., plasma antifusogenic polypeptide level) is maintained at a first level for a period of time greater 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, eighth, or last dose, wherein the first level is greater than the 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). In some embodiments, in a subject receiving multiple doses of a nucleic acid molecule provided herein (e.g., at least three doses) at intervals of about seven days, the level of an antifusogenic polypeptide (e.g., plasma antifusogenic polypeptide level) is maintained at a first level for a period of time greater than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eighth, or last dose, wherein the first level is greater than the level of an antifusogenic polypeptide measured immediately after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).

Delivery Methods Circular polyribonucleotides encoding the antifusogenic polypeptides described herein (eg, the polypeptides of Table 1) can be included in a pharmaceutical composition with or without a carrier.

The pharmaceutical compositions described herein can be formulated, for example, to include a carrier, e.g., a pharmaceutical carrier and/or a polymeric carrier, e.g., a liposome, and delivered to a subject in need thereof (e.g., a human or non-human agricultural or livestock animal, e.g., cow, dog, cat, horse, poultry) by known methods. Such methods include, but are not limited to, 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, phototransfection, protoplast fusion, impalfection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. Delivery methods are described, for example, in Gori et al. , Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy. July 2015, 26(7):443-451. doi:10.1089/hum. 2015.074; and Zuris et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Also described in Nat Biotechnol. 2014 Oct 30; 33(1): 73-80.

In some embodiments, the cyclic polyribonucleotides can be delivered in a "naked" delivery formulation. A naked delivery formulation delivers the cyclic polyribonucleotide to a cell without the aid of a carrier and without covalent modification of the cyclic polyribonucleotide or partial or complete encapsulation of the cyclic polyribonucleotide.

A naked delivery formulation is a formulation that does not include a carrier, does not have a covalent modification that attaches the cyclic polyribonucleotide to a moiety that aids in delivery to a cell, and is not partially or completely encapsulated. In some embodiments, a cyclic polyribonucleotide that does not have a covalent modification that attaches it to a moiety that aids in delivery to a cell can be a polyribonucleotide that is not covalently attached to a moiety that aids in delivery to a cell, such as a protein, small molecule, particle, polymer, or biopolymer. In some embodiments, the cyclic polyribonucleotide can be delivered in a delivery formulation that includes protamine or a protamine salt (e.g., protamine sulfate).

A polyribonucleotide that does not have a covalent modification attached to a moiety that aids in delivery to a cell may not contain modified phosphate groups. For example, a polyribonucleotide that does not have a covalent modification attached to a moiety that aids in delivery to a cell may not contain phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.

In some embodiments, the naked delivery formulation may not include any or all of a transfection reagent, a cationic carrier, a carbohydrate carrier, a nanoparticle carrier, or a protein carrier. For example, naked delivery formulations include phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxypropyl)-1,2-dimethylamino-2-propane ... 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamidoglycylspermidine (DOGS), N,N-distearate May not contain allyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

The naked delivery formulation may include non-carrier excipients. In some embodiments, the non-carrier excipients may include inactive ingredients that do not exhibit an active cell penetrating effect. In some embodiments, the non-carrier excipients may include a buffer, e.g., PBS. In some embodiments, the non-carrier excipients may be a solvent, non-aqueous solvent, diluent, suspending aid, surfactant, isotonicity agent, thickener, emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, dispersant, granulating agent, disintegrant, binder, buffer, lubricant, or oil.

In some embodiments, the naked delivery formulation may include a diluent, such as a parenterally acceptable diluent. The diluent (e.g., parenterally acceptable diluent) may be a liquid diluent or a solid diluent. In some embodiments, the diluent (e.g., parenterally acceptable diluent) may be an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of buffers 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 tonicity agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.

In some embodiments, the formulation includes a cell-penetrating agent. In some embodiments, the formulation is a topical formulation and includes a cell-penetrating agent. The cell-penetrating agent can include an organic compound, such as an alcohol having one or more hydroxyl functional groups. In some cases, the cell-penetrating agent includes an alcohol, such as, but not limited to, a monohydric alcohol, a polyhydric alcohol, an unsaturated aliphatic alcohol, and an alicyclic alcohol. 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 glycol (PEG)-400, ethoxylated fatty acids, or hydroxyethyl cellulose. In certain embodiments, the cell-penetrating agent includes ethanol. The cell permeation agent can include any cell permeation agent in any amount or formulation described in WO 2020/180751 or WO 2020/180752, the entireties of which are incorporated herein by reference.

In some embodiments, the pharmaceutical formulation disclosed herein, the pharmaceutical composition disclosed herein, the active pharmaceutical ingredient of the disclosed, or the drug product disclosed herein is in a parenteral nucleic acid delivery system. The parent nucleic acid delivery system may include the pharmaceutical formulation disclosed herein, the pharmaceutical composition disclosed herein, the active pharmaceutical ingredient of the disclosed, or the drug product disclosed herein, and a parenterally acceptable diluent. In some embodiments, in the parenteral nucleic acid delivery system, the pharmaceutical formulation disclosed herein, the pharmaceutical composition disclosed herein, the active pharmaceutical ingredient of the disclosed, or the drug product disclosed herein does not include any carrier.

The present disclosure further relates to a host or host cell comprising the cyclic polyribonucleotides described herein. In some embodiments, the host or host cell is a vertebrate, mammal (e.g., human), or other organism or cell.

In some embodiments, the cyclic polyribonucleotides produce reduced or no undesirable responses by the host's immune system compared to responses elicited by a reference compound, e.g., a linear polynucleotide corresponding to the described cyclic polyribonucleotide. In embodiments, the cyclic polyribonucleotides are 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).

In some embodiments, a host or host cell is contacted (e.g., delivered or administered) with a cyclic polyribonucleotide. In some embodiments, the host is a mammal, such as a human. The amount of cyclic 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 culture is determined. If growth increases or decreases in the presence of cyclic polyribonucleotide or linear, the cyclic polyribonucleotide or expression product, or both, is identified as effective in increasing or decreasing host growth.

Methods for delivering a cyclic polyribonucleotide molecule described herein to a cell, tissue, or subject include administering a pharmaceutical composition, drug substance, or drug product described herein to the cell, tissue, or subject.

In some embodiments, 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 connective tissue, muscle tissue, nervous tissue, or epithelial tissue. In some embodiments, the tissue is an organ (e.g., liver, lung, spleen, kidney, etc.).

In some embodiments, the method of delivery is an in vivo method. For example, the method of delivery of a cyclic polyribonucleotide described herein comprises parenterally administering to a subject in need thereof a pharmaceutical composition, drug substance, or drug product described herein to a subject in need thereof. As another example, the method of delivery of a cyclic polyribonucleotide to a cell or tissue of a subject comprises parenterally administering to the cell or tissue a pharmaceutical composition, drug substance, or drug product described herein. In some embodiments, the cyclic polyribonucleotide is in an amount effective to elicit a biological response in the subject. In some embodiments, the cyclic polyribonucleotide is in an amount effective to have a biological effect on a cell or tissue in the subject. In some embodiments, the pharmaceutical composition, drug substance, or drug product described herein comprises a carrier. In some embodiments, the pharmaceutical composition, drug substance, or drug product described herein comprises a diluent and does not comprise any carrier.

In some embodiments, the pharmaceutical composition, drug substance, or drug product is administered parenterally. In some embodiments, the pharmaceutical composition, drug substance, or drug product is administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, intralymphatically, subcutaneously, or intramuscularly. In some embodiments, the parenteral administration is intravenous, intramuscular, ophthalmic, subcutaneous, intradermal, or topical.

In some embodiments, a pharmaceutical composition, drug substance, or drug product described herein is administered intramuscularly. In some embodiments, a pharmaceutical composition, drug substance, or drug product described herein is administered subcutaneously. In some embodiments, a pharmaceutical composition, drug substance, or drug product described herein is administered topically. In some embodiments, a pharmaceutical composition, drug substance, or drug product is administered intratracheally.

In some embodiments, the pharmaceutical composition, drug substance, or drug product is administered by injection. Administration can be systemic or local. In some embodiments, any of the delivery methods as described herein are performed using a carrier. In some embodiments, any of the delivery methods described herein are performed without a carrier or cell permeation agent.

In some embodiments, the cyclic polyribonucleotide or a product translated from the cyclic 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 administration step. In some embodiments, the presence of the cyclic polyribonucleotide or a product translated from the cyclic polyribonucleotide is assessed in the cell, tissue, or subject before the administration step. In some embodiments, the presence of the cyclic polyribonucleotide or a product translated from the cyclic polyribonucleotide is assessed in the cell, tissue, or subject after the administration step.

