Circular polyribonucleotides encoding ornithine transcarbamylase polypeptides

Abstract

The present disclosure relates, generally, to compositions and methods for producing, purifying, and using circular RNA encoding an ornithine transcarbamylase (OTC) polypeptide. Circular polyribonucleotides described herein are particularly useful for delivering a polyribonucleotide cargo encoding an OTC polypeptide to a target cell. The composition may be administered to a subject to treat or prevent a urea cycle disorder or an OTC deficiency.

Description

CIRCULAR POLYRIBONUCLEOTIDES ENCODING ORNITHINE TRANSCARBAMYLASE POLYPEPTIDESThe Sequence Listing associated with this application is provided in XML format in lieu of paper copy, and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is “96676-419726_SeqListing. xml” The XML file is 323 KB, was created on Mar 12, 2025, and is being submitted electronically, concurrent with the filing of this specification.BackgroundOrnithine transcarbamylase (OTC) is a key enzyme involved in protein breakdown and ammonia regulation as part of the urea cycle in the liver. The enzyme catalyzes the production of L-citrulline from L-ornithine as part of the urea cycle. Thus, OTC deficiency is a genetic disorder that impacts the central nervous system resulting from the disruption of nitrogen regulation that produces toxic levels of ammonia (hyperammonemia) in the blood. Thus, patients suffering from OTC deficiency may exhibit a variety of symptoms including excessive vomiting, poor appetite, lethargy, memory problems or coma, with the disorder eventually becoming life threatening.Treatments may include dietary protein restriction to limit ammonia accumulation, drugs targeting nitrogen removal from the blood, and in severe cases liver transplant. Thus, novel and effective therapies are still needed, including therapies focused on improving OTC expression, or OTC replacement Challenges with these OTC based therapies include delivery to target cells in the liver.Many modes of delivery exist for the delivery of therapeutic polynucleotides or proteins in a wide variety of therapeutic fields However, many delivery modalities are often ineffective For example, delivery of short polypeptides often results in short half-life and rapid clearance of the polypeptides Accordingly, a need exists for improved compositions and methods for the treatment of OTC deficiency by delivering a therapeutic OTC polypeptide to the liver.Summary of the InventionThe disclosure generally relates to a circular polyribonucleotide including a polyribonucleotide cargo encoding an ornithine transcarbamylase (OTC) polypeptideIn some embodiments the polyribonucleotide cargo includes an expression sequence encoding one or more OTC polypeptide.In some embodiments, the circular polyribonucleotide described herein includes a splice junction at a 5’ exon fragment or a 3’ exon fragment.In some embodiments, the polyribonucleotide cargo includes one or more non-coding sequences. In some embodiments, the polyribonucleotide cargo includes a non-coding sequence operably linked to the expression sequence encoding the OTC polypeptide In some embodiments, the one or more noncoding sequences includes an internal ribosomal entry site (IRES) sequence or spacer sequences. In some embodiments, the IRES sequence is operably linked to the expression sequence encoding the OTC polypeptide.In some embodiments, the polyribonucleotide cargo further includes the spacer sequence between the IRES and the 3’ exon fragment or the 5' exon fragment. In some embodiments, thepolyribonucleotide cargo further includes the spacer sequence between the OTC expression sequence and the 3’ exon fragment. In some embodiments, the polyribonucleotide further includes the spacer sequence adjacent to the 5’ exon or the 3’ exon fragment. In some embodiments, the spacer sequence is at least about 50 ribonucleotides in length. In some embodiments, the spacer sequence is from about 50 to about 650 ribonucleotides in length. In some embodiments, the spacer sequence comprises a polyA-T, polyA-U sequence, or polyA-C sequence.In some embodiments, the circular polyribonucleotide is at least about 2,000 ribonucleotides in length In some embodiments, the circular polyribonucleotide is from about 2,000 to about 3,000 ribonucleotides in length.The disclosure further relates to a linear polyribonucleotide including, from 5’ to 3’, (a) a 3’ intron fragment; (b) a 3’ splice site; (c) a 3’ exon fragment; (d) a polyribonucleotide cargo encoding an OTC polypeptide; (e) a 5’ exon fragment; (f) a 5’ splice site; and (g) a 5’ intron fragment.In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding one or more OTC polypeptides. In some embodiments, polyribonucleotide cargo includes an IRES operably linked to the expression sequence encoding the OTC polypeptide. In some embodiments, the linear polyribonucleotide further includes one or more spacer sequences. In some embodiments, the spacer sequence is at least 50 ribonucleotides in length. In some embodiments, the spacer sequence is at least 50 to 650 ribonucleotides in length. In some embodiments, the spacer sequence comprises a polyA-T, or polyA-U sequence.In some embodiments, the linear polyribonucleotide is at least about 2,200 ribonucleotides in length. In some embodiments, the linear polyribonucleotide is about 2,200 to 3,300 ribonucleotides in lengthIn some embodiments, the disclosure relates to a DNA vector encoding the linear polyribonucleotide as described herein. In some embodiments, the disclosure relates to a hepatocyte comprising the circular polyribonucleotide as described herein.In some embodiments, the disclosure relates to a method of producing a circular polyribonucleotide from the linear polyribonucleotide as described above, the method including providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to produce the circular polyribonucleotideIn some embodiments, the disclosure relates to a pharmaceutical composition including the circular polyribonucleotide as described herein, the linear polyribonucleotide as described herein, the DNA vector as described herein, or the hepatocyte as described herein, and a diluent, carrier, or excipient.In some embodiments, the disclosure relates to a method of treating OTC deficiency in a subject in need thereof, including administering a therapeutically effective amount of the pharmaceutical composition as described hereinIn some embodiments, the disclosure relates to a method of treating a urea cycle disorder in a subject in need thereof, including administering a therapeutically effective amount of the pharmaceutical composition as described herein.DefinitionsTo facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the disclosure. In case of conflict, the present document, including definitions, will control. Methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.T erms such as "a", "an," and "the" are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The term "or" is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or.” The terminology herein is used to describe specific embodiments, but their usage is not to be taken as limiting, except as outlined in the claims.As used herein, any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.As used herein, the term “about” refers to a value that is within ± 10% of a recited value.As used herein, the term “administering" refers to providing one or more pharmaceutical compositions described herein to a patient or a subject. By way of example and not limitation, composition administration, e.g., injection, may be performed by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (rd ) injection, intraperitoneal (i p ) injection, or intramuscular (i.m ) injection One or more such routes may be employed Parenteral administration may be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route In an embodiment, a composition of the present disclosure may comprise engineered cells or host cells expressing nucleic acid sequences described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount that is effective to treat or prevent inflammatory or urea cycle disorder or an OTC deficiency.The terms “administered in combination” or “co-administration” or “co-administering” or “coproviding” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, orother parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments may be partially additive, wholly additive, or greater than additive. The delivery may be such that an effect of the first treatment delivered is still detectable when the second is delivered.In some embodiments, the first treatment and second treatment may be administered simultaneously (e.g., at the same time), in the same or in separate compositions, or sequentially.Sequential administration refers to administration of one treatment before (e.g., immediately before, less than 5, 10, 15, 30, 45, 60 minutes; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 or more hours; 4, 5, 6, 7, 8, 9 or more days; 1, 2, 3, 4, 5, 6, 7, 8 or more weeks before) administration of an additional, e g., secondary, treatment. The order of administration of the first and secondary treatment may also be reversed.As used herein, the term “carrier” is a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., a circular polyribonucleotide) into a cell by a covalent modification of the circular polyribonucleotide, via a partially or completely encapsulating agent, or a combination thereof. Non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride-modified phytoglycogen or glycogen-type material), nanoparticles (e.g., a nanoparticle that encapsulates or is covalently linked binds to the circular polyribonucleotide), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., a protein covalently linked to the circular polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or transfection reagent).As used herein, the terms “circular polyribonucleotide,” “circular RNA,” and “circRNA” are used interchangeably and mean a polyribonucleotide molecule that has a structure having no free ends (i.e., no free 3’ or 5’ ends), for example a polyribonucleotide molecule that forms a circular or end-less structure through covalent or non-covalent bonds. The circular polyribonucleotide may be, e.g., a covalently closed polyribonucleotide.As used herein, the term “circularization efficiency” is a measurement of resultant circular polyribonucleotide versus its non-circular starting material.As used herein, the term “derived from” in the context of a nucleic acid, i.e., for a nucleic acid “derived from” (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares e g at least 60%, 70%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived. The skilled person is aware that sequence identity is typically calculated for the same types of nucleic acids, i.e., for DNA sequences or for RNA sequences. Thus, it is understood, if a DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, in a first step the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by U throughout the sequence) Thereafter, the sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined. Preferably, a nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g., in order to increase RNA stability even further and / or to prolong and / or increase protein production. In the context of amino acid sequences, the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares e.g. at least 60%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence from which it is derived.The term “diluent” means a vehicle including an inactive solvent in which a composition described herein (e.g., a composition including a circular polyribonucleotide) may be diluted or dissolved. A diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof A diluent can be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and 1,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, or powdered sugar.As used herein, the terms “disease,” “disorder,” and “condition” each refer to a state of sub-optimal health, for example, a state that is or would typically be diagnosed or treated by a medical professionalAs used herein, the term “expression sequence” is a nucleic acid sequence that encodes a product, e g., a peptide or polypeptide (e.g., an OTC polypeptide). An exemplary expression sequence that codes for a peptide or polypeptide can include a plurality of nucleotide triads, each of which can code for an amino acid and is termed as a “codon.”As used herein, the phrase “codon degenerate variant” when used with reference to a nucleic acid sequence refers to a nucleic acid sequence that differs from the referenced sequence, but that encodes a polypeptide having the same amino acid sequence as that encoded by the referenced sequence.As used herein, the term “fragment” with respect to a polypeptide or a nucleic acid sequence, e.g., an OTC polypeptide or a nucleic acid sequence encoding an OTC polypeptide, refers to a continuous, less than a whole portion of a sequence of the polypeptide or the nucleic acid. A fragment of a polypeptide or a nucleic acid sequence encoding a polypeptide, for instance, refers to continuous, less than a whole fraction (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the entire length) of the sequence such as a sequence disclosed herein. It is understood that all the present disclosure contemplates fragments of any OTC polypeptide disclosed herein.As used herein, the term “GC content” refers to the percentage of guanosine (G) and cytosine (C) in a nucleic acid sequence. The formula for calculation of the GC content is (G+C) I (A+G+C+U) x 100% (for RNA) or (G+C) I (A+G+C+T) 100% (for DNA). Likewise, the term “uridine content” refers to the percentage of uridine (U) in a nucleic acid sequence The formula for calculation of the uridine content is (U) / (A+G+C+U) x 100%. Likewise, the term “thymidine content” refers to the percentage of thymidine (T) in a nucleic acid sequence. The formula for calculation of the thymidine content is (T) I (A+G+C+T) x 100% Likewise, the term “adenosine content” refers to the percentage of adenosine (A) in a nucleic acid sequence. The formula for calculation of the adenosine content is (A) I (A+G+C+U) x 100% (for RNA) or (A) / (A+G+C+T) x 100% (for DNA) Likewise, the term “cytidinecontent” refers to the percentage of cytidine (C) in a nucleic acid sequence. The formula for calculation of the cytidine content is (C) / (A+G+C+U) × 100% (for RNA) or (C) / (A+G+C+T) × 100% (for DNA).Likewise, the term “guanosine content” refers to the percentage of guanosine (G) in a nucleic acid sequence. The formula for calculation of the guanosine content is (G) / (A+G+C+U) × 100% (for RNA) or (G) / (A+G+C+T) × 100% (for DNA).As used herein, the term “minimum free energy” (MFE) refers to a metric that measures the lowest kilocalorie per mole of free energy for a given RNA sequence’s potential secondary structure. The calculation of the MFE is based on a loop-based thermodynamic energy model and dynamic programming algorithm of Zucker and Steigler (Zuker, M and Stiegler, P. Nucleic Acid Research 9(1): 133-148, 1981).As used herein, the term “codon adaptation index” (CAI) refers to a metric that measures the relative codon bias of a given open reading frame of RNA in terms of deviation to a reference set of genes as defined by Sharp and Li (Sharp, PM, Li, W-H. Nucleic Acids Research. 15(3): 1281-1295. 1987)As used herein, the term “average unpaired probability" (AUP) refers to a metric that summarizes a given RNA sequence’s secondary structure as introduced by Wayment-Steele et al. (Wayment-Steele, HK. Nucleic Acids Research 49(18): 10604-10617, 2021). The AUP is the average of each base position’s probability of being unpaired according to all possible pairings and corresponding thermodynamic pairing probability.As used herein, the term “minimum degradation score” (MinDegScore) refers to a metric that aggregates all per-position predicted degradation levels of given RNA sequence and associated minimum free energy secondary structure The per-position degradation levels are predicted using machine learning of thousands of experimentally determined degradation sites, as introduced in Leppek et al. (Leppek, K., Byeon, G. W., Kladwang, W., Wayment-Steele, H. K., Kerr, C., Barna, M., Das, R.Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. Nature Commun (2022) 13(1): 1536, PMID: 35318324).By “heterologous” is meant to occur in a context other than in the naturally occurring (native) context. A “heterologous” polynucleotide sequence indicates that the polynucleotide sequence is being used in a way other than what is found in that sequence’s native genome For example, a “heterologous promoter” is used to drive transcription of a sequence that is not one that is natively transcribed by that promoter; thus, a “heterologous promoter” sequence is often included in an expression construct by means of recombinant nucleic acid techniques. The term "heterologous" is also used to refer to a given sequence that is placed in a non-naturally occurring relationship to another sequence; for example, a heterologous coding or non-coding nucleotide sequence is commonly inserted into a genome by genomic transformation techniques, resulting in a genetically modified or recombinant genome.As used herein, the term “impurity” is an undesired substance present in a composition, e g, a pharmaceutical composition as described herein. In some embodiments, an impurity is a process-related impurity In some embodiments, an impurity is a product-related substance other than the desired product in the final composition, e.g., other than the active drug ingredient, e.g., circular polyribonucleotide, as described herein. As used herein, the term “process-related impurity” is a substance used, present, or generated in the manufacturing of a composition, preparation, or product that is undesired in the finalcomposition, preparation, or product other than the linear polyribonucleotides or circular polyribonucleotide described herein. In some embodiments, the process-related impurity is an enzyme used in the synthesis or circularization of polyribonucleotides. As used herein, the term “product-related substance” is a substance or byproduct produced during the synthesis of a composition, preparation, or product, or any intermediate thereof. In some embodiments, the product-related substance is deoxyribonucleotide fragments. In some embodiments, the product-related substance is deoxyribonucleotide monomers. In some embodiments, the product-related substance is one or more of: derivatives or fragments of polyribonucleotides described herein, e g, fragments of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acids, monoribonucleic acids, diribonucleic acids, or triribonucleic acids.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. An intron fragment may be a 5’ portion of an intron (e.g., a 5’ portion of a catalytic intron) or a 3’ portion of an intron (e.g., a 3’ portion of a catalytic intron), such that the 5’ intron fragment and the 3’ intron fragment, together, form a functional intron, such as a functional intron capable of catalytic self-splicing. The term intron fragment is meant to refer to an intron split into two portions. The term intron fragment is not meant to state, imply, or suggest that the two portions or halves are equal in length. The term intron fragment is used synonymously with the term split-intron and may be used instead of the term “half-intron.”As used herein, the term “linear counterpart" is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence identity) as a circular polyribonucleotide and having two free ends (i.e., the uncircularized version (and its fragments) of the circularized polyribonucleotide). In some embodiments, the linear counterpart (e g., a pre-circularized version) is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence identity) and same or similar nucleic acid modifications as a circular polyribonucleotide and having two free ends (i.e., the uncircularized version (and its fragments) of the circularized polyribonucleotide). In some embodiments, the linear counterpart is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence (e g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence identity) and different or no nucleic acid modifications as a circular polyribonucleotide and having two free ends (i e, the uncircularized version (and its fragments) of the circularized polyribonucleotide). In some embodiments, a fragment of the polyribonucleotide molecule that is the linear counterpart is any portion of linear counterpart polyribonucleotide molecule that is shorter than the linear counterpart polyribonucleotide molecule. In some embodiments, the linear counterpart further includes a 5’ cap. In some embodiments, the linear counterpart further includes a poly adenosine tail. In some embodiments, the linear counterpart further includes a “spacer” or “spacer sequence”, which, as used herein refers to a region of a gene that is 5’ or 3’ of the coding region of a gene As used herein, the term “3’ spacer” or “3’ spacer sequence” is an untranslated region that is 3’ of the coding region of a gene. As used herein, the term “5’ spacer” or “5’ spacer sequence” is an untranslated region that is 5’ of the coding region of a gene. The untranslated region may or may not be transcribed in a cell In some embodiments, the untranslated region may comprise translation enhancing sequences or mRNA stability enhancing sequences which are transcribed but not translated. In some embodiments, the linear counterpart further includes a 3’ spacer sequence.In some embodiments, the linear counterpart further includes a 5' spacer sequence. In addition, a “spacer” or “spacer sequence” may refer to any contiguous, non-coding nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Exemplary spacer sequences include, but are not limited to, polyX sequences as described herein, repetitive or random non-coding DNA or RNA sequences located 3’ or 5’ to open reading frames, or 3’ or 5’ untranslated regions (UTRs). Any spacer sequence deemed appropriate by the skilled artisan for the polyribonucleotides described herein are contemplated by this disclosure.As used interchangeably herein, the terms “polyX” and “polyX sequence” refer to an untranslated, contiguous region of any nucleic acid molecule and consisting of individual adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) residues, or some combination thereof For example, in some embodiments, a polyA sequence may be sequence of adenine residues. In other embodiments, a polyA-T sequence is a combination of adenine and thymine residues. In other embodiments, a poly A-U sequence may be a combination of adenine and uracil residues. In some embodiments, a polyA-G sequence is a combination of adenine and guanine residues. In some embodiments, a polyG-C sequence is a combination of guanine and cytosine residues In some embodiments a poly A-C sequence is a combination of adenine and cytosine residues Any combination of nucleotides may be used, including combinations with two to four different nucleotides, and may be referred to as, for example “polyATC sequence” or “polyATOG" sequence. In some embodiments, a polyX sequence may be at least about 50 nucleotides to about 700 nucleotides in length, at least about 60 nucleotides to about 600 nucleotides in length, at least about 70 nucleotides to about 500 nucleotides in length, at least about 80 nucleotides to about 400 nucleotides in length, at least about 90 nucleotides to about 300 nucleotides in length, at least about 100 nucleotides to about 200 nucleotides in length In some embodiments, the polyX sequence may be at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 nucleotides in length.In some embodiments, the polyX sequence may comprise shorter sequences of 2-5 nucleotides in length. In some embodiments, a polyX sequence may be located 3’ to (e.g, downstream of) an open reading frame (e.g, an open reading frame encoding an OTC polypeptide), and the polyX sequence may be 3’ to a termination element (e.g., a stop codon) such that the polyX sequence is not translated. In some embodiments, a polyX sequence may be located 3’ to a termination element and a 3’ spacer sequence. In some embodiments the polyX sequence may be located 5’ (e.g upstream of) or 3’ to a spacer sequence. Such polyX sequences located on either side of a spacer sequence my serve to “connect” spacer sequences to other nucleotide elements.As used herein, the terms “linear RNA,” “linear polyribonucleotide,” and “linear polyribonucleotide molecule” are used interchangeably and mean polyribonucleotide molecule having a 5’ and 3’ end. One or both of the 5’ and 3’ ends may be free ends or joined to another moiety. Linear RNA includes RNA that has not undergone circularization (e g, is pre-circularized) and can be used as a starting material for circularization through, for example, splint ligation, or chemical, enzymatic, ribozyme- or splicing-catalyzed circularization methods.As used herein, the term “modified ribonucleotide” means a nucleotide with at least one modification to the sugar, the nucleobase, or the internucleoside linkage.As used herein, the term “naked delivery” is a formulation for delivery to a cell without the aid of a carrier and without covalent modification to a moiety that aids in delivery to a cell. A naked delivery formulation is free from any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, naked delivery formulation of a circular polyribonucleotide is a formulation that includes a circular polyribonucleotide without covalent modification and is free from a carrier.As used herein, the terms “nicked RNA,” “nicked linear polyribonucleotide,” and “nicked linear polyribonucleotide molecule” are used interchangeably and mean a polyribonucleotide molecule having a 5’ and 3' end that results from nicking or degradation of a circular RNA.The term “pharmaceutical composition” is intended to also refer to the circular or linear polyribonucleotide included within a pharmaceutical composition that can be used for the treatment of the human or animal body by therapy. It is thus meant to be equivalent to “a polyribonucleotide for use in therapy”. A pharmaceutical composition may thus comprise any of the circular or linear polyribonucleotides described herein, a cell population comprising any of the circular or linear polyribonucleotides described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers as described herein, diluents or excipients Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.The term “polynucleotide,” as used herein, means a molecule including one or more nucleic acid subunits, or nucleotides, and can be used interchangeably with “nucleic acid” or “oligonucleotide” A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide can include a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides in which the sugar is ribose. Polyribonucleotides or ribonucleic acids, or RNA, can refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose As used herein, a polyribonucleotide sequence that recites thymine (T) is understood to represent uracil (U).As used herein, the term “polyribonucleotide cargo” herein includes any sequence including at least one polyribonucleotide. In embodiments, the polyribonucleotide cargo includes one or multiple expression sequences, wherein each expression sequence encodes a polypeptide. In embodiments, the polyribonucleotide cargo includes one or multiple noncoding sequences, such as a polyribonucleotide having regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo includes a combination of expression and noncoding sequences In embodiments, the polyribonucleotide cargo includes one or more polyribonucleotide sequence described herein, such as one or multiple regulatory elements, internal ribosomal entry site (IRES) elements, or spacer sequences.As used herein, the elements of a nucleic acid are “operably connected” or “operably linked” if they are positioned on the vector such that they can be transcribed to form a linear RNA that can then be circularized into a circular RNA using the methods provided herein.“Polydeoxyribonucleotides,” “deoxyribonucleic acids,” and “DNA” mean macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds.“Polyribonucleotides,” “ribonucleic acids,” and “RNA” mean macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. A nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide means a deoxyribonucleoside polyphosphate, such as, e.g., a deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, and may include detectable tags, such as protein tags, luminescent tags or markers (e.g., fluorophores). A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, 0, G, T or U, or complementary to a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C, T or U, or variant thereof). In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. In some cases, a polynucleotide is a short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA(mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In some cases, a polynucleotide molecule is circular. In other embodiments, a polynucleotide molecule may be linear and may have various lengths. A nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 50 kb, or more A polynucleotide can be isolated from a cell or a tissue Embodiments of polynucleotides include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.Embodiments of polynucleotides, e.g., polyribonucleotides or polydeoxyribonucleotides, may include one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s), 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-( carboxy hydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Non-limiting examples of such modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e g., alpha-thiotriphosphate and beta-thiotriphosphates). In embodiments, nucleicacid molecules are modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone. In embodiments, nucleic acid molecules contain amine -modified groups, such as amino allyl 1-dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N-hydroxysuccinimide esters (NHS). Alternatives to standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure can provide higher density in bits per cubic mm, higher safety (resistant to accidental or purposeful synthesis of natural toxins), easier discrimination in photo-programmed polymerases, or lower secondary structure. Such alternative base pairs compatible with natural and mutant polymerases for de novo or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul; 8(7):612-4, which is herein incorporated by reference for all purposes. The specific examples and types of modifications provided herein are representative of the modifications contemplated by the present disclosure. Thus the present disclosure contemplates all or any modifications known in the art, and may be made independently or in combination with others, and based on the specific parameters of the polynucleotides, target cells and tissues.As used herein, “polypeptide”, or “peptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing A polypeptide can be a single molecule or a multi-molecular complex such as a dimer, trimer, or tetramer They can also include single chain or multichain polypeptides such as antibodies or insulin and can be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.As used herein, the term “prevent” means to reduce the likelihood of developing a disease, disorder, or condition (e.g., a urea cycle disorder, or an OTO deficiency), or alternatively, to completely or partially reduce the severity or frequency of symptoms in a subsequently developed disease or disorder A therapeutic agent may be administered to a subject who is at increased risk of developing a urea cycle disorder or OTC deficiency, relative to a member of the general population in order to prevent the development of, or lessen the severity of, the disease or condition. A therapeutic agent may be administered as a prophylactic, e.g., before development of any symptom or manifestation of a viral infection.As used herein, the term “regulatory element” is a moiety, such as a nucleic acid sequence, that modifies expression of an expression sequence within the circular or linear polyribonucleotide Exemplary regulatory elements include, but are not limited to, promoters, termination sequences, enhancers and other expression control elements known in the art.As used herein, the term “RNA equivalent” refers to an RNA sequence that is the RNA equivalent of a DNA sequence. An RNA equivalent of a DNA sequence therefore refers to a DNA sequence in which each of the thymidine (T) residues is replaced by a uridine (U) residue. The disclosure specificallycontemplates that any of these DNA sequences may be converted to the corresponding RNA sequence and included in an RNA molecule described herein.A “signal sequence” refers to a polypeptide sequence, e.g., between 10 and 100 amino acids in length, that is present at the N-terminus of a polypeptide sequence of a nascent protein which targets the polypeptide sequence to the mitochondrial pathway. In some embodiments, a signal sequence polypeptide contemplates a human sequence, non-human sequence, or a combination thereof, for the mitochondrial targeting sequence.As used herein, the term “sequence identity” is determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm Sequences are referred to as "substantially identical” or “essentially similar” when they share at least a certain minimal percentage of sequence identity when optimally aligned (e g., when aligned by programs such as GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) 12 (proteins). For nucleotides the default scoring matrix used is nwsgapdna, and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity are determined, e.g., using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc, 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively, or additionally, percent identity is determined by searching against databases, e.g., using algorithms such as FAST A, BLAST, etc Sequence identity refers to the sequence identity over the entire length of the sequence Methods of alignment of sequences for comparison are well-known in the art Alignment may also be performed by inspection and manual alignment, in some embodiments, the polypeptides described herein may be at least 75%, 80%, 85%, 90%, 98% 99% or 100% identical to a reference polypeptide, or a fragment thereof, e g, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids may also be described with reference to a starting nucleic acid, e.g., they may be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid or a fragment thereof, e g, as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters When one molecule is said to have a certain percentage of sequence identity With a larger molecule, it means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned.As used herein, the term "subject" refers to an organism, such as an animal, plant, or microbe. In embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In embodiments, the subject is a human In embodiments, the subject is a non-human mammal In embodiments, the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, bison, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit) In embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g, ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g.,pigeons, doves), or