Preparations In some embodiments of the present disclosure, the cyclic polyribonucleotides described herein can be formulated in compositions, such as agricultural, animal, or pharmaceutical compositions, for delivery to cells, plants, invertebrates, non-human vertebrates, or human subjects. In some embodiments, the cyclic polyribonucleotides are formulated in pharmaceutical compositions. In some embodiments, the compositions include cyclic polyribonucleotides and a diluent, carrier, adjuvant, or combinations thereof. In certain embodiments, the compositions include cyclic polyribonucleotides described herein and a carrier or a diluent without any carrier. In some embodiments, the compositions including cyclic polyribonucleotides and a diluent without any carrier are used for naked delivery of cyclic polyribonucleotides to a subject.

The pharmaceutical composition may optionally include one or more additional active substances, e.g., therapeutically and/or prophylactically active substances. The pharmaceutical composition may optionally include an inactive substance that serves as a vehicle or medium for the compositions described herein (e.g., a composition comprising a cyclic polyribonucleotide, such as any one of the inactive ingredients approved by the U.S. Food and Drug Administration (FDA) and listed in the Inactive Ingredients Database). The pharmaceutical composition of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical products 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 inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspending aids, surfactants, isotonicity agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof.

Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to provide compositions suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of skill in the art can design and/or perform such modifications, if any, with no more than routine 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 cows, 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.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the steps of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients and then, as necessary and/or desired, portioning, shaping and/or packaging the product.

In some embodiments, the reference standard for the amount of cyclic polyribonucleotide molecules present in the formulation 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), 100% (w/w), 101% (w/w), 102% (w/w), 103% (w/w), 104% (w/w), 105% (w/w), 106% (w/w), 107% (w/w), 108% (w/w), 109% (w/w), 110% (w/w), 111% (w/w), 112% (w/w), 113% (w/w), 114% (w/w), 115% (w/w), 116% (w/w), 117% (w/w), 118% (w/w), 119% (w/w), 120% (w/w), 121% (w/w), 122% (w/w), 123% (w/w), 124% (w/w), 125% (w/w), 126% (w/w), 127% (w/w), 128% (w/w), 129% (w/w), 130% (w/w), 131% (w/w), 132% (w/w), 13 4% (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.

In some embodiments, the reference standard for the amount of linear polyribonucleotide molecule present in the formulation is 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, Present at less than 200ng/ml, 300ng/ml, 400ng/ml, 500ng/ml, 600ng/ml, 1μg/ml, 10μg/ml, 50μg/ml, 100μg/ml, 200μg/ml, 300μg/ml, 400μg/ml, 500μg/ml, 600μg/ml, 700μg/ml, 800μg/ml, 900μg/ml, 1mg/ml, 1.5mg/ml, or 2mg/ml.

In some embodiments, the reference standard for the amount of linear polyribonucleotide molecules present in the formulation is 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) or less of linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical formulation.

In some embodiments, a reference standard for the amount of nicked polyribonucleotide molecules present in the formulation is no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), or 15% (w/w) of nicked polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical formulation.

In some embodiments, the reference standard for the amount of combination of nicked and linear polyribonucleotide molecules present in the formulation is 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) or less of the combination of nicked and linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is an intermediate pharmaceutical formulation of a final circular polyribonucleotide drug product. In some embodiments, the pharmaceutical formulation is an active pharmaceutical ingredient or active ingredient (API). In some embodiments, the pharmaceutical formulation is a drug product for administration to a subject.

In some embodiments, the preparation of cyclic polyribonucleotides is further processed (before, during or after reduction of the linear RNA) to substantially remove DNA, protein contaminants (e.g., cellular proteins such as host cell proteins or protein process impurities), endotoxins, mononucleotide molecules, and/or process-related impurities.

In some embodiments, the pharmaceutical formulations disclosed herein can include (i) a compound disclosed herein (e.g., a cyclic polyribonucleotide); (ii) a buffer; (iii) a non-ionic surfactant; (iv) a tonicity agent; and/or (v) a stabilizer. In some embodiments, the pharmaceutical formulations disclosed herein are stable liquid pharmaceutical formulations. In some embodiments, the pharmaceutical formulations disclosed herein include protamine or a protamine salt (e.g., protamine sulfate).

Preservatives The compositions or pharmaceutical compositions provided herein may contain material for a single dose or may contain material for multiple doses (e.g., a "multi-dose" kit). Polyribonucleotides may be present in either linear or cyclic form. The compositions or pharmaceutical compositions may contain one or more preservatives, such as thiomersal or 2-phenoxyethanol. Preservatives may be used to prevent microbial contamination during use. Suitable preservatives include benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, edetate disodium, sorbic acid, Onamer M, or other agents known to those skilled in the art. In ophthalmic products, for example, such preservatives may be used at levels of 0.004% to 0.02%. In the compositions described herein, preservatives, such as benzalkonium chloride, may be used at levels of 0.001% to less than 0.01% by weight, for example, 0.001% to 0.008% by weight, preferably about 0.005% by weight.

Polyribonucleotides may be susceptible to RNases, which may be abundant in the surrounding environment. The compositions provided herein may include reagents that inhibit RNase activity, thereby preventing degradation of polyribonucleotides. In some cases, the compositions or pharmaceutical compositions include any RNase inhibitor known to those of skill in the art. Alternatively or additionally, the polyribonucleotides in the compositions provided herein, as well as the cell permeation agent and/or the pharma- ceutically acceptable diluent or carrier, vehicle, excipient, or other reagent, may be prepared in an RNase-free environment. The compositions may be formulated in an RNase-free environment.

In some cases, the compositions provided herein may be sterile. The compositions may be formulated as a sterile solution or suspension in a suitable vehicle as known in the art. The compositions may be sterilized by conventional sterilization techniques known in the art, for example, the compositions may be sterile filtered.

Salts In some cases, the compositions or pharmaceutical compositions provided herein include one or more salts. To control osmolality, physiological salts, such as sodium salts, may be included in the compositions provided herein. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, and the like. In some cases, the compositions are formulated with one or more pharma- ceutically acceptable salts. The one or more pharma-ceutically acceptable salts may include those of inorganic ions, such as, for example, sodium, potassium, calcium, magnesium ions. Such salts may include salts of 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. Polyribonucleotides may exist in either linear or cyclic form.

Buffer/pH
The compositions or pharmaceutical compositions provided herein may 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. The buffers are in some cases within the range of 5-20 mM.

The compositions or pharmaceutical compositions provided herein may 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 compositions or pharmaceutical compositions may have a pH of about 7. Polyribonucleotides may exist in either linear or circular form.

Detergents/Surfactants The compositions or pharmaceutical compositions provided herein may contain, depending on the intended route of administration, one or more detergents and/or surfactants, such as polyoxyethylene sorbitan ester surfactants (commonly referred to as "Tween®"), e.g., polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), such as linear EO/PO block copolymers, sold under the trade name DOWFAX™; octoxynols which may vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, such as octoxynol-9 (Triton® X-100, or t-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL) (Octylphenoxy)polyethoxyethanol ... CA-630/NP-40); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (known as Brij® surfactants), such as triethylene glycol monolauryl ether (Brij® 30); and sorbitan esters (commonly known as “SPAN®”), such as sorbitan trioleate (Span® 85) and sorbitan monolaurate, octoxynol (such as octoxynol-9 (Triton® X-100) or t-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide ("CTAB"), or sodium deoxycholate. One or more detergents and/or surfactants may be included only in minor amounts. In some examples, the composition may include less than 1 mg/ml each of octoxynol-10 and polysorbate 80. Non-ionic surfactants may be used herein. Surfactants may be classified by their "HLB" (hydrophilic/lipophilic balance). In some examples, surfactants have an HLB of at least 10, at least 15, and/or at least 16. Polyribonucleotides may be included in linear or cyclic form.

Diluents In some embodiments, the compositions of the present disclosure comprise a cyclic polyribonucleotide and a diluent. In some embodiments, the compositions of the present disclosure comprise a linear polyribonucleotide and a diluent.

The diluent can be a non-carrier excipient. The non-carrier excipient serves as a vehicle or medium for the composition, such as the cyclic polyribonucleotides described herein. The non-carrier excipient serves as a vehicle or medium for the composition, such as the linear polyribonucleotides described herein. Non-limiting examples of non-carrier excipients include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonicity agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. A non-carrier excipient can be any of the inactive ingredients listed in the Inactive Ingredient Database that are approved by the United States Food and Drug Administration (FDA) and do not exhibit cell-penetrating effects. A non-carrier excipient can be, for example, any inactive ingredient suitable for administration to non-human animals that are suitable for veterinary use. Modifications of compositions suitable for administration to humans to make the compositions suitable for administration to various animals are well understood and a veterinary pharmacologist of ordinary skill can design and/or make such modifications with only routine experimentation, if any.

In some embodiments, the cyclic polyribonucleotides can be delivered as a naked delivery formulation, such as one that includes a diluent. A naked delivery formulation delivers the cyclic polyribonucleotides to cells without a carrier and without modification or partial or complete encapsulation of the cyclic polyribonucleotides, capped polyribonucleotides, or complexes thereof.

A naked delivery formulation is a formulation that does not include a carrier, where the cyclic polyribonucleotide does not have a covalent modification attached to a moiety that aids in delivery to a cell, or where there is no partial or complete encapsulation of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide does not have a covalent modification attached to a moiety that aids in delivery to a cell, and is a polyribonucleotide that is not covalently attached to a protein, small molecule, particle, polymer, or biopolymer. The cyclic polyribonucleotide does not have a covalent modification attached to a moiety that aids in delivery to a cell, and does not include modified phosphate groups. For example, the cyclic polyribonucleotide does not have a covalent modification attached to a moiety that aids in delivery to a cell, and does not include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.

In some embodiments, the naked delivery formulation does not include any or all of a transfection reagent, a cationic carrier, a carbohydrate carrier, a nanoparticle carrier, or a protein carrier. In certain embodiments, the naked delivery formulation is selected from the group consisting of phytoglycogen octenylsuccinate, phytoglycogen β-dextrin, anhydride-modified phytoglycogen β-dextrin, lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-β-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3- (2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N,N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamidoglycylspermidine (DOGS), N, Does not contain N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

In some embodiments, the naked delivery formulation includes a non-carrier excipient. In some embodiments, the non-carrier excipient includes an inactive ingredient that does not exhibit a cell-penetrating effect. In some embodiments, the non-carrier excipient includes a buffer, e.g., PBS. In some embodiments, the non-carrier excipient is a solvent, non-aqueous solvent, diluent, suspending aid, surfactant, isotonicity agent, thickener, emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, dispersant, granulating agent, disintegrant, binder, buffer, lubricant, or oil.

In some embodiments, the naked delivery formulation includes a diluent. The diluent can be a liquid diluent or a solid diluent. In some embodiments, the diluent is an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of buffers 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 isotonic agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.

Carrier In some embodiments, the compositions of the present disclosure comprise a cyclic polyribonucleotide and a carrier. In some embodiments, the compositions of the present disclosure comprise a linear polyribonucleotide and a carrier.

In certain embodiments, the composition comprises a cyclic polyribonucleotide as described herein in a vesicle or other membrane-based carrier. In certain embodiments, the composition comprises a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier.

In other embodiments, the composition comprises a cyclic polyribonucleotide in or via a cell, vesicle, or other membrane-based carrier. In other embodiments, the composition comprises a linear polyribonucleotide in or via a cell, vesicle, or other membrane-based carrier. In one embodiment, the composition comprises a cyclic polyribonucleotide in a liposome or other similar vesicle. In one embodiment, the composition comprises a linear polyribonucleotide in a liposome or other similar vesicle. Liposomes are spherical vesicular structures composed of a mono- or multi-membrane lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible, non-toxic, 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 a review).

Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for the preparation of multilamellar lipid vesicles are known in the art (see, e.g., U.S. Pat. No. 6,693,086, the teachings of which related to multilamellar lipid vesicle preparation are incorporated herein by reference). When lipid membranes are mixed with an aqueous solution, vesicle formation can occur spontaneously, but it can also be promoted by applying force in the form of shaking, by using a homogenizer, sonicator, or extrusion device (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679, for a review). Extruded lipids can be prepared by extrusion through reduced size filters as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which regarding the preparation of extruded lipids are incorporated herein by reference.

In certain embodiments, the compositions of the present disclosure include cyclic polyribonucleotides and lipid nanoparticles, e.g., lipid nanoparticles, as described herein. In certain embodiments, the compositions of the present disclosure include linear polyribonucleotides and lipid nanoparticles. Lipid nanoparticles are another example of carriers that provide a biocompatible and biodegradable delivery system for the cyclic polyribonucleotide molecules described herein. Lipid nanoparticles are another example of carriers that provide a biocompatible and biodegradable delivery system for the linear polyribonucleotide molecules described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the properties of SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLN consists of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, the two components increase drug encapsulation efficiency, facilitate surface modification, and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122.

Further non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), protein carriers (e.g., proteins covalently attached to cyclic polyribonucleotides or proteins covalently attached to linear polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin. Non-limiting examples of cationic carriers include lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxypropyl)-1,2-dimethylamino-2 ... [0036] Examples of suitable glycylspermidine include N,N-diisopropyl-N,N-dimethyl-N-propanaminium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). Non-limiting examples of protein carriers include human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

Exosomes may also be used as drug delivery vehicles for the circular RNA compositions or preparations described herein. Exosomes may also be used as drug delivery vehicles for the linear polyribonucleotide compositions or preparations described herein. For review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; doi. org/10.1016/j. apsb. 2016.02.001.

Ex vivo differentiated erythrocytes may also be used as a carrier for the circular RNA compositions or preparations described herein. Ex vivo differentiated erythrocytes may also be used as a carrier for the linear polyribonucleotide compositions or preparations described herein. See, for example, WO 2015/073587; WO 2017/123646; WO 2017/123644; WO 2018/102740; WO 2016/183482; WO 2015/153102; WO 2018/151829; WO 2018/009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28):10131-10136; U.S. Pat. No. 9,644,180; Huang et al. 2017. Nature Communications 8:423; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28):10131-10136. For example, fusosomal compositions as described in WO 2018/208728 may also be used as carriers for delivering the circular polyribonucleotide molecules described herein. For example, fusosomal compositions as described in WO 2018/208728 may also be used as carriers for delivering the linear polyribonucleotide molecules described herein.

Virosomes and virus-like particles (VLPs) can also be used as carriers for delivering the circular polyribonucleotide molecules described herein to target cells. Virosomes and virus-like particles (VLPs) can also be used as carriers for delivering the linear polyribonucleotide molecules described herein to target cells.

Plant nanovesicles and plant messenger packs (PMPs), such as those described in International Patent Publication Nos. WO 2011/097480, WO 2013/070324, WO 2017/004526, or WO 2020041784, may also be used as carriers for delivering the circular RNA compositions or preparations described herein. Plant nanovesicles and plant messenger packs (PMPs) may also be used as carriers for delivering the linear polyribonucleotide compositions or preparations described herein.

Microbubbles may also be used as a carrier for delivering the circular polyribonucleotide molecules described herein. Microbubbles may also be used as a carrier for delivering the linear polyribonucleotide molecules described herein. See, for example, U.S. Pat. No. 7,115,583; Beeri, R. et al., Circulation. 2002 Oct 1; 106(14): 1756-1759; Bez, M. et al., Nat Protoc. 2019 Apr; 14(4): 1015-1026; Hernot, S. et al., Adv Drug Deliv Rev. 2008 Jun 30; 60(10): 1153-1166; Rychak, J. J. et al. , Adv Drug Deliv Rev. 2014 Jun;72:82-93. In some embodiments, the microbubbles are albumin-coated perfluorocarbon microbubbles.

The carriers comprising cyclic polyribonucleotides described herein may comprise a plurality of particles. The particles may have a median article size of 30-700 nanometers (e.g., 30-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 100-500, 50-500, or 200-700 nanometers). The size of the particles may be optimized to favor deposition of the payload comprising cyclic polyribonucleotides into cells. Deposition of cyclic polyribonucleotides into specific cell types may favor different particle sizes. For example, the particle size may be optimized for deposition of cyclic polyribonucleotides into antigen-presenting cells. The particle size may be optimized for deposition of cyclic polyribonucleotides into dendritic cells. Additionally, the particle size may be optimized for deposition of cyclic polyribonucleotides into draining lymph node cells.

Lipid Nanoparticles The compositions, methods, and delivery systems provided by the present disclosure may, in certain embodiments, use any suitable carrier or delivery modality, including lipid nanoparticles (LNPs) as described herein. The lipid nanoparticles, in certain embodiments, include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or amphoteric lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers as described in Table 5 of WO2019217941, which is incorporated herein by reference in its entirety); and one or more sterols (e.g., cholesterol).

Lipids (e.g., lipid nanoparticles) that can be used in nanoparticle formation include, for example, those described in Table 4 of WO2019217941, which is incorporated by reference - for example, a lipid-containing nanoparticle can include one or more of the lipids in Table 4 of WO2019217941. The lipid nanoparticle can include additional elements, for example, a polymer, for example, a polymer described in Table 5 of WO2019217941, which is incorporated by reference.

In some embodiments, the conjugated lipid, if present, is a PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradecyl)glycerol), PEG-glyceryl ester (PEG-glyceryl)-2,3-dimyristoylglycerol (PEG-DMG) ... or PEG-glyceryl ester (PEG-glyceryl)-2,3-dimyristoylglycerol (PEG-DMG)). The polyoxyalkylene glycols may include one or more of N-(carbonyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those listed in Table 2 of WO2019051289 (incorporated by reference), as well as combinations of the above.

In some embodiments, sterols that may be incorporated into the lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009/127060 or U.S. Patent Publication No. 2010/0130588, which are incorporated by reference. Further exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, which are incorporated by reference herein.