Psitaciformes (e.g., parrots). In embodiments, the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc. In embodiments, the subject is an invertebrate agricultural pest or an invertebrate that is parasitic on an invertebrate or vertebrate host. Alternatively, " Patient” or “subject” as used herein may further refer to a mammalian subject diagnosed with or suspected of having an OTC deficiency. In some embodiments, the term “patient” refers to a mammalian subject with a higher than average likelihood of developing an OTC deficiency Exemplary patients may be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents and other mammalians that may benefit from the therapies disclosed herein Exemplary human patients may be male and / or female “Patient in need thereof’ or “subject in need thereof” is referred to herein as a patient diagnosed with or suspected of having a disease or disorder, for instance, but not restricted to a urea cycle disorder, or an OTC deficiency.As used herein, the term “OTC polypeptide” refers to a polypeptide, such as a polypeptide of between 10 and 500 amino acids, which encode the ornithine transcarbamylase enzyme that catalyzes the reaction between carbamoyl phosphate and ornithine to form citrulline as part of the urea cycle (Figure 2). OTC polypeptide includes, for example, the polypeptide of Figure 1, Table 1, and may be encoded by any of the DNA or RNA sequences of Figure 1, Table 1, all of which may be codon degenerate variants encoding the polypeptide sequence of Figure 1, T able 1. Furthermore, Figure 1, T able 1 contemplates any RNA sequences not shown of any DNA sequences shown in the table. An OTC polypeptide includes a polypeptide as well as any biologically active fragments thereof (e.g., a fragment of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids). In some embodiments, an OTC polypeptide includes a polypeptide having (e.g., at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or at least about 100%) sequence identity to SEQ ID NO: 1. In some embodiments, an OTC polypeptide includes a polypeptide having (e.g., at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or at least about 100%) sequence identity to any one of SEQ ID Nos: 1 to 5 An OTC polypeptide also refers to a polynucleotide (e.g., polyribonucleotide, e.g., circular polyribonucleotide encoding an OTC polypeptide (e.g., a polypeptide of Figure 1, Table 1) or a biologically active fragment thereof. In some embodiments, the OTC polypeptide sequence contemplated is human sequence, non-human sequence, or combinations thereof.Also used herein, the term “means for expressing an OTC polypeptide” includes any of the DNA or RNA sequences of the figures or tables herein that express an OTC polypeptide, and equivalents thereof. An OTC polypeptide may be any OTC polypeptide as described herein, and functionally equivalent variants thereof. Thus, in one embodiment is a means for expressing an OTC polypeptide from a circular polyribonucleotide In another embodiment is a means for expressing an OTC polypeptide from a linear polyribonucleotide Another embodiment is a means for expressing an OTC polypeptide from a polynucleotide. Equivalents of all sequences described herein are contemplated by this disclosure.As used herein, the terms “treat” and “treating" refer to a prophylactic or therapeutic treatment of a urea cycle disorder or OTC deficiency, in a subject. The effect of treatment may include reversing, alleviating, reducing severity of, curing, inhibiting the progression of, reducing the likelihood of recurrenceof the disease or one or more symptoms or manifestations of OTC deficiency, stabilizing (i e., not worsening) the state of the OTC deficiency in the absence of any therapeutic treatment.As used herein, the term “termination element” is a moiety, such as a nucleic acid sequence, that terminates translation of the expression sequence in the circular or linear polyribonucleotide.As used herein, the term “translation efficiency” is a rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency may be expressed as an amount of protein or peptide produced per given amount of transcript that codes for the protein or peptide, e g, in a given period of time, e g, in a given translation system, e g, a cell-free translation system, such as for example, the rabbit reticulocyte lysate system.As used herein, the term “translation initiation sequence” is a nucleic acid sequence that initiates translation of an expression sequence in the circular or linear polyribonucleotide.As used herein, a "vector" means a piece of DNAthat is synthesized (e.g., using PCR), or that is taken from a virus, plasmid, or cell of a higher organism into which a foreign DNA fragment may be or has been inserted for cloning or expression purposes. In some embodiments, a vector may be stably maintained in an organism. A vector may include, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, or a multiple cloning site (MCS). The term includes linear DNA fragments (e g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like. In one embodiment, a DNA vector encodes any of the linear polypeptides described herein. In one embodiment, the vectors provided herein include a multiple cloning site (MCS). In another embodiment, the vectors provided herein do not include an MCS.Brief Description of the DrawingsFigure 1: depicts Table 1, describing exemplary sequences for the OTC polypeptide described herein, and codon optimized DNA sequences encoding the OTC polypeptide, including a consensus codon optimized DNA sequence. The table contemplates any RNA sequences corresponding to the DNA sequences in the table that also encode the OTC polypeptide. The table also includes the RNA sequences for the OTC polypeptide for circRNA candidates.Figure 2: describes the reaction of the OTC enzyme with carbamoyl phosphate and ornithine to form citrulline.Figure 3: describes circularization of a linear RNA polyribonucleotide through the use of selfsplicing introns to obtain a circularized polyribonucleotide constructFigure 4: depicts Table 2, describing exemplary 5’ and 3’ spacer sequences.Figure 5: depicts Table 4, describing exemplary linear and circular polyribonucleotide sequences as described herein.Figure 6: describes an exemplary plasmid map for expression of OTC-containing polyribonucleotides described herein.Figure 7: describes the evaluation of codon optimized OTC constructs for expression efficiency. Figure 7A describes the Minimum Free Energy (MFE) and Codon Adaptation Index (CAI) that was usedto screen constructs for optimal codon usage, maximal translation rate, and minimal MFE to modulate translation rate and promote circRNA stability. Figure 7B describes the Average Unpaired Probability (AUP) calculations that were used to design codon optimized OTC sequences with minimal global AUP and optimized local AUP at 573’ ORF ends. Figure 7C describes the MinDegScore used to screen constructs to minimize degradation and extend the half-life of the OTC constructs.Figure 8: describes results of varying spacer sequences on mRN A stability and expression. Figure 9: describes expression of OTC polypeptides using circRNA candidates in HEK293 cells and quantified by JESS: Simple Western blotsFigure 10: describes the effect of spacer sequences on expression of OTC polypeptides from circular RNA.Figure 11: describes a scatterplot of the sequence optimization data in vitro for OTC expression in human hepatocytes compared to HEK293T cells. The data compiled includes the MFE / CAI screen of Figure 7A (grey squares), the AUP calculations of Figure 7B (white circles), the MinDegScore of Figure 7C (grey circles), and the spacer screen of Figure 8 (black triangles).Figure 12: describes the effect of the IFN type 1 response on protein expression for OTC polypeptides from eRNA and modified mRNA in mice liver.Figure 13: describes the IFNa levels in mouse plasma with different human OTC (hOTC) eRNA obtained from the sequence optimization and human OTC modified mRNA endogenous sequence (TriLink). Results show that using improved sequences (SEQ ID NO: 178, 193, and 194) resulted in lower plasma of IFNa compared to eRNA v1.0 (SEQ ID 192) and modified mRNA (SEQ ID 190), indicating lower induction of the immune response.Figure 14: describes Liver hOTC protein expression in mice 3 days post-injection with different human OTC eRNA obtained from the sequence optimization and human OTC modified mRNA endogenous sequence (TriLink). Results presented as fold change to eRNA Version 1 (SEQ ID 192).Figure 15: describes the quantification of liver hOTC protein with JESS detection following a single dose in WT mice with LNP-encapsulated eRNA expressing hOTC which led to persistent expression of human OTC protein in mouse liver for 14 days.Figure 16: shows OTC protein derived from eRNA is able to reduce urinary orotic acid (Urea cycle Biomarker) to normal levels in Spf-ash OTC deficient mice over the duration of 8 daysFigure 17: shows circular RNA constructs in LNPs encoding hOTC were dosed in vivo to measure the effects of increased dosage and the use of dexamethasone on constructs having a low uridine content.Detailed DescriptionThe present disclosure describes a circular polyribonucleotide (circular RNA) encoding an OTC polypeptide, composition, and methods of use thereof. Circular polyribonucleotides described herein are particularly useful for delivering a polyribonucleotide cargo encoding an OTC polypeptide to a target cell The circular polyribonucleotide described herein may include a polyribonucleotide cargo encoding a polypeptide of Figure 1, Table 1. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding an OTC polypeptide. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding an OTC polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of SEQ ID NO: 1. In someembodiments, the polyribonucleotide cargo comprises an expression sequence encoding an OTC polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of any one of SEQ ID NOs: 1 to 5. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding an OTC polypeptide from a polyribonucleotide corresponding to a DNA sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 6-95.The circular polyribonucleotide described herein may be produced from a precursor, such as a linear deoxyribonucleotide, a linear polyribonucleotide, a circular deoxyribonucleotide, or a circular polyribonucleotide.A circular polyribonucleotide may include, for example, a splice junction joining a 5’ exon fragment and a 3' exon fragment.The linear RNA molecules described herein a polyribonucleotide encoding an OTC polypeptide. 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 OTC polypeptide (e.g., polyribonucleotide cargo encoding an IRES operably linked to an expression sequence encoding an OTC polypeptide); (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment. The catalytic intron fragments and splice sites may then allow the linear polyribonucleotide to self-splice after expression, thus forming a circular polyribonucleotide encoding an OTC polypeptide.Also featured are methods of using a circular polyribonucleotide as described herein. For example, the circular polyribonucleotide may be formulated as a composition (e.g., a pharmaceutical composition) for administration to a subject, e.g., a human subject. The pharmaceutical composition may be administered in one or more doses of the composition The composition may be administered to the subject to treat or prevent a urea cycle disorder or an OTC deficiency.Each of the DNA sequences described herein include the RNA equivalent sequence, as would be understood by one skilled in the art. Likewise, all RNA sequences described herein include the DNA equivalent sequence, as would be understood by one skilled in the art.PolynucleotidesThe disclosure features circular polyribonucleotides encoding an OTC polypeptide, and composition and uses thereof, methods of making circular polyribonucleotides encoding an OTC polypeptide. In some embodiments, a circular polyribonucleotide is produced from a linear polyribonucleotide (e.g., by self-splicing compatible ends of the linear polyribonucleotide). In some embodiments, a linear polyribonucleotide is transcribed from a deoxyribonucleotide template (e.g., a vector, a linearized vector, or a cDNA). Accordingly, the disclosure features deoxyribonucleotides, linear polyribonucleotides, and circular polyribonucleotides and compositions thereof, useful in the production of circular polyribonucleotides encoding an OTC polypeptideTemplate DeoxyribonucleotidesThe present disclosure features a template deoxyribonucleotide for making a circular RNA as described herein. In embodiments, the deoxyribonucleotide includes the following, operably linked in a 5’-to-3’ orientation: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) apolyribonucleotide cargo encoding an OTC polypeptide; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment. In embodiments, the deoxyribonucleotide includes further elements, e.g., outside of or between any of elements (A), (B), (C), (D), (E), (F), or (G). In embodiments, any of the elements (A), (B), (C), (D), (E), (F), or (G) is separated from each other by a spacer sequence, as described herein.In embodiments, the deoxyribonucleotide is, for example, a circular DNA vector, a linearized DNA vector, or a linear DNA (e.g., a cDNA, e.g., produced from a DNA vector).In some embodiments, the deoxyribonucleotide further includes an RNA polymerase promoter operably linked to a sequence encoding a 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 virus promoter, or an SP3 promoter.In some embodiments, the deoxyribonucleotide includes a multiple-cloning site (MCS).In some embodiments, the deoxyribonucleotide is used to produce circular RNA with the size range of about 100 to about 20,000 nucleotides. In some embodiments, the circular RNA is at least about 100, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1,900, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, or about 20,000 nucleotides in size. In some embodiments, the circular RNA is no more than about 20,000, about 15,000 about 10,000, about 9,000, about 8,000, about 7,000, about 6,000, about 5,000, about 4,000, about 3,000, or about 2,000 nucleotides in size. In other embodiments, the circular RNA is no more than about 4,000, about 4,100, about 4,200, about 4,300, about 4,400, about 4,500, about 4,600, about 4,700, about 4,800, about 4,900, about 5,000, about 5,100, about 5,200, about 5,300, about 5,400, about 5,500, about 5,600, about 5,700, about 5,800, about 5,900, about 6,000, about 6,100, about 6,200, about 6,300, about 6,400, about 6,500, about 6,600, about 6,700, about 6,800, about 6,900 or about 7,000.Linear PolyribonucleotidesThe present disclosure also features linear polyribonucleotides encoding an OTC polypeptide. In other embodiments, the linear polyribonucleotide comprises one or more non-coding sequence or an expression sequence. In other embodiments, the one or more non-coding sequence is operably linked to the expression sequence encoding the OTC polypeptide described herein. In one embodiment, the noncoding sequence comprises an internal ribosomal entry (IRES) sequence or spacer sequences. The linear polyribonucleotide may be used to create a circular polyribonucleotide, e.g., by ligating or splicing (e.g., self-splicing) the linear polyribonucleotide to produce the circular polyribonucleotide. In embodiments, the linear polyribonucleotide includes the following, operably linked in a 5’-to-3’ orientation: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotidecargo encoding an OTC polypeptide; (E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment, as described in Figure 3. In embodiments, the linear polyribonucleotide includes further elements, e.g., outside of or between any of elements (A), (B), (C), (D), (E), (F), or (G). For example, any of elements (A), (B), (C), (D), (E), (F), or (G) may be separated by a spacer sequence, as described herein.In certain embodiments, provided herein is a method of generating linear RNA encoding an OTC polypeptide by performing transcription in a cell-free system (e.g., in vitro transcription) using a deoxyribonucleotide (e g, a vector, linearized vector, or cDNA) encoding an OTC polypeptide provided herein as a template (e.g., a vector, linearized vector, or cDNA provided herein with an RNA polymerase promoter positioned upstream of the region that codes for the linear RNA).In embodiments, a deoxyribonucleotide template is transcribed to produce a linear RNA containing the components described herein. For example, any of the deoxyribonucleotide sequences of Figure 1, Table 1 may be transcribed to produce a linear polyribonucleotide sequence encoding the OTC polypeptide as described herein. Upon expression, the linear polyribonucleotide produces a splicingcompatible polyribonucleotide, which may be self-spliced in order to produce a circular polyribonucleotide. In some embodiments, the circular polyribonucleotide comprises a splice junction at a 5’ exon fragment or a 3’ exon fragment.In some embodiments, the linear polyribonucleotide is from 50 to 6,000, 100 to 4,000, 200 to 3,000, 300 to 2,000 (e.g., 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, or 6,000) ribonucleotides in length. In embodiments, the linear polyribonucleotide is, e g., at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, or at least 5,000 ribonucleotides in length. In other embodiments the linear polyribonucleotide is at least about 2,000, about 2,100, about 2,200, about 2,300, about 2,400, about 2,500, about 2,600, about 2,700, about 2,800, about 2,900, about 3,000, about 3,100, about 3,200, about 3,300, about 3,400, about 3,500, about 3,700, about 3,800, about 3,900, about 4,000 ribonucleotides in length. In some embodiments, the linear polyribonucleotide is about 2,220 to about 3,300 ribonucleotides in length.Circular PolyribonucleotidesIn some embodiments, the disclosure describes a circular polyribonucleotide comprising an expression sequence encoding an OTC polypeptide. In other embodiments, the circular polyribonucleotide comprises one or more non-coding sequence. In other embodiments, the one or more non-coding sequence is operably linked to the expression sequence encoding the OTC polypeptide described herein In one embodiment, the non-coding sequence comprises an internal ribosomal entry (IRES) sequence or spacer sequences. In embodiments, the circular polyribonucleotide includes an IRES operably linked to an expression sequence encoding an OTC polypeptide. The circular polyribonucleotide may include a splice junction, e.g, joining a 5’ exon fragment and a 3’ exon fragment. The circular polyribonucleotide may include any one or more of the elements described herein. In some embodiments, the circular polyribonucleotide includes any feature or any combination of features asdisclosed in International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In embodiments, the circular polynucleotide further includes a polyribonucleotide cargo. In embodiments, the polyribonucleotide cargo includes an expression (or coding) sequence, a non-coding sequence, or a combination of an expression (coding) sequence and a non-coding sequence. In some embodiments, the polyribonucleotide cargo includes an expression (coding) sequence encoding a polypeptide. In some embodiments, the polyribonucleotide includes an IRES operably linked to an expression sequence encoding a polypeptide In some embodiments, the polyribonucleotide includes an IRES operably linked to an expression sequence encoding an OTO polypeptide as described herein. In some embodiments, the IRES is located upstream of the expression sequence. In other embodiments, the IRES is located downstream of the expression sequence In some embodiments, the circular polyribonucleotide further includes one or more spacer sequence between the IRES and the 3’ exon fragment or the 5’ exon fragment.The spacer sequence may be, e.g., at least 3 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length ribonucleotides in length. In some embodiments, the spacer region may be, e.g., from 50 to 1000 ribonucleotides in length. In some embodiments, the spacer is about 60 to about 750, about 70 to about 650, about 80 to about 600, about 90 to about 550, about 100 to about 500, about 150 to about 450, about 200 to about 400, ribonucleotides in length. In some embodiments the spacer is about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1,000 ribonucleotides in length In some embodiments, the spacer sequence is a polyX sequence. In some embodiments, the spacer sequence comprises a polyA sequence. In some embodiments, the spacer sequence comprises a polyA-C sequence. In some embodiments, the spacer sequence comprises a polyA-G sequence. In some embodiments, the spacer sequence comprises a polyA-T sequence. In some embodiments, the spacer sequence comprises a polyA-U sequence In some embodiments, the spacer sequence includes a random sequence. In some embodiments, a first annealing region and a second annealing region are joined, thereby forming a circular polyribonucleotideIn some embodiments, the circular RNA is produced by a deoxyribonucleotide template or a corresponding linear RNA described herein. In some embodiments, the circular RNA is produced by any of the methods described herein.In some embodiments, the circular polyribonucleotide is at least about 50 ribonucleotides, at least about 75 ribonucleotides, at least about 100 ribonucleotides, at least about 200 ribonucleotides, at least about 300 ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 1,000 ribonucleotides, at least about 2,000 ribonucleotides, at least about 3,000 ribonucleotides, at least about 4,000 ribonucleotides, or at least about 5,000 ribonucleotides.In some embodiments, the circular polyribonucleotide is between about 500 ribonucleotides and about 5,000 ribonucleotides, between about 600 and about 3,000 ribonucleotides, or between about 700 and about 2,000 ribonucleotides. In some embodiments, the circular polyribonucleotide is about 1,000 ribonucleotides, about 1,100 ribonucleotides, about 1,200 ribonucleotides, about 1,300 ribonucleotides,about 1,400 ribonucleotides, about 1,500 ribonucleotides, about 1,600 ribonucleotides, about 1,700 ribonucleotides, about 1,800 ribonucleotides, about 1,900 ribonucleotides, about 2,000 ribonucleotides, about 2,100 ribonucleotides, about 2,200 ribonucleotides, about 2,300 ribonucleotides, about 2,400 ribonucleotides, about 2,500 ribonucleotides, about 2,600 ribonucleotides, about 2,700 ribonucleotides, about 2,800 ribonucleotides, about 2,900 ribonucleotides, about 3,000 ribonucleotides, about 3,100 ribonucleotides, about 3,200 ribonucleotides, about 3,300 ribonucleotides, about 3,400 ribonucleotides, about 3,500 ribonucleotides, about 3,600 ribonucleotides, about 3,700 ribonucleotides, about 3,800 ribonucleotides, about 3,900 ribonucleotides, or about 4,000 ribonucleotides In some embodiments, the circular polyribonucleotide is about 2,000 to about 3,000 ribonucleotides in length.As a result of its circularization, the circular polyribonucleotide may include certain characteristics that distinguish it from linear RNA. For example, the circular polyribonucleotide may be less susceptible to degradation by exonuclease as compared to linear RNA. As such, the circular polyribonucleotide may be more stable than a linear RNA, especially when incubated in the presence of an exonuclease. The increased stability of the circular polyribonucleotide compared with linear RNA may make circular polyribonucleotide more useful as a cell transforming reagent to produce polypeptides and can be stored more easily and for longer than linear RNA. The stability of the circular polyribonucleotide treated with exonuclease may be tested using methods standard in the art which determine whether RNA degradation has occurred (e.g., by gel electrophoresis). Moreover, unlike linear RNA, the circular polyribonucleotide may be less susceptible to dephosphorylation when the circular polyribonucleotide is incubated with phosphatase, such as calf intestine phosphatase.The circular polyribonucleotides described herein and compositions or pharmaceutical compositions thereof, may be used in therapeutic and veterinary methods of dosing to produce a level of circular polyribonucleotide, a level of binding to a target, or a level of protein in a plurality of cells after providing the plurality with at least one or more doses of circular polyribonucleotide. In some embodiments, the circular polyribonucleotide is non-immunogenic in a mammal, e.g, a human. In some embodiments, the circular polyribonucleotide is capable of replicating or replicates in a cell from an aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (horses, cows, pigs, chickens etc ), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, 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 a cell that includes the circular polyribonucleotide described herein, wherein the cell is a cell from an aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammalian cell, e.g, a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (horses, cows, pigs, chickens etc.), a human cell, a cultured cell, a primary cell or a cell line, a stem cell, a progenitor cell, a differentiated cell, a germ cell, a cancer cell (e.g., tumorigenic, metastatic), a non-tumorigenic cell (normal cells), a fetal cell, an embryonic cell, an adult cell, a mitotic cell, a non-mitotic cell, a tissue specific cell, for example, a hepatic cell or hepatocyte, an intestinal cell, or any combination thereof. In some embodiments, the cell is modified to include the circular polyribonucleotide.In some embodiments, the circular polyribonucleotide includes sequences for expression products. In some embodiments, the circular polyribonucleotide includes a binding site for binding to a target In some embodiments, the circular polyribonucleotide is provided to a plurality of cells via any a dosing regimen described herein. In some embodiments, the circular polyribonucleotide as described herein induces a response or response level in a subject. In some embodiments, the expression products encoded by the sequences included in the circular polyribonucleotide are expressed in one or more of cells in the plurality of cells.In some embodiments, the circular polyribonucleotide has a half-life of at least that of a linear counterpart, e.g., linear expression sequence, or linear polyribonucleotide. In some embodiments, the circular polyribonucleotide has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell for at least about 1 hour, e g., at least 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell for from about 1 hour to about 60 days, e.g, about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell post division In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a dividing cell for at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a dividing cell of from about 10 minutes to about 60 days, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 60 days.In some embodiments, the circular polyribonucleotide modulates a cellular function, e.g., transiently, or long term. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer In certain embodiments, the cellular function is stably altered, such as a modulation that persists for from about 1 hour to about 60 days, e.g., from about 1 hour to about 30 days, e.g., for at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 60 days.Elements of PolynucleotidesThe polynucleotides (e.g., circular polyribonucleotides) or polyribonucleotide cargo described herein may include any one or more non-coding or coding sequences, or any one or more of the elements described herein and an expression sequence encoding an OTC polypeptide.Polyribonucleotide CargoA polyribonucleotide cargo described herein includes any sequence including at least one polyribonucleotide. In some embodiments, the polyribonucleotide cargo includes an expression sequence, a non-coding sequence, or an expression sequence and a non-coding sequence. In some embodiments, the polyribonucleotide cargo includes an expression sequence encoding an OTC polypeptide. In some embodiments, the polyribonucleotide cargo includes an IRES operably linked to an expression sequence encoding an OTC polypeptide. In some embodiments, the polyribonucleotide cargo includes an expression sequence that encodes an OTC polypeptide that has a biological effect on a subject.A polyribonucleotide cargo may, for example, include at least about 40 nucleotides, at least about 50 ribonucleotides, at least about 75 ribonucleotides, at least about 100 ribonucleotides, at least about 200 ribonucleotides, at least about 300 ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 1,000 ribonucleotides, at least about 2,000 ribonucleotides, or at least about 5,000 ribonucleotides. In some embodiments, the polyribonucleotide cargo includes from about 1-20,000 ribonucleotides, about 1-10,000 ribonucleotides, about 1-5,000 ribonucleotides, about 100-20,000 ribonucleotide, about 100-10,000 ribonucleotides, about 100-5,000 ribonucleotides, about 500-20,000 ribonucleotides, about 500-10,000 ribonucleotides, about 500-5,000 ribonucleotides, about 500-2,000 ribonucleotides, about 1,000-20,000 ribonucleotides, about 1,000-10,000 ribonucleotides, about 1,000-5,000 ribonucleotides, or about 1,000-3,000 ribonucleotides. In some embodiments the polyribonucleotide cargo may include at least about 1,000 ribonucleotides, about 1,025 ribonucleotides, about 1,050 ribonucleotides, about 1,075 ribonucleotides, about 1,100 ribonucleotides, about 1,125 ribonucleotides, about 1,150 ribonucleotides, about 1,175 ribonucleotides, about 1,200 ribonucleotides, about 1,225 ribonucleotides, about 1,250 ribonucleotides, about 1,275 ribonucleotides, about 1,300 ribonucleotides, about 1,325 ribonucleotides, about 1,350 ribonucleotides, about 1,375 ribonucleotides, about 1,400 ribonucleotides, about 1,425 ribonucleotides, about 1,450 ribonucleotides, about 1,475 ribonucleotides, about 1,500 ribonucleotides, about 1,525 ribonucleotides, about 1,550 ribonucleotides, about 1,1575 ribonucleotides, about 1,600 ribonucleotides, about 1,625 ribonucleotides, about 1,650 ribonucleotides, about 1,675 ribonucleotides, about 1,700 ribonucleotides, about 1,725 ribonucleotides, about 1,750 ribonucleotides, about 1,775 ribonucleotides, about 1,800 ribonucleotides, about 1,825 ribonucleotides, about 1,825 ribonucleotides, about 1,850 ribonucleotides, about 1,875 ribonucleotides, about 1,900 ribonucleotides, about 1,925 ribonucleotides, about 1,950 ribonucleotides, about 2,000 ribonucleotides, about 2,225 ribonucleotides, about 2,250 ribonucleotides, about 2,275 ribonucleotides, about 2,300 ribonucleotides, about 2,325 ribonucleotides, about 2,350 ribonucleotides, about 2,375 ribonucleotides, about 2,400 ribonucleotides, about 2,425 ribonucleotides, about 2,450 ribonucleotides, about 2,475 ribonucleotides, about 2,500 ribonucleotides, about 2,525 ribonucleotides, about 2,550ribonucleotides, about 2,575 ribonucleotides, about 2,600 ribonucleotides, about 2,625 ribonucleotides, about 2,650 ribonucleotides, about 2,675 ribonucleotides, about 2,725 ribonucleotides, about 2,750 ribonucleotides, about 2,775 ribonucleotides, about 2,800 ribonucleotides, about 2,825 ribonucleotides, about 2,850 ribonucleotides, about 2,875 ribonucleotides, about 2,900 ribonucleotides, about 3,000 ribonucleotides, about 3,025 ribonucleotides, about 3,050 ribonucleotides, about 3,075 ribonucleotides, about 3,100 ribonucleotides, about 3,200 ribonucleotides, about 3,225 ribonucleotides, about 3,250 ribonucleotides, about 3,275 ribonucleotides, about 3,300 ribonucleotides, about 3,400 ribonucleotides, about 3,425 ribonucleotides, about 3,450 ribonucleotides, about 3,475 ribonucleotides, or about 3,500 ribonucleotides. In some embodiments, polyribonucleotide cargo may comprise at least about 1,025 ribonucleotides, about 1,039 ribonucleotides, about 1,046 ribonucleotides, about 1,051 ribonucleotides, about 1,062 ribonucleotides, about 1,079 ribonucleotides, about 1,088 ribonucleotides, about 2,004 ribonucleotides, about 2,253 ribonucleotides, about 2,445 ribonucleotides, about 2,582 ribonucleotides, about 2.791 ribonucleotides, about 2,936 ribonucleotides, about 3,140 ribonucleotides, or about 3,389 ribonucleotides.In some embodiments, the polyribonucleotide cargo includes one or multiple expression (or coding) sequences, wherein each expression (or coding) sequence encodes a polypeptide (e.g., an OTC polypeptide). In embodiments, the polyribonucleotide cargo includes one or multiple noncoding sequences. In embodiments, the polyribonucleotide cargo consists entirely of non-coding sequence(s) In embodiments, the polyribonucleotide cargo includes a combination of expression (or coding) and noncoding sequences.In some embodiments, the polyribonucleotide includes any feature, or any combination of features as disclosed in International Patent Publication No WO2019 / 118919, which is hereby incorporated by reference in its entirety.Polypeptide expression sequencesIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the circular polyribonucleotide) includes one or more expression (or coding) sequences, wherein each expression sequence encodes an OTC polypeptide. In some embodiments, the circular polyribonucleotide includes two, three, four, five, six, seven, eight, nine, ten or more expression (or coding) sequences.Each encoded OTC polypeptide may be linear or branched. In various embodiments, the polypeptide has a length from about 5 to about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less than about 200 amino acids.Polypeptides included herein may include naturally occurring polypeptides 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 OTC polypeptide) For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a polypeptide described herein or anaturally occurring polypeptide. In some instances, the polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to a protein of interest.In embodiments, polypeptides include multiple polypeptides, e.g., multiple copies of one polypeptide sequence, or multiple different polypeptide sequences. In embodiments, multiple polypeptides are connected by linker amino acids or spacer amino acids.In embodiments, the polynucleotide cargo includes 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 a consensus SRRxFLK “twin-arginine” motif (SEQ ID NO: 211), which serves to translocate a folded protein containing such a Tat signal peptide across a lipid bilayer. See also, e.g., the Signal Peptide Database publicly available at www[dot]signalpeptide[dot]de. Signal peptides are also useful for directing a protein to specific organelles; see, e.g., the experimentally determined and computationally predicted signal peptides disclosed in the Spdb signal peptide database, publicly available at proline. bic.nus.edu.sg / spdb.In some embodiments, the expression (or coding) sequence includes a poly-A sequence (e.g., at the 3’ end of an expression sequence). In some embodiments, the length of a poly-A sequence is greater than 10 nucleotides in length. In one embodiment, the poly-A sequence is greater than 15 nucleotides in length (e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A sequence is designed according to the descriptions of the poly-A sequence in