In some embodiments, the lipid particles include an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits particle aggregation, and a sterol. The amounts of these components can be independently varied to achieve desired properties. For example, in some embodiments, the lipid nanoparticles include an ionizable lipid in an amount of about 20 mol% to about 90 mol% of the total lipid (in other embodiments, it can 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 of about 5 mol% to about 30 mol% of the total lipid, a conjugated lipid in an amount of about 0.5 mol% to about 20 mol% of the total lipid, and a sterol in an amount of about 20 mol% to about 50 mol% of the total lipid. The ratio of total lipid to nucleic acid can be varied as needed. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10:1 to about 30:1.

In some embodiments, the lipid to nucleic acid ratio (mass/mass ratio; w/w ratio) can range from about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and nucleic acid can be adjusted to obtain a desired N/P ratio, e.g., an N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the total lipid content of the lipid nanoparticle formulation can range from about 5 mg/ml to about 30 mg/mL.

Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivery of the compositions described herein, e.g., the nucleic acids described herein (e.g., RNA (e.g., circular polyribonucleotides, linear polyribonucleotides)), include the following:
In certain embodiments, an LNP comprising formula (i) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In certain embodiments, an LNP comprising formula (ii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In certain embodiments, an LNP comprising formula (iii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In certain embodiments, an LNP comprising formula (v) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, an LNP comprising formula (vi) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, an LNP comprising formula (viii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In certain embodiments, an LNP comprising formula (ix) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In the formula,
X ¹ is O, NR ¹ or a direct bond, X ² is a C2-5 alkylene, X ³ is C(═O) or a direct bond, R ¹ is H or Me, R ³ is a C1-3 alkyl, R ² is a C1-3 alkyl, or R ² together with the nitrogen atom to which it is attached and 1 to 3 carbon atoms of X ² form a 4-, 5-, or 6-membered ring, or X ¹ is NR ¹ , R ¹ and R ² together with the nitrogen atom to which they are attached form a 5-, or 6-membered ring, or R ² together with R ³ and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y ¹ is a C2-12 alkylene, and Y ² is
is selected from
n is 0 to 3, R ⁴ is C1 to 15 alkyl, and Z ¹ is C1 to 6 alkylene or a direct bond;
^Z2 is
(in any orientation) or absent, with the proviso that when Z ¹ is a direct bond and Z ² is absent;
R ⁵ is a C5-9 alkyl or a C6-10 alkoxy, R ⁶ is a C5-9 alkyl or a C6-10 alkoxy, W is methylene or a direct bond, R ⁷ is H or Me, or a salt thereof, provided that R ³ and R ² are C2 alkyl, X ¹ is O, X ² is a linear C3 alkylene, X ³ is C(=0), Y ¹ is a linear Ce alkylene, and (Y ² )n-R ⁴ is
and R ⁴ is a linear C5 alkyl, Z ¹ is a C2 alkylene, Z ² is absent, W is methylene, and R ⁷ is H, then R ⁵ and R ⁶ are not Cx alkoxy.

In some embodiments, LNPs comprising formula (xii) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In some embodiments, an LNP comprising formula (xi) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv).

In some embodiments, an LNP comprising formula (xv) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, LNPs comprising a formulation of formula (xvi) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

In certain embodiments, the lipid compounds used to form lipid nanoparticles for delivery of the compositions described herein, e.g., the nucleic acids described herein (e.g., RNA (e.g., circular polyribonucleotides, linear polyribonucleotides)), are made by one of the following reactions:

In some embodiments, LNPs comprising formula (xxi) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxi) are LNPs described by WO2021113777 (e.g., lipids of formula (1), such as lipids in Table 1 of WO2021113777).
In the formula,
each n is independently an integer from 2 to 15; L ₁ and L ₃ are each independently -OC(O)- ^* or -C(O)O- ^* , where " ^* " indicates the point of attachment to R ₁ or R ₃ ;
_R1 and _R3 are each independently 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, R 2 is a linear or branched C 9 -C 20 alkyl or C 9 -C 20 alkenyl, optionally substituted by one or more substituents selected from the group consisting of alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl _, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl _{, heterocyclylcarbonyl} , alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, _{alkylsulfoxidealkyl} , alkylsulfonyl, and _{alkylsulfonealkyl} ; and
is selected from the group consisting of:

In some embodiments, LNPs comprising formula (xxii) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxii) are LNPs described by WO2021113777 (e.g., lipids of formula (2), such as lipids in Table 2 of WO2021113777).
In the formula,
each n is independently an integer from 1 to 15;
R ₁ and R ₂ each independently represent
is selected from the group consisting of:

_R3 is
is selected from the group consisting of:

In some embodiments, LNPs comprising formula (xxiii) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxiii) are LNPs described by WO2021113777 (e.g., lipids of formula (3), such as lipids in Table 3 of WO2021113777).
In the formula,
X is selected from -O-, -S-, or -OC(O)- ^* , where ^* indicates the point of attachment to _R1 ;
_R1 is
is selected from the group consisting of
_R2 is
is selected from the group consisting of:

In some embodiments, the compositions described herein (e.g., nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) or proteins) are provided in LNPs that include an ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), as described, for example, in Example 1 of U.S. Pat. No. 9,867,888, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadecanedioate (LP01), for example, as synthesized in Example 13 of WO 2015/095340, which is incorporated by reference in its entirety. In some embodiments, the ionizable lipid is di((Z)-nona-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), for example, as synthesized in Examples 7, 8, or 9 of U.S. Patent Application Publication No. 2012/0027803, which is incorporated by reference in its entirety. In certain embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, as synthesized in Examples 14 and 16 of WO 2010/053572, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is an imidazole cholesterol ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,14,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., structure (I) from WO 2020/106946, which is incorporated herein by reference in its entirety.

In some embodiments, the ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that may exist in a positively charged or neutral form depending on the pH, or an amine-containing lipid that may be readily protonated. In some embodiments, the cationic lipid is a lipid that is capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine groups that are positively charged. In some embodiments, the lipid particles include cationic lipids in a formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structured lipids (e.g., sterols), PEG, cholesterol, and polymer-conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. Exemplary cationic lipids disclosed herein may have an effective pKa greater than 6.0. In some embodiments, the lipid nanoparticles may include a second cationic lipid having an effective pKa different from the first cationic lipid (e.g., higher than the first effective pKa). The lipid nanoparticles may comprise 40-60 mol percent cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and a therapeutic agent, e.g., a nucleic acid as described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) encapsulated within or associated with the lipid nanoparticles. In some embodiments, the nucleic acid is formulated simultaneously with the cationic lipids. The nucleic acid may be adsorbed to the surface of the LNPs, e.g., LNPs comprising cationic lipids. In some embodiments, the nucleic acid may be encapsulated within the LNPs, e.g., LNPs comprising cationic lipids. In some embodiments, the lipid nanoparticles may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulations are biodegradable. In some embodiments, lipid nanoparticles comprising one or more lipids described herein, e.g., formula (i), (ii), (vii) and/or (ix), encapsulate 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 the RNA molecules.