[0202] -

[0204] of International Patent Publication No. WO2019 / 118919A1, which is incorporated herein by reference in its entirety In some embodiments, the expression sequence lacks a poly-A sequence (e g, at the 3’ end of an expression sequence).In some embodiments, a circular polyribonucleotide includes a polyA, lacks a polyA, or has a modified polyA to modulate one or more characteristics of the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide lacking a polyA or having modified polyA improves one or more functional characteristics, e.g., immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response), half-life, and / or expression efficiency.OTC PolypeptidesThe disclosure provides circular polyribonucleotides that encode at least one expression sequence encoding an OTC polypeptide. In some embodiments, the OTC polypeptide encodes the ornithine transcarbamylase enzyme which facilitates the reaction shown in Figure 2.In some embodiments, the OTC polypeptide is a polypeptide or a variant thereof including an amino acid sequence selected from a sequence of Figure 1, Table 1. In some embodiments, the OTC polypeptide is a polypeptide including a contiguous stretch of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acids of SEQ ID NO: 1. In some embodiments, the OTC polypeptide is a polypeptide including a contiguous stretch of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the amino acids of a sequence in Figure 1, Table 1. In some embodiments, the OTC polypeptide is a polypeptide including a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequenceidentity to a sequence of Figure 1, Table 1. In some embodiments, the OTC polypeptide is a variant of a sequence in Figure 1, Table 1 that includes no more than one, two, three, four, five, six, seven, eight, nine, or ten mutations (e.g., substitutions, deletions, or insertions). In some embodiments, the circular polyribonucleotide includes an expression sequence encoding more than one OTC polypeptide.In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding an OTC polypeptide. In some embodiments, the OTC polypeptide is encoded by any of the deoxyribonucleotide sequences of Figure 1, Table 1 and SEQ ID NO: 1-92. In some embodiments, the OTC polypeptide is encoded by the ribonucleotide sequences corresponding to any of the deoxyribonucleotide sequences of Table 1. In other embodiments, the OTC polypeptide is encoded by the ribonucleotide sequences of Table 1. In some embodiments, the OTC polypeptide is encoded by a deoxyribonucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence of Figure 1, Table 1, or SEQ ID NOs: 6-95 In other embodiments, the OTC polypeptide is encoded by a ribonucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any of the ribonucleotide sequences of Figure 1, Table 1 or SEQ ID NOs: 94-95 In some embodiments, the OTC polypeptide may be encoded by the consensus sequence of SEQ ID NO: 93, where “N” represents any nucleotide or ribonucleotide.In some embodiments, the GC content of a nucleic acid sequence encoding an OTC polypeptide is at least 40% (e.g., at least 41%, at least 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%). In some embodiments, the GC content of a nucleic acid sequence encoding an OTC polypeptide is at most 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% or 55%. In some embodiments, the GC content of a nucleic acid sequence encoding an OTC polypeptide is 41% to 55%, 42% to 55%, 43% to 55%, 44% to 55%, 45% to 53%, 46% to 53%, and 47% to 53%In some embodiments, the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding an OTC polypeptide is more than 10% (e.g., more than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding an OTC polypeptide is at most 60% (e.g., at most 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 43%, 41%, or 40%) In some embodiments, the uridine content (for RNA) or the thymidine content (for DNA) of a nucleic acid sequence encoding an OTC polypeptide is 21% to 28%, 20% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21% to 23%, or 22% to 23%.The GC content of an expression sequence encoding the OTC polypeptide refers to the GC content of the expression sequence that exclusively encodes the OTC polypeptide with no other coding regions that encode polypeptides other than the OTC polypeptide. Likewise, the uridine content or thymidine of an expression sequence encoding the OTC polypeptide refers to the uridine content of the expression sequence that exclusively encodes the OTC polypeptide with no other coding regions that encode polypeptides other than the OTC polypeptide. In some embodiments, the calculation of the GC content or the uridine (or thymidine) content of the expression sequence encoding the OTC polypeptide only takes into account the continuous nucleic acid sequence that starts in a 5’ to 3’ direction from the first nucleoside of the start codon of the open reading frame that encodes the OTC polypeptide to the lastnucleoside of the stop codon of the same open reading frame. In other embodiments, the calculation of the GC content or the uridine (or thymidine) content of the expression sequence encoding the OTC polypeptide only takes into account the continuous nucleic acid sequence that starts in a 5’ to 3’ direction from the first nucleoside of the codon that encodes the N-terminal end amino acid residue of the OTC polypeptide to the last nucleoside of the codon that encodes the C-terminal end amino acid residue of the OTC polypeptide.In some embodiments, the nucleic acid sequence encoding the OTC polypeptide has a uridine content of more than 15% In some embodiments, the uridine content of a nucleic acid sequence encoding an OTC polypeptide is more than 10% (e.g., more than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%) In some embodiments, the uridine content of a nucleic acid sequence encoding an OTC 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 of a nucleic acid sequence encoding an OTC polypeptide is about 20% to 28%, 21% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21% to 23%, or 22% to 23%. In some embodiments, the nucleic acid sequence encoding the OTC polypeptide has a uridine content of 20% to 28%.Multiple OTC PolypeptidesIn some embodiments, the circular polyribonucleotide encodes multiple expression sequences each encoding an OTC polypeptide (e.g., two or more, such as 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, or 5 to 10 expression sequences).In some embodiments, the circular polyribonucleotide encodes two or more (e g, 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, or 5 to 10) copies of the same OTC polypeptide.In some embodiments, the circular polyribonucleotide encodes two or more (e.g, 2, 3, 4, 5, 6, 7, 8, 9, or 10) different (e.g., sharing less than 100% sequence identity) OTC polypeptides.Wherein a circular polyribonucleotide encodes two or more OTC polypeptides, the OTC polypeptides may be encoded in a single open reading frame or multiple open reading frames.In some embodiments, the disclosure provides a circular polyribonucleotide including an open reading frame (e g, an open reading frame operably linked to an IRES) that includes two or more expression sequences, where each expression sequence encodes an OTC polypeptide.In some embodiments, the disclosure provides a circular polyribonucleotide including a first open reading frame encoding a first OTC polypeptide (e.g, operably linked to a first IRES) and a second open reading frame encoding a second OTC polypeptide (e.g., operably linked to a second IRES).Internal Ribosomal Entry SitesIn some embodiments, a circular polyribonucleotide described herein includes one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences). In embodiments, the IRES is located between a heterologous promoter and the 5’ end of a coding sequence.A suitable IRES element to include in a polyribonucleotide includes an RNA sequence capable of engaging a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, or at least 900 nucleotides In some embodiments, the IRES element is at least about 350 nucleotides to about 900 nucleotides, at least about 450 nucleotides to about 850 nucleotides, at least about 500 nucleotides to about 750 nucleotides, at least about 700 nucleotides, at least about 727 nucleotides, at least about 741 nucleotides, at least about 756 nucleotides, at least about 779 nucleotides, or at least about 794 nucleotides.In some embodiments, the IRES element is derived from the DNA of an organism including, but not limited to, a virus, a mammal, and a Drosophila. Such viral DNA may be derived from, but is not limited to, picomavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA from which an IRES element is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster.In some embodiments, the IRES sequence is an IRES sequence of Taura syndrome virus, T riatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, fuman poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus (AEV), Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-I, Human BCL2, Human BiP, Human c-IAPI, Human c-myc, Human elF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-I, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Salivirus, Cosavirus, Parechovirus, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Human c-src, Human FGF-I, Simian picomavirus, Turnip crinkle virus, Aichivirus, Crohivirus, Echovirus 11, an aptamer to elF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 / 2). In yet another embodiment, the IRES is an IRES sequence of Coxsackievirus B3 (CVB3). In a further embodiment, the IRES is an IRES sequence of Encephalomyocarditis virus In a further embodiment, the IRES is an IRES sequence of Theiler's encephalomyelitis virus In some embodiments, the IRES sequence has sequence identity of 90% or more with one of the foregoing IRES sequences.The IRES sequence may have a modified sequence in comparison to the wild-type IRES sequence. In some embodiments, when the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be modified such that it is a cytosineresidue. For example, the IRES sequence may be a CVB3 IRES sequence wherein the terminal adenosine residue is modified to cytosine residue. In some embodiments, the modified CVB3 IRES may have the nucleic acid sequence of:TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATC ACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGAT CAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCA ATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGT AACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCA CCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATG GGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGA ATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAA CTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGT GACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATAT ATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGA ATACAGCAAC (SEQ ID NO: 96)In some embodiments, the IRES sequence is an Enterovirus 71 (EV71) IRES. In some embodiments, the terminal guanosine residue of the EV71 IRES sequence is modified to a cytosine residue. In some embodiments, the modified EV71 IRES may have the nucleic acid sequence of:UUAAAACAGCUGUGGGUUGUCACCCACCCACAGGGUCCACUGGGCGCUAGUACACUGGU AUCUCGGUACCUUUGUACGCCUGUUUUAUACCCCCUCCCUGAUUUGCAACUUAGAAGCAACGCAA ACCAGAUCAAUAGUAGGUGUGACAUACCAGUCGCAUCUUGAUCAAGCACUUCUGUAUCCCCGGAC CGAGUAUCAAUAGACUGUGCACACGGUUGAAGGAGAAAACGUCCGUUACCCGGCUAACUACUUC GAGAAGCCUAGUAACGCCAUUGAAGUUGCAGAGUGUUUCGCUCAGCACUCCCCCCGUGUAGAUC AGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCC UAUGGGGUAACCCAUAGGACGCUCUAAUACGGACAUGGCGUGAAGAGUCUAUUGAGCUAGUUAG UAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACAUACCCUUAAUCCAAAGGG CAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCCGUGUUUCUUUUU AUUCUUGUAUUGGCUGCUUAUGGUGACAAUUAAAGAAUUGUUACCAUAUAGCUAUUGGAUUGGC CAUCCAGUGUCAAACAGAGCUAUUGUAUAUCUCUUUGUUGGAUUCACACCUCUCACUCUUGAAAC GUUACACACCCUCAAUUACAUUAUACUGCUGAACACGAAGCGGCCACC (SEQ ID NO: 188)In some embodiments, the IRES sequence is an Enterovirus 71 (EV71) IRES. In some embodiments, the modified EV71 IRES may have the nucleic acid sequence of:TTAAAACAGCTGTGGGTTGTCACCCACCCACAGGGTCCACTGGGCGCTAGTACACTGGTAT CTCGGTACCTTTGTACGCCTGTTTTATACCCCCTCCCTGATTTGCAACTTAGAAGCAACGCAAACCAG ATCAATAGTAGGTGTGACATACCAGTCGCATCTTGATCAAGCACTTCTGTATCCCCGGACCGAGTAT CAATAGACTGTGCACACGGTTGAAGGAGAAAACGTCCGTTACCCGGCTAACTACTTCGAGAAGCCTA GTAACGCCATTGAAGTTGCAGAGTGTTTCGCTCAGCACTCCCCCCGTGTAGATCAGGTCGATGAGTC ACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCTATGGGGTAACCCAT AGGACGCTCTAATACGGACATGGCGTGAAGAGTCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTG AATGCGGCTAATCCTAACTGCGGAGCACATACCCTTAATCCAAAGGGCAGTGTGTCGTAACGGGCA ACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTGTATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTGGCCATCCAGTGTCAAACAGAGCTATTGTA TATCTCTTTGTTGGATTCACACCTCTCACTCTTGAAACGTTACACACCCTCAATTACATTATACTGCTG AACACGAAGCC (SEQ ID NO: 196).In some embodiments, the IRES sequence is a synthetic IRES. A “synthetic IRES" is an IRES that is modified relative to a wild type IRES in order to modulate its structure and / or activity. For example, in some embodiments, an IRES that is modified to incorporate an aptamer sequence is a synthetic IRES.In some embodiments, the polyribonucleotide includes at least one IRES flanking at least one (e g, 2, 3, 4, 5 or more) expression sequence In some embodiments, the IRES flanks both sides of at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the polyribonucleotide includes one or more IRES sequences on one or both sides of each expression sequence, leading to separation of the resulting peptide(s) and or polypeptide(s). For example, a polyribonucleotide described herein may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.In some embodiments, a polyribonucleotide described herein includes an IRES (e.g., an IRES operably linked to a coding region). For example, the polyribonucleotide may include any IRES as described in Chen et al. Nature Biotechnology 41:262-272, 2023; Chen et al. Mol. Cell 81(20):4300-18, 2021; Jopling et al. Oncogene 20:2664-70, 2001; Baranick et a / . 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; International Publication No. W02020 / 198403; International Publication No. WO2021 / 263124; and International Publication No WO2022 / 271965, each of which is hereby incorporated by reference in their entirety.Signal SequencesIn some embodiments, an OTC polypeptide expressed from a circular polyribonucleotide disclosed herein includes a mitochondrial protein, for example, a protein that naturally includes a signal sequence, or one that does not usually encode a signal sequence but is modified to contain one. In some embodiments, the OTC polypeptide encoded by the circular polyribonucleotide includes a mitochondrial targeting signal. For example, the signal may be the naturally encoded mitochondrial targeting signal for a mitochondrial protein. In another example, the signal may be a modified mitochondrial signal for a mitochondrial protein. In other embodiments, the OTC polypeptide encoded by the circular polyribonucleotide does not include a mitochondrial signal. In some embodiments, the mitochondrial targeting signal is a sequence that is human sequence, non-human sequence, or a combination thereof.In some embodiments, the signal sequence is MLFNLRILLNNAAFRNGHNFMVRNFRCGQPLQ; SEQ ID NO: 97 In some embodiments, the signal sequence is MTRILTAFKVVRTLKTGFGFTNVTAHQKWKFSRPGIRL; SEQ ID NO: 98. In some embodiments, the signal sequence is MLFNLRILLNNAALRNGHSFVVRNFRCGQPLQ; SEQ ID NO: 99. In some embodiments, the signal sequence is MLSNLRILLNNAALRKGHTSVVRHFWCGKPVQ; SEQ ID NO: 100. In some embodiments, the signal sequence is MLSNLRILLNKAALRKAHTSMVRNFRYGKPVQ; SEQ IDNO: 101. Any appropriate signal sequence for directing the OTC polypeptide to the mitochondria is contemplated hereinIn some embodiments, a circular polyribonucleotide encodes multiple copies of the same OTC polypeptide (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more). In some embodiments, at least one copy of the OTC polypeptide includes a signal sequence and at least one copy of the OTC polypeptide does not include a signal sequence. In some embodiments, a circular polyribonucleotide encodes plurality of an OTC polypeptides (e.g., a plurality of different OTC polypeptides or a plurality of an OTC polypeptides having less than 100% sequence identity), where at least one of the plurality of an OTC polypeptides includes a signal sequence and at least one copy of the plurality of an OTC polypeptides does not include a signal sequence.In some embodiments, the signal sequence is a wild-type signal sequence that is present on the N-terminus of the corresponding wild-type OTC polypeptide, e.g., when expressed endogenously. In some embodiments, the signal sequence is heterologous to the OTC polypeptide, e.g., is not present when the wild-type OTC polypeptide is expressed endogenously. A polyribonucleotide sequence encoding a polypeptide may be modified to remove the nucleotide sequence encoding a wild-type signal sequence and / or add a sequence encoding a heterologous signal sequence.A polypeptide encoded by a polyribonucleotide (e.g., an OTC polypeptide) may include a signal sequence that directs the OTC polypeptide to the mitochondrial pathway. In some embodiments, the signal sequence may direct the OTC polypeptide to reside in certain organelles (e g., the mitochondria, endoplasmic reticulum, Golgi apparatus, or endosomes). In some embodiments, the signal sequence directs the OTC polypeptide to be transported to the mitochondria in the cell. For mitochondrial proteins, the signal sequence may be cleaved after translocation, resulting in a mature protein In some embodiments, a circular polyribonucleotide includes at least one cleavage sequence (e.g. a MPP (mitochondrial processing protease) or MIP (mitochondrial intermediate peptidase)). In certain embodiments, the signal sequence of a protein is a short sequence at the N-terminal of the polypeptide. In the OTC polypeptide described herein, the signal sequence directs the OTC polypeptide to the mitochondria, where it is subsequently cleaved upon transport to its mature form.In some embodiments, the signal is a transit peptide signal In some embodiments, the signal has an amino acid sequence of at least 90% sequence identity to SEQ ID NO: 97 In some embodiments, the signal has an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 97. In some embodiments, the signal has an amino acid sequence of at least 99% sequence identity to SEQ ID NO: 97. In some embodiments, the signal has an amino acid sequence of 100% sequence identity to SEQ ID NO: 97.In some embodiments, the signal is a transit peptide signal In some embodiments, the signal has an amino acid sequence of at least 90% sequence identity to SEQ ID NOs: 97 to 101. In some embodiments, the signal has an amino acid sequence of at least 95% sequence identity to SEQ ID NOs: 97 to 101. In some embodiments, the signal has an amino acid sequence of at least 99% sequence identity to SEQ ID NOs: 97 to 101. In some embodiments, the signal has an amino acid sequence of 100% sequence identity to SEQ ID NOs: 97 to 101.Regulatory ElementsIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes one or more regulatory elements. In some embodiments, the polyribonucleotide includes a regulatory element, e.g., a sequence that modifies expression of an expression sequence within the polyribonucleotide.A regulatory element may include a sequence that is located adjacent to an expression sequence that encodes an expression product. A regulatory element may be linked operatively to the adjacent sequence A regulatory element may increase an amount of product expressed as compared to an amount of the expressed product when no regulatory element exists. In addition, one regulatory element can increase an amount of products expressed for multiple expression sequences attached in tandem. Hence, one regulatory element can enhance the expression of one or more expression sequences Exemplary regulatory elements include, but are not limited to, promoter sequences, enhancer sequences, cis-regulatory elements, repressor sequences, or silencer sequences Multiple regulatory elements are well-known to persons of ordinary skill in the art, and any appropriate regulatory elements may be used with the polyribonucleotides described herein.In some embodiments, the regulatory element is a translation modulator. A translation modulator can modulate translation of the expression sequence in the polyribonucleotide. A translation modulator can be a translation enhancer or suppressor. In some embodiments, the polyribonucleotide includes at least one translation modulator adjacent to at least one expression sequence. In some embodiments, the polyribonucleotide includes a translation modulator adjacent each expression sequence. In some embodiments, the translation modulator is present on one or both sides of each expression sequence, leading to separation of the expression products, e g, peptide(s) and or polypeptide(s)In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site. Further examples of regulatory elements are described, e.g., in paragraphs