Exemplary ionizable lipids that may be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of WO 2019051289, which is incorporated herein by reference. Further exemplary lipids include, but are not limited to, one or more of the following formulas: X of U.S. Patent Application Publication No. 2016/0311759; I of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016/0376224; I, II, or III of U.S. Patent Application Publication No. 20160151284; I, IA, II, or IIA of U.S. Patent Application Publication No. 20170210967; I-c of U.S. Patent Application Publication No. 20150140070; A of U.S. Patent Application Publication No. 2013/0178541. ; U.S. Patent Application Publication No. 2013/0303587 or U.S. Patent Application Publication No. 2013/0123338, I; U.S. Patent Application Publication No. 2015/0141678, I; U.S. Patent Application Publication No. 2015/0239926, II, III, IV, or V; U.S. Patent Application Publication No. 2017/0119904, I; WO 2017/117528, I or II; U.S. Patent Application Publication No. 2012/0149894, A; U.S. Patent Application Publication No. 2015/0057373, A; WO 201 No. 3/116126; U.S. Patent Application Publication No. 2013/0090372; U.S. Patent Application Publication No. 2013/0274523; U.S. Patent Application Publication No. 2013/0274504; U.S. Patent Application Publication No. 2013/0053572; WO 2013/016058; WO 2012/162210; U.S. Patent Application Publication No. 2008/042973 I; U.S. Patent Application Publication No. 2012/01287670 I, II, III, or the like. or IV; I or II of U.S. Patent Application Publication No. 2014/0200257; I, II, or III of U.S. Patent Application Publication No. 2015/0203446; I or III of U.S. Patent Application Publication No. 2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of U.S. Patent Application Publication No. 2014/0308304; U.S. Patent Application Publication No. 2013/0338210; I, II, III, or IV of WO 2009/132131; U.S. Patent Application Publication No. A of US Patent Application Publication No. 2012/01011478; I or XXXV of US Patent Application Publication No. 2012/0027796; XIV or XVII of US Patent Application Publication No. 2012/0058144; of US Patent Application Publication No. 2013/0323269; I of US Patent Application Publication No. 2011/0117125; I, II, or III of US Patent Application Publication No. 2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of U.S. Patent Application Publication No. 2011/0076335; I or II of U.S. Patent Application Publication No. 2006/008378; I of U.S. Patent Application Publication No. 2013/0123338; I or X-A-Y-Z of U.S. Patent Application Publication No. 2015/0064242; XVI, XVII, or XVIII of U.S. Patent Application Publication No. 2013/0022649; I of U.S. Patent Application Publication No. 2013/0116307 , II, or III; I, II, or III of U.S. Patent Application Publication No. 2013/0116307; I or II of U.S. Patent Application Publication No. 2010/0062967; I to X of U.S. Patent Application Publication No. 2013/0189351; I of U.S. Patent Application Publication No. 2014/0039032; V of U.S. Patent Application Publication No. 2018/0028664; I of U.S. Patent Application Publication No. 2016/0317458; I of U.S. Patent Application Publication No. 2013/0195920; 5 of U.S. Patent Application Publication No. 10,221,127 , 6, or 10; III-3 of WO 2018/081480; I-5 or I-8 of WO 2020/081938; 18 or 25 of U.S. Patent Application Publication No. 9,867,888; A of U.S. Patent Application Publication No. 2019/0136231; II of WO 2020/219876; 1 of U.S. Patent Application Publication No. 2012/0027803; OF-02 of U.S. Patent Application Publication No. 2019/0240349; 23 of U.S. Patent Application Publication No. 10,086,013; Miao cKK-E12/A6 of WO 2010/053572; C12-200 of Dahlman et al (2017); 7C1 of Dahlman et al (2017); 304-O13 or 503-O13 of Whitehead et al; TS-P4C2 of U.S. Pat. No. 9,708,628; I of WO 2020/106946; I of WO 2020/106946; and (1), (2), (3) or (4) of WO 2021/113777. Exemplary lipids further include any one of the lipids in Tables 1 to 16 of WO 2021/113777.

In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), for example, as described in Example 9 of WO2019051289A9, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is the lipid ATX-002, for example, as described in Example 10 of WO2019051289A9, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13,16-diene-1-amine (compound 32), for example, as described in Example 11 of WO2019051289A9, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is compound 6 or compound 22, for example, as described in Example 12 of WO2019051289A9, which is incorporated herein by reference in its entirety.

Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl dioleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diercoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidyl phospholipids include phatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It will be appreciated that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Further exemplary lipids include, in certain embodiments, but are not limited to, those described in Kim et al., incorporated herein by reference. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386. In some embodiments, such lipids include plant lipids that have been shown to improve hepatic transfection with mRNA (e.g., DGTS).

Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO 2017/099823 or U.S. Patent Publication No. 2018/0028664, the entire contents of which are incorporated herein by reference.

In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II, or IV of U.S. Patent Publication No. 2018/0028664, which is incorporated herein by reference in its entirety. The non-cationic lipid may comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to neutral lipid is in the range of about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

In some embodiments, the lipid nanoparticles do not contain any phospholipids.

In some aspects, the lipid nanoparticles may further comprise components such as sterols to provide membrane integrity. One exemplary sterol that may be used in the lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs, such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2 ^, -hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs, such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO 2009/127060 and U.S. Patent Publication No. 2010/0130588, each of which is incorporated herein by reference in its entirety.

In some embodiments, components that provide membrane integrity, such as sterols, may comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%). In some embodiments, such components are 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.

In some embodiments, the lipid nanoparticles may include polyethylene glycol (PEG) or conjugated lipid molecules. These are generally used to inhibit aggregation and/or provide steric stabilization of the lipid nanoparticles. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, e.g., a (methoxypolyethylene glycol)-conjugated lipid.

Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Further exemplary PEG-lipid conjugates are described in, for example, U.S. Pat. Nos. 5,885,613, 6,287,591, U.S. Patent Application Publication No. 2003/0077829, 2005/0077829, and 2006/0029624. No. 175682, U.S. Patent Application Publication No. 2008/0020058, U.S. Patent Application Publication No. 2011/0117125, U.S. Patent Application Publication No. 2010/0130588, U.S. Patent Application Publication No. 2016/0376224, U.S. Patent Application Publication No. 2017/0119904, and U.S. Patent Application No. 099823, all of which are incorporated herein by reference in their entireties. In some embodiments, the PEG-lipid is represented by Formula III of U.S. Patent Application Publication No. 2018/0028664, the entireties of which are incorporated herein by reference. , III-a-I, III-a-2, III-b-1, III-b-2, or V. In some embodiments, the PEG-lipid is of Formula II of US Patent Publication No. 20150376115 or US Patent Publication No. 2016/0376224, both of which are incorporated by reference in their entireties. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. PEG-lipids include PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[ω]-methyl-poly(ethylene glycol)ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises
The present invention includes a structure selected from the following:

In some embodiments, lipids conjugated with molecules other than PEG may be used in place of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (GPL) conjugates may be used in place of or in addition to PEG-lipids.

Exemplary conjugated lipids, i.e., PEG-lipid, (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, all of which are incorporated herein by reference in their entireties.

In some embodiments, the PEG or conjugated lipid may comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEG or conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. The molar ratios of ionizable lipid, non-cationic lipid, sterol, and PEG/conjugated lipid may be varied as needed. For example, the lipid particle may comprise 30-70% ionizable lipid per mole or total weight of the composition, 0-60% cholesterol per mole or total weight of the composition, 0-30% non-cationic lipid per mole or total weight of the composition, and 1-10% conjugated lipid per mole or total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid per mole or total weight of the composition, 40-50% cholesterol per mole or total weight of the composition, and 10-20% non-cationic lipid per mole or total weight of the composition. In certain other embodiments, the composition is 50-75% ionizable lipid per mole or total weight of the composition, 20-40% cholesterol per mole or total weight of the composition, and 5-10% non-cationic lipid per mole or total weight of the composition and 1-10% conjugated lipid per mole or total weight of the composition. The composition may contain 60-70% ionizable lipid per mole or total weight of the composition, 25-35% cholesterol per mole or total weight of the composition, and 5-10% non-cationic lipid per mole or total weight of the composition. The composition may also contain up to 90% ionizable lipid per mole or total weight of the composition and 2-15% non-cationic lipid per mole or total weight of the composition. Formulations may also be used that contain, for example, 8-30% ionizable lipid per mole or total weight of the composition, 5-30% noncationic lipid per mole or total weight of the composition, and 0-20% cholesterol per mole or total weight of the composition; 4-25% ionizable lipid per mole or total weight of the composition, 4-25% noncationic lipid per mole or total weight of the composition, 2-25% cholesterol per mole or total weight of the composition, 10-35% conjugated lipid per mole or total weight of the composition, and 5% cholesterol per mole or total weight of the composition; The lipid nanoparticle formulation may comprise 2-30% ionizable lipid per mole or total weight of the composition, 2-30% non-cationic lipid per mole or total weight of the composition, 1-15% cholesterol per mole or total weight of the composition, 2-35% conjugated lipid per mole or total weight of the composition, and 1-20% cholesterol per mole or total weight of the composition; or up to 90% ionizable lipid per mole or total weight of the composition and 2-10% non-cationic lipid per mole or total weight of the composition, or 100% cationic lipid per mole or total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol, and PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol, and PEGylated lipid in a molar ratio of 60:38.5:1.5.

In some embodiments, the lipid particles include an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, where the molar ratio of lipids ranges from 20-70 mole percent for the ionizable lipid, with a target of 40-60; the molar percent of the non-cationic lipid ranges from 0-30, with a target of 0-15; the molar percent of the sterol ranges from 20-70, with a target of 30-50; and the molar percent of the PEGylated lipid ranges from 1-6, with a target of 2-5.

In one embodiment, the lipid particles comprise ionizable lipid/non-cationic lipid/sterol/conjugated lipid in a molar ratio of 50:10:38.5:1.5.

In one aspect, the present disclosure provides a lipid nanoparticle formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

In some embodiments, one or more additional compounds may also be included. The compounds may be administered separately or the additional compounds may be included in the lipid nanoparticles of the present invention. In other words, the lipid nanoparticles may contain other compounds in addition to the nucleic acid or at least a second nucleic acid different from the first nucleic acid. Without being limited thereto, the other additional compounds may 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, extracts made from biological materials, or any combination thereof.

In some embodiments, the LNPs comprise a biodegradable, ionizable lipid. In some embodiments, the LNPs comprise (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate (also referred to as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,l2Z)-octadeca-9,l2-dienoate)) or another ionizable lipid. See, e.g., the lipids in WO 2019/067992, WO 2017/173054, WO 2015/095340, and WO 2014/136086, and the references provided therein. In some embodiments, the terms cationic and ionizable with respect to LNP lipids are synonymous, e.g., ionizable lipids are cationic depending on the pH.

In some embodiments, the average LNP diameter of the LNP formulation may be tens of nanometers to hundreds of nanometers, e.g., as measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be about 40 nm to about 150 nm, e.g., about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In certain embodiments, the mean LNP diameter of the LNP formulation may be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the mean LNP diameter of the LNP formulation may be about 80 nm. In certain embodiments, the mean 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 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

The LNPs may be relatively homogeneous in some cases. The polydispersity index may be used to indicate the homogeneity of the LNPs, e.g., the size distribution of the lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow size distribution. The LNPs may have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the LNPs may be about 0.10 to about 0.20.

The zeta potential of the LNPs can be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential can represent the surface charge of the LNPs. Lipid nanoparticles with a relatively low charge, positive or negative, are generally desirable, since more highly charged species may undesirably interact with cells, tissues, and other elements in the body. In certain embodiments, the zeta potential of the LNPs may be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

The efficiency of protein and/or nucleic acid encapsulation represents the amount of protein and/or nucleic acid encapsulated or otherwise associated with the LNP after preparation compared to the initial amount provided. It is desirable for the encapsulation efficiency to be high (e.g., near 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after disintegrating the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence can be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of proteins and/or nucleic acids may be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

The LNPs may optionally include one or more coatings. In some embodiments, the LNPs may be formulated into capsules, films, or tablets having a coating. Capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

Additional exemplary lipids, formulations, methods, and characterizations of LNPs are taught by WO 2020/061457 and WO 2021/113777, each of which is incorporated herein by reference in its entirety. Additional exemplary lipids, formulations, methods, and characterizations 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, e.g., exemplary lipids and lipid derivatives in FIG. 2 of Hou et al.).

In certain embodiments, in vitro or ex vivo cell lipofection is performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using GenVoy_ILM ionizable lipid mixture (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are described in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533(2012), which is incorporated herein by reference in its entirety.

LNP formulations optimized for 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 as described herein.

Additional specific LNP formulations useful for delivery of nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) are described in U.S. Pat. Nos. 8,158,601 and 8,168,775 (both of which are incorporated by reference), including the formulations used in patisiran, sold under the name ONPATTRO.

In embodiments, a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) encoding at least a portion (e.g., an antigenic portion) of a protein or polypeptide described herein is formulated into an LNP, (a) the LNP comprises a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid, (b) the LNP has an average particle size of 80 nm to 160 nm, and (c) is a polyribonucleotide. In embodiments, the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) formulated into the LNP is a vaccine.

Exemplary administrations of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) LNPs can include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg/kg (RNA). In some embodiments, the dose of the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) antigenic compositions described herein is 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg.

Kits In some aspects, the disclosure provides kits. In some embodiments, the kit includes (a) a cyclic 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, explanatory, marketing, or other material relating to the methods described herein and/or the use of the pharmaceutical composition or cyclic polyribonucleotide for the methods described herein. The pharmaceutical composition or cyclic polyribonucleotide may include material for a single dose (e.g., a single dosage form) or may include material for multiple doses (e.g., a "multi-dose" kit).

The informational materials of the kit are not limited in form. In one embodiment, the informational materials may include information regarding the manufacture of the pharmaceutical composition, drug substance, or drug product, molecular weight, concentration, expiration date, batch or manufacturing site information of the pharmaceutical composition, drug substance, or drug product, and the like. In one embodiment, the informational materials relate to methods for administering a dosage form of the pharmaceutical composition. In one embodiment, the informational materials relate to methods for administering a dosage form of a cyclic polyribonucleotide.

In addition to the pharmaceutical compositions and cyclic polyribonucleotide dosage forms described herein, the kits may include other ingredients, such as solvents or buffers, stabilizers, preservatives, flavoring agents (e.g., bitter taste antagonists or sweeteners), fragrances, dyes or colorants for coloring or staining one or more of the ingredients in the kit, or other cosmetic ingredients, and/or second agents for treating a condition or disorder described herein. Alternatively, the other ingredients may be included in the kit, but in a different composition or container than the pharmaceutical compositions or cyclic polyribonucleotides described herein. In such embodiments, the kits may include instructions for mixing the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein and the other ingredients, or for using the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein with the other ingredients.

In some embodiments, the kit components are stored under inert conditions (e.g., under nitrogen or another inert gas such as argon). In some embodiments, the kit components are stored under anhydrous conditions (e.g., using a desiccant). In some embodiments, the components are stored in light-proof containers such as amber vials.

The dosage forms of the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein may be provided in any form, e.g., liquid, dry or lyophilized. The pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein are preferably substantially pure and/or sterile. When the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein are provided in a liquid solution, the liquid solution is preferably an aqueous solution, preferably a sterile aqueous solution. When the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein are provided as a dry form, reconstitution is generally by addition of a suitable solvent. A solvent, e.g., sterile water or buffer, can be provided in the kit as needed.

The kit may include one or more containers for the composition containing the dosage forms described herein. In some embodiments, the kit includes separate containers, dividers, or compartments for the composition and the informational material. For example, the pharmaceutical composition or the circular polyribonucleotide may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single undivided container. For example, the pharmaceutical composition or the dosage form of the nucleic acid molecule (e.g., the circular polyribonucleotide) described herein is contained in a bottle, vial, or syringe to which is attached the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., packs) of individual containers, each containing one or more unit dosage forms of the pharmaceutical composition or the circular polyribonucleotide described herein. For example, the kit includes a plurality of syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of the dosage form described herein.

The containers of the kit 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 administering the dosage form, such as a syringe, pipette, forceps, measured spoon, swab (e.g., cotton bud or wooden swab), or any such device.

The kits of the invention may include dosage forms of various strengths to provide a subject with dosages appropriate for one or more of the starter, induction, or maintenance regimens described herein. Alternatively, the kits may include scored tablets to allow the user to administer split doses as needed.

The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as the invention.

Example 1: Expression of antifusogenic polypeptides from RNA in mammalian cells This example demonstrates the expression of one or more open reading frames (ORFs) encoding one or more OC43-HR2P and EK1 peptides in Huh-7 cells.

In this example, circular RNAs are designed that encode one OC43-HR2P peptide (SEQ ID NO:289), one EK1 peptide (SEQ ID NO:288), multiple OC43-HR2P peptides, multiple EK1 peptides, and combinations of OC43-HR2P peptides, peptide analogs, and EK1 peptides. The 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 will be monitored over time in vitro.

Example 2: Inhibition of MERS-CoV S protein-mediated cell-cell fusion A MERS-CoV S protein-mediated cell-cell fusion assay is used to determine inhibition of MERS-CoV fusion with 293T target cells. This example uses 293T cells transfected with plasmid pAAV-IRES-EGFP (293T/EGFP) encoding EGFP or pAAV-IRES-MERS-EGFP (293T/MERS/EGFP) encoding MERS-CoV S protein and EGFP and cultured in DMEM containing 10% FBS at 37° C. for 48 hours. Huh-7 cells ( ^5x104 cells) expressing the MERS-CoV receptor DPP4, prepared according to Example 1, are incubated in a 96-well plate at 37°C for 5 hours, and then ^1x104 cells of 293T/EGFP or 293T/MERS/EGFP cells, respectively, are added.

After 4 hours of co-culture at 37°C, 293T/MERS/EGFP cells that were fused or not fused with Huh-7 cells (293T/EGFP cells are used as negative control) are counted under an inverted fluorescent microscope (Nikon Eclipse Ti-S). Fused cells are seen as cells that are more than two times larger than unfused cells, and the difference in the intensity of fluorescence in fused cells is compared to unfused cells. The percentage inhibition of cell-cell fusion can be calculated using the following formula: (1-(E-N)/(P-N)) x 100 "E" represents the % cell-cell fusion in the experimental group. "P" represents the % cell-cell fusion in the positive control group where no circRNA was added. "N" is the % cell-cell fusion in the negative control group where 293T/MERS/EGFP cells were replaced by 293T/EGFP cells. The 50% inhibitory concentration ( _IC50 ) can be calculated using CalcuSyn software. Co-culture can be continued for 48 hours at 37°C and measurements of, for example, syncytium formation can be performed. An intracellular S protein ELISA can be adapted to measure antiviral activity 2 days after virus challenge.