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[0161] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.Cleavage DomainsA circular polyribonucleotide of the disclosure may include a cleavage domain (e.g., a stagger element or a cleavage sequence).The term “stagger element” refers to a moiety, such as a nucleotide sequence, that induces ribosomal pausing during translation. In some embodiments, the stagger element is a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence -D(V / l)ExNPGP, where x= any amino acid (SEQ ID NO: 102). In some embodiments, the stagger element may include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereofIn some embodiments, the circular polyribonucleotide includes at least one stagger element adjacent to an expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s). In some embodiments, the stagger element is a portionof the one or more expression sequences In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and each of the one or more expression sequences is separated from a succeeding expression sequence by a stagger element on the circular polyribonucleotide. In some embodiments, the stagger element prevents generation of a single polypeptide (a) from two rounds of translation of a single expression sequence or (b) from one or more rounds of translation of two or more expression sequences. In some embodiments, the stagger element is a sequence separate from the one or more expression sequences. In some embodiments, the stagger element includes a portion of an expression sequence of the one or more expression sequencesIn some embodiments, the circular polyribonucleotide includes a stagger element. To avoid production of a continuous expression product, e.g., peptide or polypeptide, while maintaining rolling circle translation, a stagger element may be included to induce ribosomal pausing during translation. In some embodiments, the stagger element is at 3’ end of at least one of the one or more expression sequences. The stagger element can be configured to stall a ribosome during rolling circle translation of the circular polyribonucleotide. The stagger element may include, but is not limited to a 2A-like, or CHYSEL (SEQ ID NO: 103) (cis-acting hydrolase element) sequence. In some embodiments, the stagger element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP (SEQ ID NO: 104), where Xi is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and Xs is any amino acid. In some embodiments, this sequence includes a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence -D(V / I)EXNPGP (SEQ ID NO: 102), where x= any amino acid. Some nonlimiting examples of stagger elements includes GDVESNPGP (SEQ ID NO: 105), GDIEENPGP (SEQ ID NO: 106), VEPNPGP (SEQ ID NO: 107), IETNPGP (SEQ ID NO: 108), GDIESNPGP (SEQ ID NO: 109), GDVELNPGP (SEQ ID NO: 110), GDIETNPGP (SEQ ID NO: 111), GDVENPGP (SEQ ID NO: 112), GDVEENPGP (SEQ ID NO: 113), GDVEQNPGP (SEQ ID NO: 114), IESNPGP (SEQ ID NO: 115), GDIELNPGP (SEQ ID NO: 116), HDIETNPGP (SEQ ID NO: 117), HDVETNPGP (SEQ ID NO: 118), HDVEMNPGP (SEQ ID NO: 119), GDMESNPGP (SEQ ID NO: 120), GDVETNPGP (SEQ ID NO: 121), GDIEQNPGP (SEQ ID NO: 122), and DSEFNPGP (SEQ ID NO: 123).In some embodiments, the stagger element described herein cleaves an expression product, such as between G and P of the consensus sequence described herein As one non-limiting example, the circular polyribonucleotide includes at least one stagger element to cleave the expression product. In some embodiments, the circular polyribonucleotide includes a stagger element adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element after each expression sequence. In some embodiments, the circular polyribonucleotide includes a stagger element present on one or both sides of each expression sequence, leading to translation of individual peptide(s) and or polypeptide(s) from each expression sequence.In some embodiments, a stagger element includes one or more modified nucleotides or unnatural nucleotides that induce ribosomal pausing during translation. Unnatural nucleotides may include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Examples such as these are distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate / to aphosphodiester linkage / to the phosphodiester backbone), and any combination thereof that can induce ribosomal pausing during translation. Some of the exemplary modifications provided herein are described elsewhere herein.In some embodiments, the stagger element is present in the circular polyribonucleotide in other forms. For example, in some exemplary circular polyribonucleotides, a stagger element includes a termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a first translation initiation sequence of an expression succeeding the first expression sequence In some examples, the first stagger element of the first expression sequence is upstream of (5’ to) a first translation initiation sequence of the expression succeeding the first expression sequence in the circular polyribonucleotide. In some cases, the first expression sequence and the expression sequence succeeding the first expression sequence are two separate expression sequences in the circular polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence can enable continuous translation of the first expression sequence and its succeeding expression sequence.In some embodiments, the first stagger element includes a termination element and separates an expression product of the first expression sequence from an expression product of its succeeding expression sequences, thereby creating discrete expression products. In some cases, the circular polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the succeeding sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide including a stagger element of a second expression sequence that is upstream of a second translation initiation sequence of an expression sequence succeeding the second expression sequence is not continuously translated In some cases, there is only one expression sequence in the circular polyribonucleotide, and the first expression sequence and its succeeding expression sequence are the same expression sequence. In some exemplary circular polyribonucleotides, a stagger element includes a first termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a downstream translation initiation sequence In some such examples, the first stagger element is upstream of (5’ to) a first translation initiation sequence of the first expression sequence in the circular polyribonucleotide In some cases, the distance between the first stagger element and the first translation initiation sequence enables continuous translation of the first expression sequence and any succeeding expression sequences.In some embodiments, the first stagger element separates one round expression product of the first expression sequence from the next round expression product of the first expression sequences, thereby creating discrete expression products. In some cases, the circular polyribonucleotide including the first stagger element upstream of the first translation initiation sequence of the first expression sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide including a stagger element upstream of a second translation initiation sequence of a second expression sequence in the corresponding circular polyribonucleotide is not continuously translated In some cases, the distance between the second stagger element and the second translation initiation sequence is at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x greater in the corresponding circular polyribonucleotide than a distance between the first stagger element and the first translation initiation inthe circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater. In some embodiments, the distance between the second stagger element and the second translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater than the distance between the first stagger element and the first translation initiation. In some embodiments, the circular polyribonucleotide includes more than one expression sequence Examples of stagger elements are described in paragraphs

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[0175] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In some embodiments, a plurality of enzymes needed to prevent a urea cycle disorder may be encoded by a circular ribonucleotide as described herein. For example, the circular ribonucleotide may include an OTC polypeptide in addition to other enzymes related to urea cycle disorders such as argininosuccinic acid synthetase, arginase, argininosuccinase acid lyase, carbamoyl phosphate synthetase, citrin, ornithine translocase, and N-acetylglutamate synthetase. In some embodiments, the OTC polypeptide and additional urea cycle enzyme encoded by a circular ribonucleotide may be separated by an IRES between each enzyme polypeptide (e.g., each enzyme polypeptide is operably linked to a separate IRES). For example, a circular polyribonucleotide may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.In some embodiments, a plurality of an OTC polypeptides encoded by a circular ribonucleotide may be separated by an IRES between each OTC polypeptide (e g, each OTC polypeptide is operably linked to a separate IRES). For example, a circular polyribonucleotide may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence. The IRES may be the same IRES between all OTC polypeptides. The IRES may be different between different OTC polypeptides.In some embodiments, the plurality of an OTC polypeptides may be separated by a 2A selfcleaving peptide For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first OTC polypeptide, a 2A, and a second OTC polypeptideIn some embodiments, the plurality of an OTC 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 OTC polypeptide, a protease cleavage site (e.g., a furin cleavage site), and a second OTC polypeptide.In some embodiments, the plurality of an OTC polypeptides may be separated by a 2A selfcleaving peptide and a protease cleavage site (e.g., a furin cleavage site) For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first OTC polypeptide, a 2A, a protease cleavage site (e g., a furin cleavage site), and a second OTC polypeptide. A circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first OTC polypeptide, a protease cleavage site (e.g., a furin cleavage site), a 2A, and a second OTC polypeptide. A tandem 2A and furin cleavage site may be referred to as a furin-2A (which includes furin-2A or 2A-furin, arranged in either orientation).Furthermore, the plurality of OTC polypeptides encoded by the circular ribonucleotide may be separated by both IRES and 2A sequences. For example, an IRES may be between one OTC polypeptide and a second OTC polypeptide while a 2A peptide may be between the second OTC polypeptide and the third OTC polypeptide. The selection of a particular IRES or 2A self-cleaving peptide may be used to control the expression level of an OTC polypeptide under control of the IRES or 2A sequence. For example, depending on the IRES and or 2A peptide selected, expression on the polypeptide may be higher or lowerIn some embodiments, a circular polyribonucleotide may include at least one cleavage sequence In some embodiments, the cleavage sequence is adjacent to an expression sequence. In some embodiments, the cleavage sequence is between two expression sequences. In some embodiments, cleavage sequence is included in an expression sequence. In some embodiments, the circular polyribonucleotide includes between 2 and 10 cleavage sequences. In some embodiments, the circular polyribonucleotide includes between 2 and 5 cleavage sequences. In some embodiments, the multiple cleavage sequences are between multiple expression sequences; for example, a circular polyribonucleotide may include three expression sequences two cleavage sequences such that there is a cleavage sequence in between each expression sequence. In some embodiments, the circular polyribonucleotide includes a cleavage sequence, such as in an immolating circRNA or cleavable circRNA or self-cleaving circRNA. In some embodiments, the circular polyribonucleotide includes two or more cleavage sequences, leading to separation of the circular polyribonucleotide into multiple products, e.g., miRNAs, linear RNAs, smaller circular polyribonucleotide, etc.In some embodiments, a cleavage sequence includes a ribozyme RNA sequence. A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNA, but they have also been found to catalyze the aminotransferase activity of the ribosome. Catalytic RNA can be “evolved” by in vitro methods. Furthermore, ribozymes and their reaction products can regulate gene expression. In some embodiments, a catalytic RNA or ribozyme can be placed within a larger non-coding RNA such that the ribozyme is present at many copies within the cell for the purposes of chemical transformation of a molecule from a bulk volume In some embodiments, aptamers and ribozymes can both be encoded in the same non-coding RNA.In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, a polyribonucleotide may encode two or more OTC polypeptides, e.g., where the two or more OTC polypeptides are encoded by a single open-reading frame (ORF). For example, two or more OTC 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 OTC polypeptides each separated by a sequence encoding a polypeptide linker (e.g., a linker of 5-200, 5 to 100, 5 to 50, 5 to 20, 50 to 100, or 50 to 200 amino acids). The polypeptide linker may include a cleavage site, for example, a cleavage site recognized and cleaved by a protease (e.g., an endogenous protease in a subject following administration of the polyribonucleotide to that subject). In such embodiments, a single expression product including the amino acid sequence of two or more OTC polypeptides is cleaved upon expression,such that the two or more OTC polypeptides are separated following expression. Exemplary protease cleavage sites are known to those of skill in the art, for example, amino acid sequences that act as protease cleavage sites recognized by a metalloproteinase (e.g., a matrix metalloproteinase (MMP), such as any one or more of MMPs 1-28), a disintegrin and metalloproteinase (ADAM, such as any one or more of ADAMs 2, 7-12, 15, 17-23, 28-30 and 33), a serine protease (e.g., furin), urokinase-type plasminogen activator, matriptase, a cysteine protease, an aspartic protease, or a cathepsin protease In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is matriptase.In some embodiments, a circular polyribonucleotide described herein is an immolating circular polyribonucleotide, a cleavable circular polyribonucleotide, or a self-cleaving circular polyribonucleotide. A circular polyribonucleotide can deliver cellular components including, for example, RNA, IncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA In some embodiments, a circular polyribonucleotide includes miRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites; (iii) degradable linkers; (iv) chemical linkers; and / or (v) spacer sequences. In some embodiments, circRNA includes siRNA separated by (i) self-cleavable elements; (ii) cleavage recruitment sites (e.g., ADAR); (iii) degradable linkers (e.g., glycerol); (iv) chemical linkers; and / or (v) spacer sequences. Nonlimiting examples of self-cleavable elements include hammerhead, splicing element, hairpin, hepatitis delta virus (HDV), Varkud Satellite (VS), and glmS ribozymes.Translation Initiation SequencesIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes at least one translation initiation sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence operably linked to an expression sequence.In some embodiments, the polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide includes the translation initiation sequence, e.g., Kozak sequence, adjacent to an expression sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, the translation initiation sequence, e.g., Kozak sequence, is present on one or both sides of each expression sequence, leading to separation of the expression products. In some embodiments, the polyribonucleotide includes at least one translation initiation sequence adjacent to an expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs

[0163] -

[0165] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.The polyribonucleotide may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons. Translation may initiate on the first start codon or may initiate downstream of the first start codon.In some embodiments, the polyribonucleotide may initiate at a codon which is not the first start codon, e.g., AUG. Translation of the polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG. In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions. As a non-limiting example, the translation of the polyribonucleotide may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the polyribonucleotide translation may begin at alternative translation initiation sequence, CTG / CUG As another non-limiting example, the polyribonucleotide translation may begin at alternative translation initiation sequence, GTG / GUG. As another non-limiting example, the polyribonucleotide may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g., CGG, GGGGCC, CAG, CTG / CUG.Termination ElementsIn some embodiments, the polyribonucleotide described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) includes at least one termination element. In some embodiments, the polyribonucleotide includes a termination element operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination element.In some embodiments, the polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element In some embodiments, the polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product.In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, the circular polyribonucleotide includes one or more expression sequences, and the expression sequences lack a termination element, such that the circular polyribonucleotide is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of expression product, e.g., peptides or polypeptides, due to lack of ribosome stalling or fall-off In such an embodiment, rolling circle translation expresses a continuous expression product through each expression sequence. In some other embodiments, a termination element of an expression sequence can be part of a stagger element. In some embodiments, one or more expression sequences in the circular polyribonucleotide includes a termination element However, rolling circle translation or expression of a succeeding (e g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide is performed. In such instances, the expression product may fall off the ribosome when the ribosome encounters the termination element, e.g., a stop codon, and terminates translation. In some embodiments, translation is terminated while the ribosome, e.g, at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.In some embodiments, the circular polyribonucleotide includes a termination element at the end of one or more expression sequences. In some embodiments, one or more expression sequences includes two or more termination elements in succession. In such embodiments, translation is terminatedand rolling circle translation is terminated. In some embodiments, the ribosome completely disengages with the circular polyribonucleotide. In some such embodiments, production of a succeeding (e.g., second, third, fourth, fifth, etc.) expression sequence in the circular polyribonucleotide may require the ribosome to reengage with the circular polyribonucleotide prior to initiation of translation. Generally, termination elements include an in-frame nucleotide triplet that signals termination of translation, e.g., UAA, UGA, UAG. In some embodiments, one or more termination elements in the circular polyribonucleotide are frame-shifted termination elements, such as but not limited to, off-frame or -1 and + 1 shifted reading frames (e g, hidden stop) that may terminate translation Frame-shifted termination elements include nucleotide triples, TAA, TAG, and TGAthat appear in the second and third reading frames of an expression sequence. Frame-shifted termination elements may be important in preventing misreads of mRNA, which is often detrimental to the cell. In some embodiments, the termination element is a stop codon.Further examples of termination elements are described in paragraphs