Example 3: Inhibition of pseudotyped SARS-CoV-2 and MERS-CoV infection SARS or MERS pseudotyped viruses carrying the SARS-CoV-2 or MERS-CoV S protein, respectively, and a defective HIV-1 genome expressing luciferase as a reporter are prepared by co-transfecting 293T cells with plasmid pNL4-3.luc.RE (encoding an Env-defective luciferase-expressing HIV-1) and pcDNA3.1-MERS-CoV-S plasmid. To detect the inhibitory activity of the expressed peptides against infection by SARS or MERS pseudotyped viruses, ACE2-transfected 293T (293T/ACE2) cells and Huh-7 cells ( ¹⁰⁴ cells/well in 96-well plates) transfected and not transfected with the circRNA of the invention are infected with SARS or MERS-CoV pseudotyped viruses, respectively. After infection, the cultures are refed with fresh medium 12 hours after infection and incubated for an additional 72 hours. The cells are washed with PBS and lysed using the lysis reagent included in the luciferase kit (Promega). An aliquot of the cell lysate is transferred to a 96-well Costar flat-bottom luminometer plate (Corning Costar) followed by the addition of luciferase substrate (Promega). The relative light units are immediately determined in an Ultra384 luminometer (Tecan US).

Example 4: Expression of SARS-CoV-2 antifusogenic polypeptides This example demonstrates the expression of SARS-CoV-2 antifusogenic polypeptides from circular RNA.

Based on the HR2 region shown in Figure 1, several SARS-CoV-2 anti-membrane fusion polypeptides were designed (Figures 2 and 3). A circular RNA was designed to contain a nucleotide sequence encoding an IRES and a SARS-CoV-2 anti-membrane fusion polypeptide. In this example, a DNA construct was designed to contain a spacer element and a polynucleotide cargo. The construct was designed to contain a combination of an IRES and an ORF as the polynucleotide cargo. The ORF was designed to contain an IL-2 secretion signal sequence, a nucleotide sequence encoding a SARS-CoV-2 anti-membrane fusion polypeptide, and a nucleotide sequence encoding a HiBiT tag with a GGGGS peptide linker. The IRES was EMCV.

The amino acid and nucleic acid sequences of all constructs used are shown below.

The N-terminal IL-2 secretion signal is shown in uppercase (20 AA or 60 nucleotides)
Bold = Furin Bold and italics = HiBiT tag with G4S peptide linker

HR2 full length
HR2A
HR2C
HR2B
EK1

Circular RNA was generated by self-splicing using the methods described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from a DNA template containing the motifs listed above in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with DNase. Synthesized linear RNA was purified with an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription. Circular RNA encoding antifusogenic peptides was purified by urea polyacrylamide gel electrophoresis (urea-PAGE) or reverse-phase column chromatography.

To measure expression of SARS-CoV-2 antifusogenic polypeptides, 0.4 pmol of circular RNA was delivered to HEK293 cells using lipofectamine. Expression was measured 48 hours later. Various polypeptides were expressed in HEK293 cells. Total expression (ng/ml and nM) is shown in Table 2.

Example 5: Inhibition of pseudotyped SARS-CoV-2 infection This example demonstrates the inhibition of pseudotyped SARS-CoV2 infection by antifusogenic polypeptides expressed from circular RNA.

The circular RNA was designed to contain an internal ribosome entry site (IRES) and a nucleotide sequence encoding a fusion inhibitory polypeptide of SARS-CoV-2. In this example, the DNA construct was designed to contain a spacer element and a combination of the EMCV IRES and ORF as a polynucleotide cargo. The ORF was designed to contain a nucleotide sequence encoding an IL-2 secretion signal sequence and an HR2 full-length anti-membrane fusion polypeptide, as well as a nucleotide sequence encoding a HiBiT peptide tag. The ORF was also designed to contain an IL-2 secretion signal sequence and a nucleotide sequence encoding an HR2 full-length anti-membrane fusion polypeptide without the nucleotide sequence encoding a HiBiT peptide tag.

Circular RNA was generated by self-splicing using the methods described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from a DNA template containing the motifs listed above in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with DNase. Synthesized linear RNA was purified with an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription. Circular RNA encoding the HR2 full-length antifusogenic polypeptide was purified by urea polyacrylamide gel electrophoresis (urea-PAGE) or reverse-phase column chromatography.

To detect the inhibitory activity of the expressed polypeptides against infection by SARS pseudotyped viruses, ACE2-transfected 293T (293T/ACE2) cells ( ¹⁰⁴ cells/well in 96-well plates) transfected with and without circular RNA were infected with SARS-CoV-2 pseudotyped viruses, respectively. Transfection reagent alone (without circular RNA) was used as a control ("mock"). After infection, the cultures were refed with fresh medium 12 hours after infection and incubated for an additional 72 hours. Cells were washed with PBS and lysed using the lysis reagent included in the luciferase kit (Promega). Aliquots of cell lysates were transferred to 96-well Costar flat-bottom luminometer plates (Corning Costar) followed by addition of luciferase substrate (Promega). Relative light units were immediately determined in an Ultra384 luminometer (Tecan US).

The efficacy of in vitro fusion inhibition using Omicron and Delta pseudotyped viruses was determined using circular polyribonucleotides encoding the antifusogenic polypeptides described herein. HR2 full-length antifusogenic polypeptide expressed in cells (Figure 4A), as well as HR2 full-length and HR2 full-length conjugated to HiBiT-tagged antifusogenic polypeptides, were shown to inhibit fusion of Delta and Omicron strains (Figure 4A). There was no change in cell viability (measured by cellTiter glo) (Figure 4B), suggesting that the reduction in luciferase signaling (Figure 4A) was due to inhibition of viral fusion.

To measure the expression of the polypeptide in vivo, 120 pmol of circular RNA formulated in lipid nanoparticles was delivered to mice by intravenous injection. Expression was measured by Nano-Glo® HiBiT Extracellular Detection System (#N3030, Promega) 10% serum. As shown in Table 3 and Figure 5, the antifusogenic polypeptide was highly expressed at 6 hours and significantly decreased at 24 hours.

Example 6: Inhibition of pseudotyped SARS-CoV-2 infection This example demonstrates inhibition of pseudotyped SARS-CoV-2 infection using antifusogenic polypeptides.

Anti-membrane fusion polypeptides of SARS-CoV-2 were created based on the HR2, HR2A, HR2B, and HR2C regions of the SARS-CoV-2 Spike and EK1 polypeptides (Figure 2).

Functional assays were performed to measure pseudotype virus neutralization by EK-1 and HR2A polypeptides in vitro. HR2A polypeptides showed efficacy against Wuhan and Omicron strains (Figures 6A and 6B).

Further experiments with the polypeptides were performed using HR2A-C and HR2 full length polypeptides. For the negative control, IPB19, an HIV peptide, was used, and for the positive control, ACE-Fc, an antibody that directly binds to the receptor, was used. Polypeptides (starting dilution 10 μM) were prepared using 4-fold serial dilutions (8 dilutions) and HEK293 ACE2 cells. All polypeptides were shown to inhibit the Omicron and WT strains, with IC50 values shown in Table 4.

Full-length HR2 Fc fusions were also tested and shown to maintain inhibitory activity against Omicron and Wuhan strains.

The full-length HR2 polypeptide ("HR2 complete") was shown to successfully inhibit fusion of Omicron BA.4/BA.5, SARS CoV-1, and Wuhan strains (Figures 7A, 7B, and 8A-8D).

Example 7: Expression of HIV antifusogenic polypeptides This example demonstrates the expression of HIV antifusogenic polypeptides from circular RNA.

Several HIV antifusogenic polypeptides were designed (Figure 9). Circular RNAs were designed to contain a nucleotide sequence encoding an IRES and an HIV antifusogenic polypeptide. In this example, a DNA construct was designed to contain a spacer element and a polynucleotide cargo (Figure 9). The construct was designed to contain a combination of an IRES and an ORF as the polynucleotide cargo. The ORF was designed to contain 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 the sequence VSGWRLFKKIS (SEQ ID NO: 362) with a GGS or GGGGS peptide linker. The IRES was either EMCV or modified CVB3.

Circular RNA was generated by self-splicing using the methods described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from a DNA template containing the motifs listed above in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with DNase. Synthesized linear RNA was purified with an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription. Circular RNA encoding antifusogenic peptides was purified by urea polyacrylamide gel electrophoresis (urea-PAGE) or reverse-phase column chromatography.

To measure expression of HIV antifusogenic polypeptides, 0.4 pmol of circular RNA was delivered to HEK293 cells using lipofectamine. Expression was measured after 48 hours. Polypeptides were expressed in HEK293 cells as shown in Figures 10A and 10B. Expression was comparable between circular RNA and DNA plasmid as shown in Figures 11A and 11B. Total expression (ng/mL and nM) is shown in Figure 12 for the various polypeptides.

HIV Polypeptide and Nucleic Acid Sequences T20
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 313)
tatacttctctgatccactctctgatcgaggaatctcagaaccagcaggagaagaacgaacgaacaggaactgctggaactggataagtgggcttctctgtggaactggttc (using EMCV IRES) (SEQ ID NO: 363)
or tacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaagaacgagcaggagctgctggagctggacaagtggggccagcctgtggaactggttc (using modified CVB3 IRES) (SEQ ID NO: 364)
T1249
WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 318)
tggcaggagtgggaacagaagatcactgctctgctggaacaggctcagattcagcaggaaaagaacgaatacgaactgcagaagctggataagtgggcttctctgtgggagtggttc (using EMCV IRES) (SEQ ID NO: 365)
or tggcaggagtgggagcagaagatcaccgccctgctggagcaggccagatccagcaaggaagaagaacgagtacgagctgcagaagctggacaagtgggccagcctgtgggagtggttc (using modified CVB3 IRES) (SEQ ID NO: 366)
T1144
TTWEAWDRAIAAEYAARIEALLRALQEQQEKNEAALREL (SEQ ID NO: 316)
actacttgggaagcttgggatagagctatcgctgaatacgctgctagaattgaagctctgctgagagctctgcaggaacagcaggaaaagaacgaagctgctctgagagaactg (using EMCV IRES) (SEQ ID NO: 367)
or accacctgggaggcctgggaccggggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagcagcaagagaagaacgaggccgccctgcgggagctg (using modified CVB3 IRES) (SEQ ID NO: 368)
1144-2-0
TTWEAWDRAIAAEYAARIEALLRALQEQQEKNEAARLELDKWASLWNWF (SEQ ID NO: 317)
accacctgggaggcctgggacgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcag gagcagcaagagaagaacgaggccgccctgcgggagctggacaagtggggccagcctgtggaactggttc (modified CVB3 using IRES) (SEQ ID NO: 369)
T_2410
MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL (SEQ ID NO: 314)
atgacctggatggagtgggaccgggagatcaacaattacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaaggaagagctgctggagctg (using modified CVB3 IRES) (SEQ ID NO: 370)
T_2410_2.0
MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 315)
atgacctggatggagtgggaccgggagatcaacaattacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaaggaagacgaagcaggagctgctggagctggacaagtggggccagcctgtggaactggttc (using modified CVB3 IRES) (SEQ ID NO: 371)
1249_2.0
TTWQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 319)
accacctggcaggagtgggagcagaagatcaccgccctgctggagcaggccagatccagcaaggaagaagaacgagtacgagctgcagaagctggacaagtgggccagcctgtgggagtggttc (using modified CVB3 IRES) (SEQ ID NO: 372)
290676
TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELREL (SEQ ID NO: 323)
accacctgggaggcctgggaccggggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagccaggagcagcaagagaagaacgaggccgagctgcgggagctg (using modified CVB3 IRES) (SEQ ID NO: 373)
290676-2-0
TTWEAWDRAIAAEYAARIEALIRASQEQQEKNEAELRELDKWASLWNWF (SEQ ID NO: 324)
accacctgggaggcctgggaccggggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagccaggagcagcaagagaagaagaacgaggccgagctgcgggagctggacaagtgggccagcctgtggaactggttc (using modified CVB3 IRES) (SEQ ID NO: 374)
2635
TTWEAWDRAIAAEYAARIEALIRAAQEQQEKNEAALREL (SEQ ID NO: 320)
accacctgggaggcctgggaccggggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcgggagctg (using modified CVB3 IRES) (SEQ ID NO: 375)
2635_2.0
TTWEAWDRAIAAEYAARIEALIRAAQEQQEKNEAARLELDKWASLWNWF (SEQ ID NO: 321)
accacctgggaggcctgggaccggggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcgggagctggacaagtgggccagcctgtggaactggttc (using modified CVB3 IRES) (SEQ ID NO: 376)
2635_3.0
MTWEAWDRAIAAEYAARIEALIRAAQEQQEKNEAARLELDKWASLWNWF (SEQ ID NO: 322)
atgacctgggaggcctgggacgggccatcgccgagtacgccgctcggatcgaggccctgatccggggccgcccagg agcagcaagagaagaacgaggccgcactgcgggagctggacaagtggggccagcctgtggaactggttc (SEQ ID NO: 377)

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications, and that this application is intended to cover any variations, uses, or adaptations of the invention which generally follow the principles of the invention and include such departures from the invention which are within known or customary practice in the art to which the invention pertains, may be applied to the essential features previously described, and which fall within the scope of the claims. Other embodiments are within the scope of the claims.

Claims (36)

A circular polyribonucleotide comprising a polyribonucleotide cargo encoding an antifusogenic polypeptide. The circular polyribonucleotide of claim 1, wherein the polyribonucleotide cargo comprises an expression sequence encoding the antifusogenic polypeptide. The circular polyribonucleotide of claim 1 or 2, comprising a splice junction joining a 5' exon fragment and a 3' exon fragment. The circular polyribonucleotide of claim 2 or 3, wherein the polyribonucleotide cargo comprises an IRES operably linked to the expression sequence encoding the antifusogenic polypeptide. The circular polyribonucleotide of claim 4, further comprising a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The circular polyribonucleotide of claim 5, wherein the spacer region is at least 5 ribonucleotides in length. The circular polyribonucleotide of claim 6, wherein the spacer region is 5 to 500 ribonucleotides in length. The circular polyribonucleotide according to any one of claims 5 to 7, wherein the spacer region comprises a poly A, poly A-C, poly A-U, or poly A-G sequence. The circular polyribonucleotide according to any one of claims 1 to 8, which is at least 500 ribonucleotides in length. The circular polyribonucleotide of claim 9, which is 500 to 20,000 ribonucleotides in length. A linear polyribonucleotide comprising, from 5' to 3', (A) a 3' 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' intron fragment. The linear polyribonucleotide of claim 11, wherein the polyribonucleotide cargo comprises an expression sequence encoding the antifusogenic polypeptide. 13. The linear polyribonucleotide of claim 12, wherein the polyribonucleotide cargo comprises an IRES operably linked to the expression sequence encoding the antifusogenic polypeptide. The linear polyribonucleotide of any one of claims 11 to 13, wherein the cyclic polyribonucleotide further comprises a spacer region between one or more of (A), (B), (C), (D), (E), (F), and (G). The linear polyribonucleotide of claim 14, wherein the spacer region is at least 5 ribonucleotides in length. The linear polyribonucleotide of claim 15, wherein the spacer region is 5 to 500 ribonucleotides in length. The linear polyribonucleotide of any one of claims 14 to 16, wherein the spacer region comprises a polyA, polyA-C, polyA-U, or polyA-G sequence. A linear polyribonucleotide according to any one of claims 11 to 17, which is at least 500 ribonucleotides in length. The linear polyribonucleotide of claim 18, which is 500 to 20,000 ribonucleotides in length. A DNA vector encoding the linear polyribonucleotide according to any one of claims 11 to 19. A method for expressing an anti-membrane fusion polypeptide in a cell, comprising providing the cell with a circular polyribonucleotide according to any one of claims 1 to 10, a linear polyribonucleotide according to any one of claims 11 to 19, or a DNA vector according to claim 20 under conditions suitable for expressing the anti-membrane fusion polypeptide. A method for producing a circular polyribonucleotide from a linear polyribonucleotide according to any one of claims 11 to 20, comprising providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to produce the circular polyribonucleotide. A pharmaceutical composition comprising a circular polyribonucleotide according to any one of claims 1 to 10, a linear polyribonucleotide according to any one of claims 11 to 19, or a DNA vector according to claim 20, and a diluent, carrier, or excipient. A method for expressing an antifusogenic polypeptide in a subject, comprising administering a first dose of the pharmaceutical composition of claim 23 in an amount sufficient to result in a serum concentration of the antifusogenic polypeptide in the subject of at least 500 ng/mL. 25. The method of claim 24, further comprising administering a second dose of the pharmaceutical composition. 26. The method of claim 25, wherein the second dose is administered at least one day after the first dose of the pharmaceutical composition. 27. The method of claim 26, wherein the second dose is administered 1 to 90 days after the first dose of the pharmaceutical composition. The method of any one of claims 25 to 27, wherein the second dose is administered before the serum concentration of the antifusogenic polypeptide is less than about 500 ng/mL in the serum of the subject. 29. The method of claim 28, wherein the subject is maintained at a serum concentration of the antifusogenic polypeptide of at least 500 ng/mL. The method according to any one of claims 24 to 29, for treating or preventing a viral infection in the subject. 31. The method of claim 30, wherein the pharmaceutical composition is administered to the subject in an amount and for a duration sufficient to treat or prevent the viral infection. The method according to any one of claims 24 to 31, which reduces viral entry. A circular polyribonucleotide comprising a polyribonucleotide cargo encoding multiple antifusogenic polypeptides. 34. The circular polyribonucleotide of claim 33, wherein the polyribonucleotide cargo comprises an expression sequence encoding the antifusogenic polypeptide. The circular polyribonucleotide according to claim 33 or 34, wherein the antifusogenic polypeptides are directed against the same virus. The circular polyribonucleotide of claim 33 or 34, wherein the antifusogenic polypeptide is directed against two or more viruses.