[0169] -

[0170] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.Spacer SequencesIn some embodiments, the polyribonucleotide described herein includes one or more spacer sequences. A spacer or spacer sequence refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacers may be present in between any of the nucleic acid elements described herein. Spacer may also be present within a nucleic acid element as described hereinFor example, wherein a nucleic acid includes any two or more of the following elements: (A) a 3' catalytic intron fragment; (B) a 3’ splice site; (C) a 3’ exon fragment; (D) a polyribonucleotide cargo; ( E) a 5’ exon fragment; (F) a 5’ splice site; and (G) a 5' catalytic intron fragment; a spacer region may be present between any one or more of the elements. Any of elements (A), (B), (C), (D), (E), (F), or (G) may be separated by a spacer sequence, as described herein. For example, there may be a spacer between (A) and (B), between (B) and (C), between (C) and (D), between (D) and (E), between (E) and (F), or between (F) and (G)In some embodiments, the polyribonucleotide further includes a first spacer sequence between the 5’ exon fragment of (C) and the polyribonucleotide cargo of (D). The spacer may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, the polyribonucleotide further includes a second spacer sequence between the polyribonucleotide cargo of (D) and the 5’ exon fragment of (E).A spacer sequence may be used to separate an IRES from adjacent structural elements to maintain the structure and function of the IRES or the adjacent element A spacer can be specifically engineered depending on the IRES In some embodiments, an RNA folding computer software, such as RNAFold, can be utilized to guide designs of the various elements of the vector, including the spacers. Thus, in one embodiment, the spacer sequence is between the IRES and the 3’ exon fragment or the 5’ exon fragment. In other embodiments, the spacer sequence is between the OTC expression sequenceand the 3’ exon fragment. In other embodiments, the spacer sequence is adjacent to the 5’ exon fragment or the 3’ exon fragment.The spacer may be, e.g., at least 3 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, each spacer sequence is at least 3 (e g., at least 10, at least 15, at least 20) ribonucleotides in length. Each spacer region may be, e.g., from 5 to 800 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or 800) ribonucleotides in length. In some embodiments, the spacer sequence is at least about 50 ribonucleotides in length. In some embodiments, the spacer sequence is from about 50 to about 650 ribonucleotides in lengthIn some embodiments, the first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence may include a polyX sequence. In some embodiments the first spacer sequence and the second spacer sequence, or the first spacer sequence and the second spacer sequence, may include a polyA sequence. The first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence, may include a polyA-C sequence. In some embodiments, the first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence includes a polyA-G sequence. In some embodiments, the first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence includes a polyA-T or polyA-U sequence. In some embodiments, the first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence includes a random sequence.Spacers may also be present within a nucleic acid region described herein. For example, a polynucleotide cargo region may include one or multiple spacers. Spacers may separate regions within the polynucleotide cargoIn some embodiments, the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 70 nucleotides in length. In some embodiments, the spacer sequence is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length. In some embodiments, the spacer sequence is no more than 800, 700, 600, 500, 400, 300, 300, 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the spacer sequence is from 20 to 70 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides in length.The spacer sequences may be polyX sequences, polyA sequences, poly A-T sequences, poly A-U sequences, polyA-0 sequences, polyA-G sequences, polyG, polyC sequences, poly-U sequences, polyC-A sequences or random sequences.Exemplary spacer sequences are described in paragraphs

[0293] -

[0302] of International Patent Publication No WO2019 / 118919, (referred to as untranslated regions (UTRs)), which is hereby incorporated by reference in its entirety.In some embodiments, the polyribonucleotide includes 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 about 50 nucleotides in length. In another embodiment, the 5’ spacer sequence is at least 100 nucleotides in length In a further embodiment, the 5’ spacer sequence is at least about 200 nucleotidesin length. In other embodiment, the 5' spacer sequence is at least 300 nucleotides in length. In another embodiment, the 5’ spacer sequence is at least about 400 nucleotides in length. In another embodiment, the 5’ spacer sequence is at least about 500 nucleotides in length. In other embodiment, the 5’ spacer sequence is at least about 600 nucleotides in length. In other embodiment, the 5’ spacer sequence is at least 700 nucleotides in length. In one embodiment, the 5’ space is a polyX sequence. 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 includes a polyA-G sequence In some embodiments, the 5’ spacer sequence includes a polyA-T sequence In some embodiments, the 5’ spacer sequence includes a polyA-U sequence. In some embodiments, the 5’ spacer sequence includes a random sequence.In some embodiments, the polyribonucleotide includes a 3’ spacer sequence (e.g., between the 3’ annealing region and the polyribonucleotide cargo). In some embodiments, the polyribonucleotide includes 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 about 50 nucleotides in length. In another embodiment, the 3’ spacer sequence is at least 100 nucleotides in length. In a further embodiment, the 3’ spacer sequence is at least about 200 nucleotides in length. In other embodiment, the 3’ spacer sequence is at least 300 nucleotides in length. In another embodiment, the 3’ spacer sequence is at least about 400 nucleotides in length. In another embodiment, the 3’ spacer sequence is at least about 500 nucleotides in length In other embodiment, the 3’ spacer sequence is at least about 600 nucleotides in length. In other embodiment, the 3’ spacer sequence is at least 700 nucleotides in length. In one embodiment, the 3’ spacer sequence is a polyX sequence. In one embodiment, the 3’ spacer sequence is a polyA sequence In another embodiment, the 3’ spacer sequence is a polyA-C sequence In some embodiments, the 3’ spacer sequence includes a polyA-G sequence. In some embodiments, the 3’ spacer sequence includes a polyA-T sequence. In some embodiments, the 3’ spacer sequence includes a polyA-U sequence. In some embodiments, the 3’ spacer sequence includes a random sequence.In one embodiment, the polyribonucleotide includes a 5’ spacer sequence, but not a 3’ spacer sequence. In another embodiment, the polyribonucleotide includes a 3’ spacer sequence, but not a 5’ spacer sequence. In another embodiment, the polyribonucleotide includes neither a 5’ spacer sequence, nor a 3’ spacer sequence In another embodiment, the polyribonucleotide does not include an IRES sequence. In a further embodiment, the polyribonucleotide does not include an IRES sequence, a 5’ spacer sequence or a 3’ spacer sequence.In some embodiments, the spacer sequence includes at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides, at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least 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 700ribonucleotides, at least about 800 ribonucleotides, at least about 900 ribonucleotides, or at least about 1000 ribonucleotides.Untranslated RegionsIn some embodiments, a circular polyribonucleotide includes untranslated regions. UTRs of a genomic region including a gene may be transcribed but not translated. In some embodiments, a UTR may be included upstream of the translation initiation sequence of an expression sequence described herein In some embodiments, a UTR may be included downstream of an expression sequence described herein. In some instances, one UTR for a first expression sequence is the same as or continuous with or overlapping with another UTR for a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, e.g, ZKSCAN1.Exemplary UTR are described in paragraphs

[0197] -

[0201] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety.In some embodiments, a circular polyribonucleotide includes a polyA sequence. Exemplary polyA sequences are described in paragraphs

[0202] -

[0205] of International Patent Publication No. WO2019 / 118919, which is hereby incorporated by reference in its entirety. In some embodiments, a circular polyribonucleotide lacks a polyA sequence.In some embodiments, a circular polyribonucleotide includes a UTR with one or more stretches of Adenosines and Uridines embedded within. These AU rich signatures may increase turnover rates of the expression product.Introduction, removal, or modification of the UTR AU rich elements (AREs) may be useful to modulate the stability, or immunogenicity (e.g., the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide. When engineering specific circular polyribonucleotides, one or more copies of an ARE may be introduced to the circular polyribonucleotide and the copies of an ARE may modulate translation and / or production of an expression product.Likewise, AREs may be identified and removed or engineered into the circular polyribonucleotide to modulate the intracellular stability and thus affect translation and production of the resultant protein.It should be understood that any UTR from any gene may be incorporated into the respective flanking regions ofthe circular polyribonucleotide.In some embodiments, a circular polyribonucleotide lacks a 5’ UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 3’ UTR and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a polyA sequence and is competent for protein expression from its one or more expression sequences In some embodiments, the circular polyribonucleotide lacks a termination element and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks an internal ribosomal entry site and is competent for protein expression from its one or more expression sequences In some embodiments, the circular polyribonucleotide lacks a cap and is competent for protein expression from its one or more expression sequences In some embodiments, the circular polyribonucleotide lacks a 5’ UTR, a 3’ UTR, and an IRES, and is competentfor protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide includes one or more of the following sequences: a sequence that encodes one or more miRNAs, a sequence that encodes one or more replication proteins, a sequence that encodes an exogenous gene, a sequence that encodes a therapeutic, a regulatory element (e.g., translation modulator, e.g., translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids that targets endogenous genes (e.g., siRNA, IncRNAs, shRNA), and a sequence that encodes a therapeutic mRNA or protein.In some embodiments, a circular polyribonucleotide lacks a 5’ UTR In some embodiments, the circular polyribonucleotide lacks a 3’ UTR. In some embodiments, the circular polyribonucleotide lacks a polyX sequence. In some embodiments, the circular polyribonucleotide lacks a termination element. In some embodiments, the circular polyribonucleotide lacks an internal ribosomal entry site. In some embodiments, the circular polyribonucleotide lacks degradation susceptibility by exonucleases. In some embodiments, the fact that the circular polyribonucleotide lacks degradation susceptibility can mean that the circular polyribonucleotide is not degraded by an exonuclease, or only degraded in the presence of an exonuclease to a limited extent, e.g., that is comparable to or similar to in the absence of exonuclease. In some embodiments, the circular polyribonucleotide is not degraded by exonucleases. In some embodiments, the circular polyribonucleotide has reduced degradation when exposed to exonuclease. In some embodiments, the circular polyribonucleotide lacks binding to a cap-binding protein. In some embodiments, the circular polyribonucleotide lacks a 5’ cap.In some embodiments, the linear or circular polyribonucleotide includes a 5’ UTR encoded by any of the deoxyribonucleotide sequences of Table 2. In some embodiments, linear or circular polyribonucleotide includes a 5’ UTR having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of Table 2. In other embodiments, the linear or circular polyribonucleotide includes a 5’ UTR having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any of the ribonucleotide sequences encoded by the deoxyribonucleotide sequences of Table 2.In some embodiments, the linear or circular polyribonucleotide includes a 3’ UTR encoded by any of the deoxyribonucleotide sequences of Table 2. In some embodiments, the linear or circular polyribonucleotide includes a 3’ UTR having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of Table 2. In other embodiments, the linear or circular polyribonucleotide includes a 3’ spacer sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any of the ribonucleotide sequences encoded by the deoxyribonucleotide sequences of Table 2.Protein-Binding SequencesIn some embodiments, a circular polyribonucleotide includes one or more protein binding sites that allow a protein, e.g., a ribosome, to bind to an internal site in the RNA sequence. By engineering protein binding sites, e.g., ribosome binding sites, into the circular polyribonucleotide, the circular polyribonucleotide may evade or have reduced detection by the host’s immune system, have modulated degradation, or modulated translation, by masking the circular polyribonucleotide from components of the host’s immune system.In some embodiments, a circular polyribonucleotide includes at least one immunoprotein binding site, for example to evade immune responses, e.g., CTL (cytotoxic T lymphocyte) responses. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and aids in masking the circular polyribonucleotide as exogenous. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and aids in hiding the circular polyribonucleotide as exogenous or foreign.T raditional mechanisms of ribosome engagement to linear RNA involve ribosome binding to the capped 5' end of an RNA From the 5' end, the ribosome migrates to an initiation codon, whereupon the first peptide bond is formed. According to the present disclosure, internal initiation (i.e., cap-independent) of translation of the circular polyribonucleotide does not require a free end or a capped end. Rather, a ribosome binds to a non-capped internal site, whereby the ribosome begins polypeptide elongation at an initiation codon. In some embodiments, the circular polyribonucleotide includes one or more RNA sequences including a ribosome binding site, e.g., an initiation codonNatural 5' UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG (SEQ ID NO: 124), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5’ UTRs also have been known to form secondary structures which are involved in elongation factor binding.In some embodiments, a circular polyribonucleotide encodes a protein binding sequence that binds to a protein. In some embodiments, the protein binding sequence targets or localizes the circular polyribonucleotide to a specific target In some embodiments, the protein binding sequence specifically binds an arginine-rich region of a protein.In some embodiments, the protein binding site includes, but is not limited to, a binding site to the protein such as ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, ELAVL1, ELAVL3, FAM120A, FBL, 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, RBFOX2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF1, 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 RNA.ModificationsA polyribonucleotide (e g., circular polyribonucleotide) as described herein may include one or more substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences, in particular, the parent polyribonucleotide, are included within the scope of this disclosure.In some embodiments, a circular polyribonucleotide includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, polyA sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). In some embodiments, the first isolated nucleic acid includes messenger RNA (mRNA) In some embodiments, the polyribonucleotide includes at least one nucleoside selected from the group such as those described in

[0311] of International Patent Publication No. WO2019 / 118919A1, which is incorporated herein by reference in its entirety.A polyribonucleotide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro) In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.In some embodiments, a polyribonucleotide includes at least one N(6)methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the m6A modification can reduce immunogenicity (e g, reduce the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide.In some embodiments, a modification may include a chemical or cellular induced modification. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.In some embodiments, chemical modifications to the ribonucleotides of a circular polyribonucleotide may enhance immune evasion The circular polyribonucleotide may be synthesized and / or modified by methods well established in the art, such as those described in " Current protocols in nucleic acid chemistry," Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc ), base modifications (e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners), removal of bases (abasic nucleotides), or conjugated bases The modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications may modulate expression, immune response, stability, subcellular localization, to name a few functional effects, of the circular polyribonucleotide. In some embodiments, the modification includes a bi-orthogonal nucleotide, e.g., an unnatural base. See for example, Kimoto et al, Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039 / c7cc06661a, which is hereby incorporated by reference.In some embodiments, sugar modifications (e g., at the 2' position or 4' position) or replacement of the sugar one or more ribonucleotides of the circular polyribonucleotide may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of circular polyribonucleotide include, but are not limited to, circular polyribonucleotide including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Circular polyribonucleotides having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the circular polyribonucleotide will include ribonucleotides with a phosphorus atom in its internucleoside backbone.Modified polyribonucleotide backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3‘ or 2'-5' to 5'-2‘. Various salts, mixed salts and free acid forms are also included. In some embodiments, the circular polyribonucleotide may be negatively or positively charged.The modified nucleotides, which may be incorporated into the polyribonucleotide, can be modified on the internucleoside linkage (e g, phosphate backbone) Herein, in the context of the polynucleotide backbone, the phrases "phosphate" and "phosphodiester" are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylenephosphonates).The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.Phosphorothioate linked to the circular polyribonucleotide is expected to reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine (a-thio-cytidine), 5'-0-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-0-(1-thiophosphate)-pseudouridine).Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.In some embodiments, a circular polyribonucleotide may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into circular polyribonucleotide, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5'-elaidic acid ester).A polyribonucleotide may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the circular polyribonucleotide, or in a given predetermined sequence region thereof. For example, in some embodiments, the circular polyribonucleotide includes one or more pseudouridine. In some embodiments, the circular polyribonucleotide includes one or more N1-methylpseudouridine. In some embodiments, the circular polyribonucleotide includes one or more 5-methoxyuridine. In some embodiments, the circular polyribonucleotide replaces uridine with pseuodouridine at all existing uridine positions or a portion of all existing uridine positions In some embodiments, the circular polyribonucleotide replaces uridine with N1 -methylpseudouridine at all existing uridine positions or a portion of all existing uridine positions. In some embodiments, the circular polyribonucleotide replaces uridine with 5-methoxyuridine at all existing uridine positions or a portion of all existing uridine positions. In some embodiments, the circular polyribonucleotide includes an inosine, which may aid in the immune system characterizing the circular polyribonucleotide as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability / reduced degradation. See for example, Yu, Z et al (2015) RNA editing by ADAR1 marks dsRNA as “self” Cell Res 25, 1283-1284, which is incorporated by reference in its entirety.In some embodiments, all nucleotides in a polyribonucleotide (or in a given sequence region thereof) are modified. In some embodiments, the modification may include an m6A, which may augment expression; an inosine, which may attenuate an immune response; pseudouridine, which may increase RNA stability, or translational readthrough (stagger element), an m5C, which may increase stability; and a 2,2,7-trimethylguanosine, which aids subcellular translocation (e.g., nuclear localization)Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e g, backbone structures) may exist at various positions in a circular polyribonucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the circular polyribonucleotide, such that the function of the circular polyribonucleotide is not substantially decreased. A modification may also be a non-coding region modification. The circular polyribonucleotide may include from about 1% to about 100% modified nucleotides (either in relation tooverall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%)Methods of CircularizationThe disclosure provides methods for producing circular polyribonucleotides encoding an OTC polypeptide (e g., a polypeptide of Figure 1, Table 1), including, e.g., recombinant technology or chemical synthesis. For example, a DNA molecule used to produce an RNA circle can include a DNA sequence of a naturally occurring original nucleic acid sequence, a modified version thereof, or a DNA sequence encoding a synthetic polypeptide not normally found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant techniques, such as site- directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.In some embodiments, a linear polyribonucleotide for circularization may be cyclized, or concatemerized. In some embodiments, the linear polyribonucleotide for circularization may be cyclized in vitro prior to formulation and / or delivery In some embodiments, the circular polyribonucleotide may be in a mixture with linear polyribonucleotides. In some embodiments, the linear polyribonucleotides have the same nucleic acid sequence as the circular polyribonucleotidesIn some embodiments, a linear polyribonucleotide for circularization is cyclized, or concatemerized using a chemical method to form a circular polyribonucleotide. In some chemical methods, the 5'-end and the 3'-end of the nucleic acid (e g., a linear polyribonucleotide for circularization) includes chemically reactive groups that, when close together, may form a new covalent linkage between the 5'-end and the 3'-end of the molecule. The 5'-end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a linear RNA molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'- / 3'-amide bondIn some embodiments, a DNA or RNA ligase is used to enzymatically link a 5'-phosphorylated nucleic acid molecule (e.g., a linear polyribonucleotide for circularization) to the 3'-hydroxyl group of a nucleic acid (e.g, a linear nucleic acid) forming a new phosphodiester linkage. In an example reaction, a linear polyribonucleotide for circularization is incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. The ligationreaction may occur in the presence of a linear nucleic acid capable of base-pairing with both the 5'- and 3'- region in juxtaposition to assist the enzymatic ligation reaction. In some embodiments, the ligation is splint ligation. For example, a splint ligase, like 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), like a single stranded RNA, can be designed to hybridize with both termini of a linear polyribonucleotide, so that the two termini can be juxtaposed upon hybridization with the single-stranded splint. Splint ligase can thus catalyze the ligation of the juxtaposed two termini of the linear polyribonucleotide, generating a circular polyribonucleotideIn some embodiments, a DNA or RNA ligase is used in the synthesis of the circular polynucleotides. In some embodiments, either the 5'-or 3'-end of the linear polyribonucleotide for circularization can encode a ligase ribozyme sequence such that during in vitro transcription, the resultant linear polyribonucleotide for circularization includes an active ribozyme sequence capable of ligating the 5'-end of the linear polyribonucleotide for circularization to the 3'-end of the linear polyribonucleotide for circularization. The ligase ribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°CIn some embodiments, a linear polyribonucleotide for circularization is cyclized 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 regions or features near the 5' terminus and / or near the 3' terminus of the linear polyribonucleotide for circularization in order to cyclize or concatemerized the linear polyribonucleotide for circularization. In another aspect, the at least one non-nucleic acid moiety may be located in or linked to or near the 5' terminus and / or the 3' terminus of the linear polyribonucleotide for circularization. The non-nucleic acid moieties contemplated may be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage, and / or a cleavable linkage. As another non-limiting example, the non-nucleic acid moiety is a ligation moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.In some embodiments, the linear polyribonucleotide for circularization is synthesized using in vitro transcription (IVT) and an RNA polymerase, where the nucleotide mixture used for IVT may contain an excess of guanosine monophosphate relative to guanosine triphosphate to preferentially produce RNA with a 5’ monophosphate; the purified IVT product may be circularized using a splint DNA.In some embodiments, a linear polyribonucleotide for circularization is cyclized or concatemerized due to a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near or linked to the 5' and 3' ends of the linear polyribonucleotide for circularization. As a non-limiting example, one or more linear polyribonucleotides for circularization may be cyclized or concatemerized by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, Van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation and antibondingIn some embodiments, a linear polyribonucleotide for circularization may include a ribozyme RNA sequence near the 5' terminus and near the 3' terminus. The ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, the peptides covalently linked to the ribozyme RNA sequence near the 5' terminus and the 3 'terminus may associate with each other causing a linear polyribonucleotide for circularization to cyclize or concatemerized. In another aspect, the peptides covalently linked to the ribozyme RNA near the 5' terminus and the 3' terminus may cause the linear primary construct or linear mRNA to cyclize or concatemerized after being subjected to ligation using various methods known in the art such as, but not limited to, protein ligation. Non-limiting examples of ribozymes for use in the linear primary constructs or linear RNA of the present invention or a non-exhaustive listing of methods to incorporate and / or covalently link peptides are described in US patent application No. US20030082768, the contents of which is here in incorporated by reference in its entirety.In some embodiments, a linear polyribonucleotide for circularization may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase). In some embodiments, the 5’ end of at least a portion of the linear polyribonucleotides includes a monophosphate moiety. In some embodiments, the population of polyribonucleotides including circular and linear polyribonucleotides is contacted with RppH prior to digesting at least a portion of the linear polyribonucleotides with a 5' exonuclease and / or a 3’ exonuclease Alternately, converting the 5' triphosphate of the linear polyribonucleotide for circularization into a 5' monophosphate may occur by a two-step reaction including: (a) contacting the 5' nucleotide of the linear polyribonucleotide for circularization with a phosphatase (e g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphateIn some embodiments, circularization efficiency of the circularization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40% In some embodiments, the circularization method provided has a circularization efficiency of between about 10% and about 100%; for example, the circularization efficiency may be about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 99%. In some embodiments, the circularization efficiency is between about 20% and about 80%. In some embodiments, the circularization efficiency is between about 30% and about 60%. In some embodiments the circularization efficiency is about 40%In some embodiments, the circular polyribonucleotide includes an internal splicing element that when replicated the spliced ends are joined together. Some examples may include miniature introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in (suptable4 enriched motifs) c / s-sequence elements proximal to back splice events such as sequences inthe 200 bp preceding (upstream of) or following (downstream from) a back splice site with flanking exons. In some embodiments, the linear polyribonucleotide includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repetitive nucleotide sequence may include repeated sequences from the Alu family of introns. In some embodiments, a splicing-related ribosome binding protein can regulate circular polyribonucleotide biogenesis (e.g., the Muscle blind and Quaking (QKI) splicing factors).In some embodiments, the linear polyribonucleotide may include canonical splice sites that flank head-to-tail junctions of the circular polyribonucleotideIn some embodiments, the linear polyribonucleotide may include a bulge-helix-bulge motif, including a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage occurs at a site in the bulge region, generating characteristic fragments with terminal 5'-hydroxyl group and 2', 3'-cyclic phosphate. Circularization proceeds by nucleophilic attack of the 5'-OH group onto the 2',3'-cyclic phosphate of the same molecule forming a 3',5'-phosphodiester bridge.In some embodiments, the linear polyribonucleotide may include a multimeric repeating RNA sequence that harbors a HPR element. The HPR includes a 2', 3'-cyclic phosphate and 5-OH termini. The HPR element self-processes the 5 - and 3-ends of the linear polyribonucleotide, thereby ligating the ends together.In some embodiments, the linear polyribonucleotide may include a sequence that mediates selfligation. In one embodiment, the linear polyribonucleotide may include a HDV sequence, e.g., HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUG CUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (SEQ ID NO: 172) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGC CGCCCGAGCC (SEQ ID NO: 173), to self-ligate. In one embodiment, the linear polyribonucleotide may include loop E sequence (e.g., in PSTVd) to self-ligate In another embodiment, the linear polyribonucleotide may include a self-circularizing intron, e.g., a 5' and 3’ slice junction, or a selfcircularizing catalytic intron such as a Group I, Group II or Group III Introns. In some embodiments, the linear polyribonucleotide may include a 5’ and 3’ sequence mediates self-splicing such as the 5’ fragment AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAG ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGA AAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA (SEQ ID NO: 174) and the 3’ fragment CCCACACGACCGTTTAGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGA TGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAG TAATTAGTAAGTT (SEQ ID NO: 175). In some embodiments, the 5’ and 3’ sequence that mediates selfsplicing to produce a post-spliced sequence of CCCACACGACCGUUUAGACGCUACGGACUUAAAAUCCGUAGCGUCUAAACGGUCGUGUGGGUUC AAGUCCCUCCACCCCCA (SEQ ID NO: 176) used in the circularized polyribonucleotide constructs described herein.In some embodiments, the linear polyribonucleotide may include a 5’ and 3’ sequence mediates self-splicing such as the 5’ fragment96676-419726LRN23-101WQ AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGGGA CAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCGACGA GCTACCAGGCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA (SEQ ID NO: 203) and the 3’ fragment GCCTGGTAGCTCGTCGGGCTCAACAAGCAAAGTTAACTAAACGCTTATCAGTTAGTTTTGCAATGGG CGGTACGTGAAGAAACTTACGTGCGTTTACCTGTCAAACTCGGGGAAGCCATTAGCGTGGTAATCCC GAACCAAGCTCC (SEQ ID NO: 204). In some embodiments, the 5’ and 3’ sequence that mediates self-splicing to produce a post-spliced sequence of GCCTGGTAGCTCGTCGGGCTCATAACCCGACGAGCTACCAGGCAAATCCACTTCCCGCCACCAAAT TAAAAAAACAATAA (SEQ ID NO: 205) used in the circularized polyribonucleotide constructs described herein.Nonlimiting examples of group I intron self-splicing sequences may include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena.In some embodiments, the polyribonucleotide includes catalytic intron fragments, such as a 3' half of Group I catalytic intron fragment and a 5' half of Group I catalytic intron fragment. The first and second annealing regions may be positioned within the catalytic intron fragments. Group I catalytic introns are self-splicing ribozymes that catalyze their own excision from mRNA, tRNA, and rRNA precursors via two-metal ion phosphoryl transfer mechanism Importantly, the RNA itself self-catalyzes the intron removal without the requirement of an exogenous enzyme, such as a ligaseIn some embodiments, the 3' half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a cyanobacterium Anabaena pre-tRNA-Leu gene, or a Tetrahymena pre-rRNA.In some embodiments, the 3' half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a Cyanobacterium Anabaena pre-tRNA-Leu gene, and the 3’ exon fragment includes the first annealing region and the 5’ exon fragment includes the second annealing region. The first annealing region may include, e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides and the second annealing region may include, e g, from 5 to 50, e g, from 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotidesIn some embodiments, the 3' half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a Tetrahymena pre-rRNA, and the 3' half of Group I catalytic intron fragment includes the first annealing region and the 5’ exon fragment includes the second annealing region. In some embodiments, the 3' exon includes the first annealing region and the 5’ half of Group I catalytic intron fragment includes the second annealing region. The first annealing region may include, e g, from 6 to 50, e g, from 10 to 16 (e g, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may include, e.g., from 6 to 50, e.g., from 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.In some embodiments, the 3' half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a cyanobacterium Anabaena pre-tRNA-Leu gene, a Tetrahymena pre-rRNA, or a T4 phage td gene.In some embodiments, the 3' half of Group I catalytic intron fragment and the 5’ Group I catalytic intron fragment are from a T4 phage td gene. The 3' exon fragment may include the first annealing region and the 5’ half of Group I catalytic intron fragment may include the second annealing region. The first annealing region may include, e.g., from 2 to 16, e.g., 10 to 16 (e.g, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may include, e.g., from 2 to 16, e.g, 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.In some embodiments, the 3' half of Group I catalytic intron fragment is the 5’ terminus of the linear polynucleotideIn some embodiments, the 5' half of Group I catalytic intron fragment is the 3’ terminus of the linear polyribonucleotide.In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5'-AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAG ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATG-3’ (SEQ ID NO: 215)In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-AAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTA GCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT-3’ (SEQ ID NO: 216)).In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 215 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 216.In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-CTTCTGTTGATATGGATGCAGTTCACAGACTAAATGTCGGTCGGGGAAGATGTATTCTTCTCATAAGA TATAGTCGGACCTCTCCTTAATGGGAGCTAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAA CTAATTTGTATGCGAAAGTATATTGATTAGTTTTGGAGTACTCG-3’ (SEQ ID NO: 217).In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-AAATAGCAATATTTACCTTTGGAGGGAAAAGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCA ATAGATTGCATCGGTTTAAAAGGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTA CCAAGTCTCAGGGGAAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGT CCTAACCACGCAGCCAAGTCCTAAGTCAACAGAT-3’ (SEQ ID NO: 218).In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 217 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 218.In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-GGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGAC AACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGG GGTAAGATTAACGACCTTATCTGAACATAATG-3’ (SEQ ID NO: 219).In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-TAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGA CAATCCCGTGCTAAATTGTAGGACT-3’ (SEQ ID NO: 220).In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 219 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 220.In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-TAAACAACTAACAGCTTTAGAAGGTGCAGAGACTAGACGGGAGCTACCCTAACGGATTCAGCCGAG GGTAAAGGGATAGTCCAATTCTCAACATCGCGATTGTTGATGGCAGCGAAAGTTGCAGAGAGAATGA AAATCCGCTGACTGTAAAGGTCGTGAGGGTTCGAGTCCCTCCGCCCCCA-3’ (SEQ ID NO: 221).In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-ACGGTAGACGCAGCGGACTTAGAAAACTGGGCCTCGATCGCGAAAGGGATCGAGTGGCAGCTCTC AAACTCAGGGAAACCTAAAACTTTAAACATTMAAGTCATGGCAATCCTGAGCCAAGCTAAAGC-3’ (SEQ ID NO: 222).In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 221 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 222.In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-TTAAACTCAAAATTTAAAATCCCAAATTCAAAATTCCGGGAAGGTGCAGAGACTCGACGGGAGCTAC CCTAACGTAAAGCCGAGGGTAAAGGGAGAGTCCAATTCTCAAAGCCTGAAGTTGCTGAAGCAACAA GGCAGTAGTGAAAGCTGCGAGAGAATGAAAATCCGTTGACTGTAAAAAGTCGTGGGGGTTCAAGTC CCCCCACCCCC-3’ (SEQ ID NO: 223)In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTTAAGTGGAAGCTCTCAA ATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGCCAAGCCCAGAAAATTTAGACTTG AGATTTGATTTTGGAG-3’ (SEQ ID NO: 224)In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 223 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 224 In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-GGCTTTCAATTTGAAATCAGAAATTCAAAATTCAGGGAAGGTGCAGAGACTCGACGGGAGCTACCCT AACGTAAAGGCGAGGGTAAAGGGAGAGTCCAATTCTTAAAGCCTGAAGTTGTGCAAGCAACAAGGC AACAGTGAAAGCTGTGGAAGAATGAAAATCCGTTGACCTTAAACGGTCGTGGGGGTTCAAGTCCCC CCACCCCC-3’ (SEQ ID NO: 225).In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTCAAGTGGAAGCTCTCAA ATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGCCAAGCCCGGAAATTTTAGAATCA AGATTTTATTTT-3’ (SEQ ID NO: 226).In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 225 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 226.In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGGGA CAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCGAAGG TCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA-3’ (SEQ ID NO: 227).In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGGGA CAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCGAAGG TCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA-3’ (SEQ ID NO: 228).In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 227 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 228.In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-ACAACAGATAACTTACTAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAAC GTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGA GAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA-3’ (SEQ ID NO: 229).In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAG GGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAG TAAGTT-3’ (SEQ ID NO: 230).In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 229 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 230.In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-AACAACAGATAACTTACTAGTTACTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTC AAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAA TGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA-3’ (SEQ ID NO: 231).In some embodiments, the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’-AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAG GGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT-3’ (SEQ ID NO: 232).In some embodiments, the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 231 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 232.In some embodiments, the Group I catalytic intron fragment is from the T4 phage nrdB gene or nrdD gene. In some embodiments, the 3' half of Group I catalytic intron fragment of includes a sequencehaving at least 80% (e g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-TTGCAAAACAAGGTTCAACGACTAGTCTTCGGACGTAGGGTCAAGCGACTCGAAATGGGGAGAATC CCTCCGGGATTGTGATATAGTCTGGACTGCATGGTAACATGCAGCAGTTCATAAGAGAACGGGTTGA GAATTAGCGAGCTCAATCGAACATACG-3’ (SEQ ID NO: 233).In some embodiments, the 3' half of Group I catalytic intron fragment of (A) includes a sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-TTGCAAAACAAGGTTCAACGACTAGTCTTCGGACGTAGGGTCAAGCGACTCGAAATGGGGAGAATC CCTCCGGGATTGTGATATAGTCTGGACTGCATGGTAACATGCAGCAGTTCATAAGAGAACGGGTTGA GAATTAGCGAGCTCAATCGAACATACG-3’ (SEQ ID NO: 234).In some embodiments, the 5' half of Group I catalytic intron fragment from the T4 phage nrdB gene. In some embodiments, the 3’ half of Group I catalytic intron fragment is from the T4 phage nrdB gene and the 5’ half of Group I catalytic intron fragment is from the T4 phage nrdB gene.In some embodiments, the 5’ half of Group I catalytic intron includes a sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-AAAATGCGCCTTTAAACGGTAACGTTTATCGAAAACTCCTTTAATTGCTGGAAAGTCCTTTATGGAAA ACTAGCAGCCAAGGTTTTGCTT-3’ (SEQ ID NO: 235).In some embodiments, the 51half of Group I catalytic intron includes a sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-AAAATGCGCCTTTAAACGGTAACGTTTATCGAAAACTCCTTTAATTGCTGGAAAGTCCTTTATGGAAA ACTAGCAGCCAAGGTTTTGCTT-3’ (SEQ ID NO: 236).In some embodiments, the 3’ half of Group I catalytic intron fragment is from the T4 phage nrdD geneIn some embodiments, the 3’ half of Group I catalytic intron fragment includes a sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-CAGTAGCTGTAAATGCCCAACGACTATCCCTGATGAATGTAAGGGAGTAGGGTCAAGCGACCCGAA ACGGCAGACAACTCTAAGAGTTGAAGATATAGTCTGAACTGCATGGTGACATGCAGCTGTTTATCCT CGTATAAATATGAATACGAGGTGAAACGATGAAATGAATTACATTGTTTCATATAAACGGGTAGAGAA GTAGCGAACTCTACTGAACACATTG-3’ (SEQ ID NO: 237).In some embodiments, the 3’ half of Group I catalytic intron fragment includes a sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-CAGTAGCTGTAAATGCCCAACGACTATCCCTGATGAATGTAAGGGAGTAGGGTCAAGCGACCCGAA ACGGCAGACAACTCTAAGAGTTGAAGATATAGTCTGAACTGCATGGTGACATGCAGCTGTTTATCCT CGTATAAATATGAATACGAGGTGAAACGATGAAATGAATTACATTGTTTCATATAAACGGGTAGAGAA GTAGCGAACTCTACTGAACACATTG-3’ (SEQ ID NO: 238).In some embodiments, the 5’ half of Group I catalytic intron fragment is from the T4 phage nrdD gene In some embodiments, the 3’ half of Group I catalytic intron fragment is from the T4 phage nrdD gene and the 5’ half of Group I catalytic intron fragment is from the T4 phage nrdD gene.In some embodiments, the 5’ half of Group I catalytic intron includes a sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-TAACGTAAGTCAAGCTCATGTAAAATCTGCCTAAAACGGGAAACTCTCACTGAGACAATCCGTTGCTA AATCAG-3’ (SEQ ID NO: 239).In some embodiments, the 5' half of Group I catalytic intron includes a sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-TAACGTAAGTCAAGCTCATGTAAAATCTGCCTAAAACGGGAAACTCTCACTGAGACAATCCGTTGCTA AATCAG-3’ (SEQ ID NO: 240).In some embodiments, the 3’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-GTACCTTTAACTTCCATAAGAACATGGAAATCATGGAAGGTAATGCCAAG-3' (SEQ ID NO: 241).In some embodiments, the 3’ exon fragment includes a sequence having at least 80% (e g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-GTACCTTTAACTTCCATAAGAACATGGAAATCATGGAAGGTAATGCCAAG-3' (SEQ ID NO: 242).In some embodiments, the 3’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-GTACCTTTAACTTCCAAAAGATACATAAAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 243.In some embodiments, the 3’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-GTACCTTTAACTTCCAAAAGATACATAAAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 244).In some embodiments, the 5’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-TTTTTATGTATCTTTTGCGT-3’ (SEQ ID NO: 245).In some embodiments, the 5’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-TTTTTATGTATCTTTTGCGT-3’ (SEQ ID NO: 246)In some embodiments, the 3’ exon fragment Includes a sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-ATGAAGTGAACACGTTATTCAGTTCAAACGGACAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 247).In some embodiments, the 3’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-ATGAAGTGAACACGTTATTCAGTTCAAACGGACAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 248).In some embodiments, the 3’ exon fragment includes a sequence having at least 80% (e g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-ATGAAGTGAACACGTTACATAAGCTTGGAATGCAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 249).In some embodiments, the 3’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’-ATGAAGTGAACACGTTACATAAGCTTGGAATGCAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 250).In some embodiments, the 5’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’- TGCATTCCAAGCTTATGAGT -3’ (SEQ ID NO: 251).In some embodiments, the 5’ exon fragment includes a sequence having at least 80% (e.g, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to 5’- TGCATTCCAAGCTTATGAGT -3’ (SEQ ID NO: 252).In some embodiments, linear polyribonucleotides for circularization may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns. Repetitive nucleic acid sequence are sequences that occur within a segment of the linear polyribonucleotide. In some embodiments, the linear polyribonucleotide includes a repetitive nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence includes poly CA or poly UG sequences. In some embodiments, the linear polyribonucleotide includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand In some embodiments, the linear polyribonucleotide includes between 1 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, and 10) repetitive nucleic acid sequences that hybridize to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, the linear polyribonucleotide includes 2 repetitive nucleic acid sequences that hybridize to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate linear polyribonucleotides hybridize to generate a single circularized polyribonucleotide, with the hybridized segments forming internal double strands. In some embodiments, the complementary sequences are found at the 5’ and 3’ ends of the linear polyribonucleotides for circularization. In some embodiments, the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.In some embodiments, chemical methods of circularization may be used to generate the circular polyribonucleotide. Such methods may include, but are not limited to click chemistry (e.g., alkyne and azide-based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereofIn some embodiments, enzymatic methods of circularization may be used to generate the circular polyribonucleotide. In some embodiments, a ligation enzyme, e.g., DNA or RNA ligase, may be used to generate a template of the circular polyribonucleotide or complement, a complementary strand of the circular polyribonucleotide, or the circular polyribonucleotideCircularization of the linear polyribonucleotide may be accomplished by methods known in the art, for example, those described in “RNA circularization strategies in vivo and in vitro” by Petkovic and Muller from Nucleic Acids Res, 2015, 43(4): 2454-2465, and “In vitro circularization of RNA” by Muller and Appel, from RNA Biol, 2017, 14(8): 1018-1027.The circular polyribonucleotide may encode a sequence and / or motif useful for replication.Exemplary replication elements are described in paragraphs

[0280] -

[0286] of International Patent Publication No WO2019 / 118919, which is hereby incorporated by reference in its entiretyIn some embodiments, linear polyribonucleotides may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns. Repetitive nucleic acid sequence are sequences that occur within a segment of the circular polyribonucleotide. In some embodiments, the linear polyribonucleotide includes a repetitive nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence includes poly CA orpoly UG sequences. In some embodiments, the linear polyribonucleotide includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate linear polyribonucleotides hybridize to generate a single circularized polyribonucleotide, with the hybridized segments forming internal double strands. In some embodiments, the complementary sequences are found at the 5’ and 3’ ends of the linear polyribonucleotides. In some embodiments, the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.Methods of making the circular polyribonucleotides described herein are described in, for example, Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); in Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); Muller and Appel, from RNA Biol, 2017, 14(8): 1018-1027; and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012). Other methods of making circular polyribonucleotides are described, for example, in International Publication No. W02023 / 044006, International Publication No.WO2022 / 247943, US Patent No. US11000547, International Publication No. WO2018 / 191722, International Publication No. WO2019 / 236673, International Publication No. W02020 / 023595, International Publication No. W02022 / 204460, International Publication No. WO2022 / 204464, International Publication No. WO2022 / 204466, and International Publication No. 2022 / 261490, the contents of each of which are herein incorporated by reference in their entirety).Additional methods of synthesizing circular polyribonucleotides are also described elsewhere (see, e.g., US Patent No. US6210931, US Patent No. US5773244, US Patent No. US5766903, US Patent No US5712128, US Patent No. US5426180, US Publication No. US20100137407, International Publication No. W01992001813, International Publication No. W02010084371, and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015); the contents of each of which are herein incorporated by reference in their entirety).In some embodiments, the circular polyribonucleotide is purified, e.g., free ribonucleic acids, linear or nicked RNA, DNA, proteins, etc are removed In some embodiments, the circular polyribonucleotides may be purified by any known method commonly used in the art. Examples of nonlimiting purification methods include, column chromatography, gel excision, size exclusion, etc.Methods of ProductionMethods of production in a cell-free systemThe disclosure also provides methods of producing a circular RNA. For example, a deoxyribonucleotide template may be transcribed in a cell-free system (e.g., by in vitro transcription) to produce a linear RNA. The linear polyribonucleotide produces a splicing-compatible polyribonucleotide, which may be self-spliced to produce a circular polyribonucleotide.In some embodiments, the disclosure provides a method of producing a circular polyribonucleotide (e.g., in a cell-free system) by providing a linear polyribonucleotide; and self-splicinglinear 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 a deoxyribonucleotide encoding the linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotideIn some embodiments, the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide, wherein the transcribing occurs in a solution under conditions suitable for splicing of the 3’ and 5' splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide. In some embodiments, the linear polyribonucleotide comprises a 5’ split-intron and a 3’ split-intron (e.g., a self-splicing construct for producing a circular polyribonucleotide). In some embodiments, the linear polyribonucleotide comprises a 5’ annealing region and a 3’ annealing region.Suitable conditions for in vitro transcriptions and / or self-splicing may include any conditions (e.g., a solution or a buffer, such as an aqueous buffer or solution) that mimic physiological conditions in one or more respects. In some embodiments, suitable conditions include between 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 between 1-1000 mM K+ions or a salt thereof such as KCI (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 between 1-1000 mM Cl’ ions or a salt thereof such as KCI (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 between 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 pM, 1-500 pM, 1-200 pM, 50- 500 pM, 100-500 pM, 100-300 pM, 0.1-100 mM, 0.1-50 mM, 0.1-20 mM, 0 1-10 mM, 0 1- 5 mM, 0.1-2 mM, 05-50 mM, 05-20 mM, 05-15 mM, 05-5 mM, 05-2 mM, or 0 1-10 mM). In some embodiments, suitable conditions include between 0.1 mM and 100 mM ribonucleoside triphosphate (NTP) (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 to 10 (e.g., pH of 5 to 9, pH of 6 to 9, or pH of 6.5 to 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 produced from a deoxyribonucleic acid, e g, 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 cellThe disclosure also provides methods of producing a circular RNA in a cell, e.g., a prokaryotic cell or a eukaryotic cell. In some embodiments, an exogenous polyribonucleotide is provided to a cell (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding for the transcription of a linear polyribonucleotide described here). The linear polyribonucleotides may be transcribed in the cell from an exogenous DNA molecule provided to the cell. The linear polyribonucleotide may be transcribed in the cell from an exogenous recombinant DNA molecule transiently provided to the cell. In one embodiment, the linear polyribonucleotide may be transcribed in the cell from an exogenous DNA molecule provided to the cell, such as for example, with a non-limiting exemplary plasmid, as illustrated in Figure 6. In some embodiments, the exogenous DNA molecule does not integrate into the cell’s genome. In some embodiments, the linear polyribonucleotide is transcribed in the cell from a recombinant DNA molecule that is incorporated into the cell’s genome.In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell including the polyribonucleotides described herein may be a bacterial cell or an archaeal cell. For example, the prokaryotic cell including the polyribonucleotides described herein may be E. coli, halophilic archaea (e.g., Haloferax volcaniii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira) spp., and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp. (e.g., Bacillus subtilis, Bacillus anthracis, Bacillus cereus), betaproteobacteria (e.g., Burkholderia), alphaproteobacterial (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 in a bioreactor.In some embodiments, the cell is a eukaryotic cell In some embodiments, the eukaryotic cell including the polyribonucleotides described herein is a unicellular eukaryotic cell. In some embodiments, the unicellular eukaryotic is a unicellular fungal cell such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp, and Pichia spp.). In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. A unicellular animal cell may be a cell isolated from a multicellular animal and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular animal cell may be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell A unicellular plant cell may be a cell isolated from a multicellular plant and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular plant cell may be dedifferentiated In some embodiments, the unicellular plant cell is from a plant callus. In embodiments, the unicellular cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algal cell, such as a unicellular green alga, a diatom, a euglenid, or a dinoflagellate. Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris spp, Protosiphon botryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas spp. In some embodiments, the unicellular eukaryotic cell is a protist cell. In some embodiments, the unicellular eukaryotic cell is a protozoan cellIn some embodiments, the eukaryotic cell is a cell of a multicellular eukaryote. For example, the multicellular eukaryote may be selected from the group consisting of a vertebrate animal, an invertebrateanimal, 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 animal. In some embodiments, the eukaryotic organism is an invertebrate animal 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 cell of a human or a cell of a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., bovids including cattle, buffalo, bison, sheep, goat, and muskox; pig; camelids including camel, llama, and alpaca; deer, antelope; and equids including horse and donkey), carnivore (e g, dog, cat), rodent (e g, rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare). In embodiments, the eukaryotic cell is a cell of a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the eukaryotic cell is a cell of an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc In embodiments, the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte. 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 in a bioreactor.Methods of purificationOne or more purification steps may be included in the methods described herein. For example, in some embodiments, the linear polyribonucleotide is substantively enriched 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 enriching the desired reaction product from one or more undesired components, such as any unreacted stating material, byproducts, enzymes, or other reaction components. For example, purification of linear polyribonucleotide following transcription in a cell-free system (e.g., in vitro transcription) may include separation or enrichment from the DNA template prior to self-splicing the linear polyribonucleotide. Purification of the circular RNA product following splicing may be used to separate or enrich the circular RNA from its corresponding linear RNA. Methods of purification of RNA are known to those of skill in the art and include enzymatic purification or by chromatography.In some embodiments, the methods of purification result in a circular polyribonucleotide that has less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) linear polyribonucleotides.In some embodiments, detection and quantification of nicked versus total RNA in the pharmaceutical composition is determined by sequencing after gel extraction of the preparation comprising the circular RNA. In some embodiments, detection and quantification of nicked versus linear RNA in the pharmaceutical composition is determined by sequencing after gel extraction of the preparation comprising the circular RNA. In some embodiments, a circular polyribonucleotide preparation comprises less than a threshold amount (e.g., where the threshold amount is a reference criterion, e.g., apharmaceutical release specification for the circular polyribonucleotide preparation) of nicked RNA, linear RNA, or combined linear and nicked RNA when evaluated as described herein. For example, the reference criterion for the amount of linear polyribonucleotide molecules present in the preparation is no more than 30%, 20%, 15%, 10%, 1%, 0.5%, or 0.1% linear polyribonucleotide molecules, or any percentage therebetween, relative to total ribonucleotide molecules in the preparation. In some embodiments, the reference criterion for the amount of nicked polyribonucleotide molecules present in the preparation is no more than 30%, 20%, 15%, 10%, 1%, 0.5%, or 0.1%, or any percentage therebetween, nicked polyribonucleotide molecules relative to total ribonucleotide molecules in the preparation In some embodiments, the reference criterion for the amount of linear and nicked polyribonucleotide molecules present in the preparation is no more than 40%, 30%, 20%, 15%, 10%, 1%, 05%, or 0.1%, or any percentage therebetween, combined linear polyribonucleotide and nicked polyribonucleotide molecules relative to total ribonucleotide molecules in the preparationIn an embodiment, a circular polyribonucleotide preparation (e.g., a circular polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of the circular polyribonucleotide preparation) has a combined linear RNA and nicked RNA content of no more than 30% (w / w), 25% (w / w), 20% (w / w), 15% (w / w), 10% (w / w), 9% (w / w), 8% (w / w), 7% (w / w), 6% (w / w), 5% (w / w), 4% (w / w), 3% (w / w), 2% (w / w), 1% (w / w), 0.5% (w / w), or 0.1% (w / w), or percentage therebetween In an embodiment, a circular polyribonucleotide preparation (e.g., a circular polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of the circular polyribonucleotide preparation) has a combined nicked RNA and linear RNA content that is as low as zero or is substantially free of nicked and linear RNA.In another aspect, a method of making a pharmaceutical composition comprises: a) providing a preparation of circular polyribonucleotide molecules, b) processing the preparation to reduce the amount of linear polyribonucleotide molecules, c) optionally evaluating the amount of linear polyribonucleotide molecules in the preparation before, during, and / or after the processing step, and d) further processing the preparation to produce a pharmaceutical composition for pharmaceutical use. In some embodiments, the further processing of step d) comprises one or more of: i) processing the preparation to substantially remove DNA and / or protein (e.g., a cell protein such as a host cell protein) and / or endotoxin; ii) evaluating the amount of DNA and / or protein (e g, a cell protein such as a host cell protein) and / or endotoxin in the preparation; Hi) formulating the preparation with a pharmaceutical excipient; and iv) optionally, concentrating the preparation.In some embodiments, a method of making a a pharmaceutical drug substance, comprises a) providing a plurality of linear polyribonucleotide molecules; b) circularizing the plurality of linear polyribonucleotide molecules to provide a preparation of circular polyribonucleotide molecules; c) evaluating the amount of linear and / or nicked polyribonucleotide molecules remaining in the preparation; and d) processing the preparation of circular polyribonucleotide molecules as a pharmaceutical drug substance if the preparation meets a reference criterion for an amount of linear and / or nicked polyribonucleotide molecules present in the preparation.In some embodiments, a method of making a pharmaceutical drug product comprises: a) providing a plurality of linear polyribonucleotide molecules; b) circularizing the plurality of linear polyribonucleotide molecules to provide a preparation of circular polyribonucleotide molecules; c)measuring the amount of linear and / or nicked polyribonucleotide molecules in the preparation; d) formulating the preparation of circular polyribonucleotide molecules as a pharmaceutical drug product if the preparation meets a reference criterion for an amount of linear and / or nicked polyribonucleotide molecules present in the preparation; and e) labelling and shipping the pharmaceutical drug product if it meets a reference criterion for the amount of linear polyribonucleotide molecules present in the pharmaceutical drug product.BioreactorsIn some embodiments, any method of producing a circular polyribonucleotide described herein may be performed in a bioreactor. A bioreactor refers to any vessel in which a chemical or biological process is carried out which involves organisms or biochemically active substances derived from such organisms. Bioreactors may be compatible with the cell-free methods for production of circular RNA described herein. A vessel for a bioreactor may include a culture flask, a dish, or a bag that may be single use (disposable), autoclavable, or sterilizable. A bioreactor may be made of glass, or it may be polymer-based, or it may be made of other materials.Examples of bioreactors include, without limitation, stirred tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibromixers, fluidized bed reactors, and membrane bioreactors The mode of operating the bioreactor may be a batch or continuous processes. A bioreactor is continuous when the reagent and product streams are continuously being fed and withdrawn from the system. A batch bioreactor may have a continuous recirculating flow, but no continuous feeding of reagents or product harvest.Some methods of the present disclosure are directed to large-scale production of circular polyribonucleotides For large-scale production methods, the method may be performed in a volume of 1 liter (L) to 50 L, or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more). In some embodiments, the method may be performed in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L.In some embodiments, a bioreactor may produce at least 1g of circular RNA. In some embodiments, a 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, or 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 time (e.g., 1-200 g per hour or per day).In some embodiments, more than one bioreactor may be utilized in series to increase the production capacity (e.g., one, two, three, four, five, six, seven, eight, or nine bioreactors may be used in series).Methods of UseIn some embodiments, a circular polyribonucleotide encoding an OTC polypeptide (e.g., a polypeptide of Table 1) is used for the treatment or prevention of a urea cycle disorder or OTC deficiency In some embodiments, a circular polynucleotide encoding an OTC polypeptide (e.g., a polypeptide of Table 1) is used for treatment of a urea cycle disorder.In some embodiments, a circular polynucleotide encoding an OTC polypeptide (e.g., a polypeptide of Table 1) may be administered to a subject to reduce the risk of an OTC deficiency.Thus in one embodiment described herein is a method of treating OTC deficiency in a subject in need thereof, comprising administering a therapeutically effective amount of any of the polyribonucleotides described herein, or a pharmaceutical composition comprising any of the polyribonucleotides described herein, or a cell comprising any of the polyribonucleotides described herein.In another embodiment described herein is a method of treating a urea cycle disorder in a subject in need thereof, comprising administering a therapeutically effective amount of any of the polyribonucleotides described herein, or a pharmaceutical composition comprising any of the polyribonucleotides described herein, or a cell comprising any of the polyribonucleotides described herein.For example, a circular polyribonucleotide as described herein may be administered to a subject or human patient (e.g., in a pharmaceutical composition). In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots) In embodiments, the subject is an invertebrate such as an arthropod (e g, insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusk.In some embodiments, the disclosure provides a method of modifying a subject by providing to the subject a composition or formulation described herein. In some embodiments, the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes or a eukaryotic or prokaryotic cell including a nucleic acid described hereinIn some embodiments, the disclosure provides a method of treating a urea cycle disorder or an OTC deficiency in a subject in need thereof by providing to the subject a composition or formulation described herein. In some embodiments, the composition or formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or formulation is or includes a eukaryotic or prokaryotic cell including a nucleic acid described herein. In some embodiments, the polyribonucleotide is provided in an amount and for a duration sufficient to treat a urea cycle disorder or an OTC deficiency in a subject, e g., in need thereofIn some embodiments, the method may be used to treat or prevent a urea cycle disorder or OTC deficiency For example, in some embodiments, the circular polyribonucleotide encodes an OTC polypeptide that that encodes an ornithine transcarbamylase enzyme in a subject. Expression of the OTC enzyme may be in an amount, or therapeutically effective amount, sufficient to increase the amountof OTC enzyme in the cells, such that there is no or reduced accumulation of nitrogen or ammonia in the cells, or as found in patient without OTC enzyme deficiency.In some embodiments, the polyribonucleotide, polyribonucleotide cargo, linear polyribonucleotide or circular polyribonucleotide described herein may be used in combination with other known treatments for urea cycle disorders or OTC deficiency, including but not limited to, dietary restrictions, hydration regimens, arginine supplementation, hemodialysis and nitrogen removal drugs, such as glycerol phenylbutyrate. In some embodiments, the polyribonucleotide, polyribonucleotide cargo, linear polyribonucleotide or circular polyribonucleotide described herein may be used in combination with other the polyribonucleotide, polyribonucleotide cargo, linear polyribonucleotide or circular polyribonucleotides encoding for another urea cycle enzyme that may be deficient. The present disclosure contemplates the use of the polyribonucleotide, polyribonucleotide cargo, linear polyribonucleotide or circular polyribonucleotide described herein with any treatments suitable for treating urea cycle disorders or OTC deficiencyMethods of DosingA method of dosing to produce a level of circular polyribonucleotide encoding an OTC polypeptide (e g., a polypeptide of Table 1) or express a level of an OTC polypeptide (e.g., a polypeptide of Table 1) in a cell after providing the cell with at least one or more doses or compositions of circular polyribonucleotide is disclosed herein. A method of dosing to produce a level of circular polyribonucleotide or express a level of an OTC polypeptide (e.g., a polypeptide of Table 1) in a subject (e.g., a mammal, e.g., a human, or a patient) after providing (e.g., administering to) the subject with at least one or more doses or compositions of circular polyribonucleotide is disclosed herein The composition includes a circular polyribonucleotide encoding an OTC polypeptide as described herein. A method of dosing can include administering two or more doses of a composition of circular polyribonucleotides, e.g., over short time period or over an extended period In some embodiments, the composition containing a circular polyribonucleotide further includes a pharmaceutically acceptable carrier or excipient. The circular polyribonucleotide encodes an OTC polypeptide, which can be expressed in a cell, e.g., following administration.The methods described herein may include administering a first dose of the pharmaceutical composition in an amount sufficient to produce enough enzyme to reduce patient ammonia levels to a non-toxic level. In another embodiment, the amount is sufficient to reduce patient nitrogen levels to a nontoxic level In another embodiment, the amount is sufficient to produce enough OTC enzyme wherein the patient cannot be diagnosed with OTC deficiency. In another embodiment, the amount is sufficient to produce the same amount of enzyme as found in normal patients. In another embodiment, the amount is sufficient to produce about 5% to about 100% the amount of enzyme as found in normal patients. In another embodiment, the amount is sufficient to produce about 50% of the amount of enzyme as found in normal patients. In another embodiment, the amount is sufficient to produce about 25% of the amount of enzyme as found in normal patients. In another embodiment, the amount is sufficient to produce about 10% the amount of enzyme as found in normal patients. In another embodiment, the amount is sufficient to produce about 5% of the amount of enzyme as found in normal patients.In some embodiments, the method may further include administering a second dose of the pharmaceutical composition. The method may further include administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more doses of the pharmaceutical composition. In some embodiments, a subsequent dose helps maintain enough enzyme to reduce patient ammonia levels to a non-toxic level. In another embodiment, the amount is sufficient to reduce patient nitrogen levels to a non-toxic level. In another embodiment, the amount is sufficient to produce enough OTC enzyme wherein the patient cannot be diagnosed with OTC deficiency. In another embodiment, the amount is sufficient to produce the same amount of enzyme as found in normal patients In another embodiment, the amount is sufficient to produce about 5% to about 100% of the amount of enzyme as found in normal patients. In another embodiment, the amount is sufficient to produce about 50% of the amount of enzyme as found in normal patients In another embodiment, the amount is sufficient to produce about 25% of the amount of enzyme as found in normal patients. In another embodiment, the amount is sufficient to produce about 10% of the amount of enzyme as found in normal patients. In another embodiment, the amount is sufficient to produce about 5% of the amount of enzyme as found in normal patients.In some embodiments, multiple doses are provided to produce a level of the composition or express a level of the OTC 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 OTC polypeptide, in a cell, tissue or subject for a period of time, for instance, for at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 21, or24 months, or at least 1, 2, 3, 4, or 5 years. In some embodiments, multiple doses may be administered at least every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks, or monthly, to produce or maintain a level of the composition, or to produce or maintain a level of the OTC polypeptide, in a cell, tissue or subject for a period of time,In some embodiments, the second dose is administered at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer) after the first dose of the pharmaceutical composition. In some embodiments, the second dose is administered at least every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, or at least one month, after the first dose of the pharmaceutical compositionIn some embodiments, the second dose is administered from 1 hour to 1 year (e.g., from 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e g, from one day to one week, e.g., two days, three days, four days, five days, six days, or one week, e.g., from one week to one month, e.g., two weeks, three weeks, or one month, e.g., from one month to one year, e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year) after the first dose of the pharmaceutical composition. In some embodiments, the second dose is administered from 1 days to 180 days (e.g., from 1 day to 90 days,from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to 7 days, from 2 days to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days, from 3 days to 90 days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3 days to 7 days, from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days to 14 days, from 4 days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days, from 5 days to 14 days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6 days to 30 days, from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to 45 days, from 7 days to 30 days, from 7 days to 14 days, from 14 days to 90 days, from 14 days to 45 days, from 14 days to 30 days, from 21 days to 90 days, from 21 days to 60 days, from 21 days to 45 days, from 21 days to 30 days, from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45 days, from 45 to 180 days, from 45 to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to 60 days, from 60 to 180 days, from 60 to 120 days, from 60 to 100 days, from 60 to 90 days, from 90 to 100 days, from 90 to 120 days, or from 90 to 180 days) after the first dose of the pharmaceutical composition. In some embodiments, the second dose is administered at least every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, or at least one month, after the first dose of the pharmaceutical composition. Subsequent doses may be administered monthyly thereafter.In some embodiments, the third dose is administered at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer) after the second dose of the pharmaceutical composition. In some embodiments, the third dose is administered at least every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, or at least one month, after the second dose of the pharmaceutical composition. Subsequent doses may be administered monthyly thereafter.In some embodiments, the third dose is administered from 1 hour to 1 year (e.g., from 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day to one week, e.g., two days, three days, four days, five days, six days, or one week, e.g, from one week to one month, e.g., two weeks, three weeks, or one month, e.g., from one month to one year, e g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year) after the second dose of the pharmaceutical composition. In some embodiments, the third dose is administered from 1 days to 180 days (e.g., from 1 day to 90 days, from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to 7 days, from 2 days to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days, from 3 days to 90 days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3 days to 7 days, from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days to 14 days, from 4 days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days, from 5 days to 14 days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6 days to 30 days, from 6days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to 45 days, from 7 days to 30 days, from 7 days to 14 days, from 14 days to 90 days, from 14 days to 45 days, from 14 days to 30 days, from 21 days to 90 days, from 21 days to 60 days, from 21 days to 45 days, from 21 days to 30 days, from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45 days, from 45 to 180 days, from 45 to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to 60 days, from 60 to 180 days, from 60 to 120 days, from 60 to 100 days, from 60 to 90 days, from 90 to 100 days, from 90 to 120 days, or from 90 to 180 days) after the second dose of the pharmaceutical composition. In some embodiments, the third dose is administered at least every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, or at least one month, after the second dose of the pharmaceutical composition. Subsequent doses may be administered monthly thereafter.In some embodiments, the second dose is administered before ammonia levels of the subject are toxic. In another embodiment, the second dose is administered before nitrogen levels of the subject are toxic.In some embodiments, the method maintains an amount of OTC polypeptide as described herein wherein the amount may be sufficient to reduce patient ammonia levels to a non-toxic level, to reduce patient nitrogen levels to a non-toxic level, to produce enough OTC enzyme wherein the patient cannot be diagnosed with OTC deficiency, to produce the same amount of enzyme as found in normal patients, to produce about 5% to about 100% of the amount of enzyme as found in normal patients, to produce about of 50% the amount of enzyme as found in normal patients, to produce about of 25% the amount of enzyme as found in normal patients, to produce about of 10% the amount of enzyme as found in normal patients, to produce about of 5% the amount of enzyme as found in normal patients and those amounts may be maintained e g, for at least one hour (e g, at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer).A method of administering multiple doses of a composition of a nucleic acid molecule described herein (e g, a circular polyribonucleotide) includes providing two or more compositions over a period of time, to a cell, tissue or subject (e.g., a mammal). According to certain embodiments, multiple doses of a composition of a nucleic acid molecule described herein may be administered to a subject over a defined time course. The methods according to this aspect of the invention include sequentially administering to a subject multiple doses of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical or veterinary composition). As used herein, “sequentially administering” means that each dose of composition of a nucleic acid molecule described herein is administered to the subject at a different point in time, e g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). In some embodiments, the present invention provides methods which include sequentially administering to the subject a single initial dose of a composition of a nucleic acid molecule described herein, followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition.The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of a composition of a nucleic acid molecule described herein. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen; the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of a composition of a nucleic acid molecule described herein, and in certain embodiments, may differ from one another in terms of frequency of administration In certain embodiments, the amount of a composition of a nucleic acid molecule described herein contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, one or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).In certain embodiments, each secondary and / or tertiary dose is administered after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of the composition of a nucleic acid molecule described herein which is administered to a subject prior to the administration of the very next dose in the sequence with no intervening doses. In certain embodiments, each secondary and / or tertiary dose is administered every day, every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the immediately preceding dose. In certain embodiments, each secondary and / or tertiary dose is administered every 0.5 weeks, 1 week, 2 weeks, 3 weeks, or 4 weeks after the immediately preceding dose. In other embodiments, each secondary and / or tertiary dose may be administered at least every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, or one month after the immediately preceding doseThe methods according to this aspect of the invention may include administering to a subject any number of secondary and / or tertiary doses of a composition of a nucleic acid molecule described herein. For example, in certain embodiments, only a single secondary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the subject. Likewise, in certain embodiments, only a single tertiary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the subjectIn certain embodiments, the frequency at which the secondary and / or tertiary doses are administered to a subject can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatmentIn some embodiments, the method includes providing (e.g., administering) at least a first composition and a second composition to the cells, tissue, or subject (e.g, a mammal, e.g, a human). In some embodiments, the method further includes providing (e.g., administering) a third composition, fourth composition, fifth composition, sixth composition, seventh composition, eighth composition, ninth composition, tenth composition, or more. In some embodiments, additional compositions are provided for the duration of the life of the cell. In some embodiments, additional compositions are provided (e.g., administered) while the cell, tissue or subject obtains a benefit from the compositionIn some embodiments, a first composition in a multiple dosing regimen includes a first amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, asecond composition in a multiple dosing regimen includes a second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, a third composition, a fourth composition, a fifth composition, a sixth composition, a seventh composition, an eighth composition, a ninth composition, a tenth composition, or more in a multiple dosing regimen includes a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein. In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e g, circular polyribonucleotide) In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide) In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide) In some embodiments, the second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, the third amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e g, circular polyribonucleotide) In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition varies by no more than 1%, 5%, 10%, 15%, 20%, or 25% of an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition is no more than 1%, 5%, 10%, 15%, 20%, or 25% less than an amount of the nucleic acid molecule (e g., circular polyribonucleotide) of the first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1-fold to 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g., a composition administered after a first composition) is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1-fold to 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a firstcomposition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g, a composition administered after a first composition) is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the nucleic acid molecule (e.g, circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e g, circular polyribonucleotide) of a subsequent composition (e.g., after a first composition of an amount of nucleic acid molecule (e.g., circular polyribonucleotide)) is from 0.1-fold to 1000-fold higher or lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition (e.g, after a first composition of an amount of nucleic acid molecule (e.g., circular polyribonucleotide)) is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher or lower than an amount of the nucleic acid molecule (e.g, circular polyribonucleotide) of a first composition. For example, a first composition includes 1-fold nucleic acid molecule (e.g., circular polyribonucleotide), a second composition includes 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition, and a third composition includes 0.2-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition. In some embodiments, the second composition includes at least 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to an amount of nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.In some embodiments, the first composition includes a higher amount of the nucleic acid molecule (e g, circular polyribonucleotide) than the second composition In some embodiments, the first composition includes a higher amount of the nucleic acid molecules (e.g., circular polyribonucleotides) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition.In some embodiments, the plurality (e g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding an OTC polypeptide, which are administered in a multiple dosing regimen as described herein, are the same compositions. In some embodiments, the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding an OTC polypeptide, which are administered in a multiple dosing regimen as described herein, are different compositions. In some embodiments, the same compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding the same OTC polypeptide. In some embodiments, the different compositions include the nucleic acid molecules (e.g., circular polyribonucleotides) encoding different OTC polypeptides, or a combination thereof.In some embodiments, a plurality of enzymes needed to prevent a urea cycle disorder may be administered in a multiple dosing regimen as described herein. Compositions of a nucleic acid molecule (e g, circular polyribonucleotide) encoding other enzymes related to prevention of urea cycle disorders may be administered with the circular polyribonucleotide expressing an OTC polypeptide described herein. Non-limiting examples of other urea cycle disorder enzymes include argininosuccinic acid synthetase, arginase, argininosuccinase acid lyase, carbamoyl phosphate synthetase, citrin, ornithine translocase, and N-acetylglutamate synthetase. In some embodiments, the plurality (e.g, two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding an OTC polypeptideand additional enzymes, which are administered in a multiple dosing regimen as described herein, are the same compositions. In some embodiments, the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding an OTC polypeptide and additional enzymes, which are administered in a multiple dosing regimen as described herein, are different compositions.In some embodiments, in a multiple dosing regimen, the method of administering the nucleic acid molecule (e.g., circular polyribonucleotide) provided herein includes administering to a subject in need thereof the nucleic acid molecule for multiple times (multiple doses), e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 100, 150, 200, or 500 times, with an interval of from 1 day to 56 days, such as about 49 days, 42 days, 35 days, 28 days, 21 days, 14 days, or 7 days. In some embodiments, in a multiple dosing regimen, the method provided herein includes administering to a subject in need thereof the nucleic acid molecule for at least 3 times, with an interval of about 7 days. In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses) provided herein, a level of the OTC polypeptide (e.g., a plasma OTC polypeptide) is maintained at a level with variation of less than 50%, 40%, 30%, 20%, or 10% for a period of longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or 20 weeks after the last dose In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses) provided herein, a level of the OTC polypeptide (e.g., a plasma OTC polypeptide level) is maintained at a first level for a period of longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first level is higher than a level of the OTC polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose). In some embodiments, in a subject that receives administration of multiple doses of the nucleic acid molecule (e g, at least 3 doses) provided herein with an interval of about 7 days, a level of the OTC polypeptide (e.g, a plasma OTC polypeptide level) is maintained at a first level for a period of longer than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first level is higher than a level of the OTC polypeptide measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).Methods of DeliveryAcircular polyribonucleotide encoding an OTC polypeptide (e.g., a polypeptide of Table 1) described herein may be included in pharmaceutical compositions with a carrier or without a carrier.Pharmaceutical compositions described herein may be formulated for example including a carrier, such as a pharmaceutical carrier and / or a polymeric carrier, e.g, a liposome, and delivered by known methods to a subject in need thereof (e.g., a human or non-human agricultural or domestic animal, e.g., cattle, dog, cat, horse, poultry). Such methods include, but 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, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof Methods of delivery are also described, e.g., in Gori et al., Delivery and Specificity of CRISPR / Cas9 Genome Editing Technologies for HumanGene 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. Nat Biotechnol. 2014 Oct 30;33(1):73-80.In some embodiments, circular polyribonucleotides may be delivered in a “naked” delivery formulation. A naked delivery formulation delivers a circular polyribonucleotide to a cell without the aid of a carrier and without covalent modification of the circular polyribonucleotide or partial or complete encapsulation of the circular polyribonucleotide.A naked delivery formulation is a formulation that is free from a carrier and wherein the circular polyribonucleotide is without a covalent modification that binds a moiety that aids in delivery to a cell and the circular polyribonucleotide is not partially or completely encapsulated. In some embodiments, a circular polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may be a polyribonucleotide that is not covalently bound to a moiety, such as a protein, small molecule, a particle, a polymer, or a biopolymer that aids in delivery to a cell. In some embodiments, circular polyribonucleotides may be delivered in a delivery formulation with protamine or a protamine salt (e.g., protamine sulfate).A polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may not contain a modified phosphate group. For example, a polyribonucleotide without covalent modification that binds to a moiety that aids in delivery to a cell may not contain phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.In some embodiments, a naked delivery formulation may be free of any or all of: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers For example, a naked delivery formulation may be free from phytoglycogen octenyl succinate, phytoglycogen betadextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N, N, N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HCI), diheptadecylamidoglycyl spermidine (DOGS), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE), N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin.A naked delivery formulation may include a non-carrier excipient In some embodiments, a noncarrier excipient may include an inactive ingredient that does not exhibit an active cell-penetrating effect. In some embodiments, a non-carrier excipient may include a buffer, for example PBS. In some embodiments, a non-carrier excipient may be a solvent, a non-aqueous solvent, a diluent, a suspension aid, a surface-active agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, apolymer, a peptide, a protein, a cell, a hyaluronidase, a dispersing agent, a granulating agent, a disintegrating agent, a binding agent, a buffering agent, a lubricating agent, or an oilIn some embodiments, a naked delivery formulation may include a diluent, such as a parenterally acceptable diluent. A diluent (e.g., a parenterally acceptable diluent) may be a liquid diluent or a solid diluent. In some embodiments, a diluent (e.g., a parenterally acceptable diluent) may be an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of an RNA solubilizing agent include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of a buffer include 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate. Examples of an isotonic agent include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.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 organic compounds such as alcohols having one or more hydroxyl function groups. In some cases, the cell-penetrating agent includes an alcohol such as, but not limited to, monohydric alcohols, polyhydric alcohols, unsaturated aliphatic alcohols, and alicyclic alcohols. The cell-penetrating agent can include one or more of methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzyl alcohol, specially denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol, menthol, polyethylene glycols (PEG)-400, ethoxylated fatty acids, or hydroxyethylcellulose In certain embodiments, the cellpenetrating agent includes ethanol. The cell-penetrating agents can include any cell-penetrating agent in any amount or in any formulation as described in W02020 / 180751 or W02020 / 180752, which are hereby incorporated by reference in their entirety.In some embodiments, the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein is in parenteral nucleic acid delivery system. The parental nucleic acid delivery system may include the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein, and a parenterally acceptable diluent. In some embodiments, the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the pharmaceutical drug substance of as disclosed, or the pharmaceutical drug product as disclosed herein in the parenteral nucleic acid delivery system is free of any carrier.The disclosure is further directed to a host or host cell including the circular polyribonucleotide described herein In some embodiments, the host or host cell is a vertebrate, mammal (e g, human), or other organism or cell.In some embodiments, the circular polyribonucleotide has a decreased, or fails to produce a, undesired response by the host’s immune system as compared to the response triggered by a reference compound, e.g., a linear polynucleotide corresponding to the described circular polyribonucleotide. In embodiments, the circular polyribonucleotide is non-immunogenic in the host. Some immune responsesinclude, 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 a host cell is contacted with (e.g, delivered to or administered to) the circular polyribonucleotide In some embodiments, the host is a mammal, such as a human. The amount of the circular polyribonucleotide or linear, expression product, or both in the host can be measured at any time after administration In certain embodiments, a time course of host growth in a culture is determined. If the growth is increased or reduced in the presence of the circular polyribonucleotide or linear, the circular polyribonucleotide or expression product or both is identified as being effective in increasing or reducing the growth of the host.A method of delivering a circular polyribonucleotide molecule as described herein to a cell, tissue, or subject, includes administering the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein to the cell, tissue, or subject.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 a connective tissue, a muscle tissue, a nervous tissue, or an epithelial tissue. In some embodiments, the tissue is an organ (e.g., liver, lung, spleen, kidney, etc.). In some embodiments, the method of delivering a circular polyribonucleotide molecule as described herein includes delivery to any tissue specific cell type, including but not limited to, bone cells, muscle cells, hepatic cells (hepatocytes), pancreatic cells, nerve cells, skin cells, fat cells, or pulmonary cells.In some embodiments, the method of delivering is an in vivo method. For example, a method of delivery of a circular polyribonucleotide as described herein includes parenterally administering to a subject in need thereof, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein to the subject in need thereof. As another example, a method of delivering a circular polyribonucleotide to a cell or tissue of a subject, includes administering parenterally to the cell or tissue the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein. In some embodiments, the circular polyribonucleotide is in an amount effective to elicit a biological response in the subject. In some embodiments, the circular polyribonucleotide is an amount effective to have a biological effect on the cell or tissue in the subject In some embodiments, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein includes a carrier. In some embodiments the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein includes a diluent and is free of any carrier.In some embodiments the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered parenterally. In some embodiments the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, intralymphaticly, subcutaneously, or intramuscularly. In some embodiments, parenteral administration is intravenously, intramuscularly, ophthalmically, subcutaneously, intradermally or topically.In some embodiments, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered intramuscularly. In some embodiments,the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered subcutaneously. In some embodiments, the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product as described herein is administered topically. In some embodiments, the pharmaceutical composition, the pharmaceutical drug substance, or the pharmaceutical drug product is administered intratracheally.In some embodiments the pharmaceutical composition, pharmaceutical drug substance or pharmaceutical drug product is administered by injection. The administration can be systemic administration or local administration In some embodiments, any of the methods of delivery as described herein are performed with a carrier. In some embodiments, any methods of delivery as described herein are performed without the aid of a carrier or cell penetrating agent.In some embodiments, the circular polyribonucleotide or a product translated from the circular polyribonucleotide is detected in the cell, tissue, or subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days after the administering step. In some embodiments, the presence of the circular polyribonucleotide or a product translated from the circular polyribonucleotide is evaluated in the cell, tissue, or subject before the administering step. In some embodiments, the presence of the circular polyribonucleotide or a product translated from the circular polyribonucleotide is evaluated in the cell, tissue, or subject after the administering step.FormulationsIn some embodiments of the present disclosure a circular polyribonucleotide described herein may be formulated in composition, e.g., a composition for delivery to a cell, a plant, an invertebrate animal, a non-human vertebrate animal, or a human subject (or patient), e g, an agricultural, veterinary, or pharmaceutical composition. In some embodiments, the circular polyribonucleotide is formulated in a pharmaceutical composition. In some embodiments, a composition includes a circular polyribonucleotide and a diluent, a carrier, an adjuvant, or a combination thereof. In a particular embodiment, a composition includes a circular polyribonucleotide described herein and a carrier or a diluent free of any carrier. In some embodiments, a composition including a circular polyribonucleotide with a diluent free of any carrier is used for naked delivery of the circular polyribonucleotide to a subject. Thus, one embodiment is a pharmaceutical composition comprising any of the linear polyribonucleotides or circular polyribonucleotides described herein, a cell comprising the polyribonucleotides, a diluent, a carrier or an excipient.Pharmaceutical compositions may optionally include one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. Pharmaceutical compositions may optionally include an inactive substance that serves as a vehicle or medium for the compositions described herein (e.g., compositions including circular polyribonucleotides, such as any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database). Pharmaceutical compositions of the present invention may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of an inactive substance include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents,dispersions, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, polymers, peptides, proteins, cells, hyaluronidases, dispersing agents, granulating agents, disintegrating agents, binding agents, buffering agents (e.g., phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures thereof.Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e g, to non-human animals, e g, non-human mammals Modification of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeysFormulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product.In some embodiments, the reference criterion for the amount of circular polyribonucleotide molecules present in the preparation is at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 99.1% (w / w), 99.2% (w / w), 99.3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w), 99.7% (w / w), 99.8% (w / w), 99.9% (w / w), or 100% (w / w) molecules of the total ribonucleotide molecules in the pharmaceutical preparation.In some embodiments, the reference criterion for the amount of linear polyribonucleotide molecules present in the preparation is the presence of no more than 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 1 pg / ml, 10 pg / ml, 50 pg / ml, 100 pg / ml, 200 µg / ml, 300 µg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, 900 pg / ml, 1 mg / ml, 1.5 mg / ml, or 2 mg / ml of linear polyribonucleotide molecules.In some embodiments, the reference criterion for the amount of linear polyribonucleotide molecules present in the preparation is no more than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical preparationIn some embodiments, the reference criterion for the amount of nicked polyribonucleotide molecules present in the preparation is no more than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), or 15% (w / w) nicked polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical preparation.In some embodiments, the reference criterion for the amount of combined nicked and linear polyribonucleotide molecules present in the preparation is no more than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) combined nicked and linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical preparation. In some embodiments, a pharmaceutical preparation is an intermediate pharmaceutical preparation of a final circular polyribonucleotide drug product. In some embodiments, a pharmaceutical preparation is a drug substance or active pharmaceutical ingredient (API) In some embodiments, a pharmaceutical preparation is a drug product for administration to a subjectIn some embodiments, a preparation of circular polyribonucleotides is (before, during or after the reduction of linear RNA) further processed to substantially remove DNA, protein contamination (e.g., cell protein such as a host cell protein or protein process impurities), endotoxin, mononucleotide molecules, and / or a process-related impurity.In some embodiments, a pharmaceutical formulation disclosed herein can include: (i) a compound (e.g., circular polyribonucleotide) disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and / or (v) a stabilizer. In some embodiments, the pharmaceutical formulation disclosed herein is a stable liquid pharmaceutical formulation. In some embodiments, the pharmaceutical formulation disclosed herein includes protamine or a protamine salt (e.g., protamine sulfate).PreservativesA composition or pharmaceutical composition provided herein can include material for a single administration, or can include material for multiple administrations (e.g., a “multidose” kit). The polyribonucleotide can be present in either linear or circular form The composition or pharmaceutical composition can include one or more preservatives such as thiomersal or 2-phenoxyethanol.Preservatives can be used to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, Onamer M, or other agents known to those skilled in the art In ophthalmic products, e.g., such preservatives can be employed at a level of from 0.004% to 0.02%. In the compositions described herein the preservative, e.g., benzalkonium chloride, can be employed at a level of from 0001% to less than 001%, e g, from 0001% to 0008%, preferably about 0005% by weight Polyribonucleotides can be susceptible to RNase that can be abundant in ambient environment. Compositions provided herein can include reagents that inhibit RNase activity, thereby preserving the polyribonucleotide from degradation. In some cases, the composition or pharmaceutical composition includes any RNase inhibitor known to one skilled in the art. Alternatively or additionally, the polyribonucleotide, and cell-penetrating agent and / or pharmaceutically acceptable diluents or carriers, vehicles, excipients, or other reagents in the composition provided herein can be prepared in RNase-free environment The composition can be formulated in RNase-free environmentIn some cases, a composition provided herein can be sterile. The composition can be formulated as a sterile solution or suspension, in suitable vehicles, known in the art. The composition can be sterilized by conventional, known sterilization techniques, e.g., the composition can be sterile filtered.Sa / tsIn some cases, a composition or pharmaceutical composition provided herein includes one or more salts. For controlling the tonicity, a physiological salt such as sodium salt can be included in a composition provided herein. Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride, or the like. In some cases, the composition is formulated with one or more pharmaceutically acceptable salts. The one or more pharmaceutically acceptable salts can include those of the inorganic ions, such as, for example, sodium, potassium, calcium, magnesium ions, and the like Such salts can include salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid. The polyribonucleotide can be present in either linear or circular form.Buffers / pHA composition or pharmaceutical composition provided herein can include one or more buffers, such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (e.g., with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers, in some cases, are included in the 5-20 mM range.A composition or pharmaceutical composition provided herein can have a pH between about 5.0 and about 8.5, between about 6.0 and about 80, between about 6.5 and about 7.5, or between about 7.0 and about 7.8. The composition or pharmaceutical composition can have a pH of about 7. The polyribonucleotide can be present in either linear or circular form.Detergents / surfactantsA composition or pharmaceutical composition provided herein can include one or more detergents and / or surfactants, depending on the intended administration route, e.g., polyoxyethylene sorbitan esters surfactants (commonly referred to as “Tweens”), e.g., polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO / PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1, 2-ethanediyl) groups, e.g., octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as “SPANs”), such as sorbitan trioleate (Span 85) and sorbitan monolaurate, an octoxynol (such as octoxynol-9 (Triton X-100) ort-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide (“CTAB”), or sodium deoxycholate. The one or more detergents and / or surfactants can be present only at trace amounts In some cases, the composition can include less than 1 mg / ml of each of octoxynol-10 and polysorbate 80. Non-ionic surfactants can be used herein. Surfactants can be classified by their “HLB” (hydrophile / lipophile balance). In some cases, surfactants have a HLB of at least 10, at least 15, and / or at least 16. The polyribonucleotide can be present in either linear or circular form.DiluentsIn some embodiments, a composition of the disclosure includes a circular polyribonucleotide and a diluent. In some embodiments, a composition of the disclosure includes a linear polyribonucleotide and a diluent.A diluent can be a non-carrier excipient. A non-carrier excipient serves as a vehicle or medium for a composition, such as a circular polyribonucleotide as described herein A non-carrier excipient serves as a vehicle or medium for a composition, such as a linear polyribonucleotide as described herein Non-limiting examples of a non-carrier excipient include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, polymers, peptides, proteins, cells, hyaluronidases, dispersing agents, granulating agents, disintegrating agents, binding agents, buffering agents (e.g., phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures thereof. A non-carrier excipient can be any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database that does not exhibit a cell-penetrating effect. A non-carrier excipient can be any inactive ingredient suitable for administration to a non-human animal, for example, suitable for veterinary use. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation.In some embodiments, the circular polyribonucleotide may be delivered as a naked delivery formulation, such as including a diluent. A naked delivery formulation delivers a circular polyribonucleotide, to a cell without the aid of a carrier and without modification or partial or complete encapsulation of the circular polyribonucleotide, capped polyribonucleotide, or complex thereof.A naked delivery formulation is a formulation that is free from a carrier and wherein the circular polyribonucleotide is without a covalent modification that binds a moiety that aids in delivery to a cell or without partial or complete encapsulation of the circular polyribonucleotide. In some embodiments, a circular polyribonucleotide without a covalent modification that binds a moiety that aids in delivery to a cell is a polyribonucleotide that is not covalently bound to a protein, small molecule, a particle, a polymer, or a biopolymer A circular polyribonucleotide without covalent modification that binds a moiety that aids in delivery to a cell does not contain a modified phosphate group. For example, a circular polyribonucleotide without a covalent modification that binds a moiety that aids in delivery to a cell does not contain phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.In some embodiments, a naked delivery formulation is free of any or all of: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers In some embodiments, a naked delivery formulation is free from phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-Dioleoyl-3-Trimethylammonium-Propane(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 HCI), diheptadecylamidoglycyl spermidine (DOGS), N, N-distearyl-N, N- dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulinIn certain embodiments, a naked delivery formulation includes a non-carrier excipient. In some embodiments, a non-carrier excipient includes an inactive ingredient that does not exhibit a cell-penetrating effect. In some embodiments, a non-carrier excipient includes a buffer, for example PBS. In some embodiments, a non-carrier excipient is a solvent, a non-aqueous solvent, a diluent, a suspension aid, a surface-active agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersing agent, a granulating agent, a disintegrating agent, a binding agent, a buffering agent, a lubricating agent, or an oilIn some embodiments, a naked delivery formulation includes a diluent. A diluent may be a liquid diluent or a solid diluent. In some embodiments, a diluent is an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of an RNA solubilizing agent include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of a buffer include 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate. Examples of an isotonic agent include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.CarriersIn some embodiments, a composition of the disclosure includes a circular polyribonucleotide and a carrier In some embodiments, a composition of the disclosure includes a linear polyribonucleotide and a carrier.In certain embodiments, a composition includes a circular polyribonucleotide as described herein in a vesicle or other membrane-based carrier. In certain embodiments, a composition includes a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier.In other embodiments, a composition includes the circular polyribonucleotide in or via a cell, vesicle or other membrane-based carrier. In other embodiments, a composition includes the linear polyribonucleotide in or via a cell, vesicle or other membrane-based carrier In one embodiment, a composition includes the circular polyribonucleotide in liposomes or other similar vesicles. In one embodiment, a composition includes the linear polyribonucleotide in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible, nontoxic, can deliver bothhydrophilic 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, 2011doi: 10.1155 / 2011 / 469679 for review).Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U. S. Pat No 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference) Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.In certain embodiments, a composition of the disclosure includes a circular polyribonucleotide and lipid nanoparticles, for example lipid nanoparticles described herein. In certain embodiments, a composition of the disclosure includes a linear polyribonucleotide and lipid nanoparticles. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a circular polyribonucleotide molecule as described herein. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a linear polyribonucleotide molecule as described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of the SLN, improve drug stability and loading capacity, and prevent drug leakage Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes. A PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell offers good biocompatibility. As such, the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390 / nano7060122.Additional non-limiting examples ...

Claims

CLAIMS1. A circular polyribonucleotide comprising a polyribonucleotide cargo encoding an ornithine transcarbamylase (OTC) polypeptide2. The circular polyribonucleotide of claim 1, wherein the polyribonucleotide cargo comprises an expression sequence encoding one or more OTC polypeptide.

3. The circular polyribonucleotide of any one of claims 1 or 2, wherein the circular polyribonucleotide comprises a splice junction at a 5’ exon fragment or a 3’ exon fragment.4 The circular polyribonucleotide of any one of claims 1 - 3, wherein the polyribonucleotide cargo comprises one or more non-coding sequences.

5. The circular polyribonucleotide of any one of claims 1 -4, wherein the polyribonucleotide cargo comprises a non-coding sequence operably linked to the expression sequence encoding the OTC polypeptide6. The circular polyribonucleotide of any one of claim 4 or 5, wherein the one or more non-coding sequences comprise an internal ribosomal entry (IRES) sequence or spacer sequences.

7. The circular polyribonucleotide of claim 6, wherein the polyribonucleotide cargo comprises the IRES sequence operably linked to the expression sequence encoding the OTC polypeptide.8 The circular polyribonucleotide of claim 6, wherein the polyribonucleotide cargo further comprises the spacer sequence between the IRES and the 3’ exon fragment or the 5’ exon fragment.

9. The circular polyribonucleotide of claim 6, wherein the polyribonucleotide cargo further comprises the spacer sequence between the OTC expression sequence and the 3’ exon fragment.

10. The circular polyribonucleotide of claim 6, wherein the polyribonucleotide further comprises the spacer sequence adjacent to the 5’ exon or the 3’ exon fragment.

11. The circular polynucleotide of any one of claims 6 – 10, wherein the spacer sequence is at least about 50 ribonucleotides in length.

12. The circular polynucleotide of claim 11, wherein the spacer sequence is from about 50 to about 650 ribonucleotides in length.

13. The circular polynucleotide of any one of claims 6 – 12, wherein the spacer sequence comprises a polyA-T polyA-C, or polyA-U sequence.

14. The circular polyribonucleotide of any one of claims 1 – 13, wherein the circular polyribonucleotide is at least about 2,000 ribonucleotides in length.

15. The circular polyribonucleotide of claim 14, wherein the circular polyribonucleotide is from about 2,000 to about 3,000 ribonucleotides in length16. 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 ornithine transcarbamylase (OTC) polypeptide; (e) a 5’ exon fragment; (f) a 5’ splice site; and (g) a 5’ intron fragment.

17. The linear polyribonucleotide of claim 16, wherein the polyribonucleotide cargo comprises an expression sequence encoding one or more OTC polypeptides.

18. The linear polyribonucleotide of any one of claims 16 or 17, wherein the polyribonucleotide cargo comprises an IRES sequence operably linked to the expression sequence encoding the OTC polypeptide.

19. The linear polyribonucleotide of claim 18, wherein the linear polyribonucleotide further comprises one or more spacer sequences20. The linear polypeptide of claim 19, wherein the spacer sequence is at least 50 ribonucleotides in length.

21. The linear polypeptide of claim 19, wherein the spacer sequence is at least 50 to 650 ribonucleotides in length.

22. The linear ribonucleotide of any one of claims 19 - 21, wherein the spacer sequence comprises a polyA-T, polyA-C, or polyA-U sequence23. The linear polyribonucleotide of any one of claims 16 – 22, wherein the linear polyribonucleotide is at least about 2,200 ribonucleotides in length.

24. The linear polyribonucleotide of claim 23, wherein the linear polyribonucleotide is about 2,200 to 3,300 ribonucleotides in length.

25. A DNA vector encoding the linear polyribonucleotide of any one of claims 16 – 24.

26. A hepatocyte comprising the circular polyribonucleotide of claims 1 – 15.

27. A method of producing a circular polyribonucleotide from the linear polyribonucleotide of any one of claims 16 – 24, the method comprising providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to produce the circular polyribonucleotide.

28. A pharmaceutical composition comprising the circular polyribonucleotide of any one of claims 1 – 15, the linear polyribonucleotide of any one of claims 16 – 24, the DNA vector of claim 25, or the hepatocyte of claim 26, and a diluent, carrier, or excipient.

29. A method of treating OTC deficiency in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 28.

30. A method of treating a urea cycle disorder in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 29.

31. The method of claims 29 or 30, wherein the therapeutically effective amount of the pharmaceutical composition may be administered once every every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks32. The method of claims 29 or 30, wherein the therapeutically effective amount of the pharmaceutical composition may be administered once every month.

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