COMPOSITIONS COMPRISING CIRCULAR POLYRIBONUCLEOTIDES AND THEIR USES

Abstract

The present invention relates to a circular polyribonucleotide for use in therapy, wherein the circular polyribonucleotide lacks a poly-A sequence and comprises an expression sequence encoding a polypeptide, and wherein the circular polyribonucleotide is adapted to be administered to a subject and provide persistent in vivo expression of the polypeptide by translating the polypeptide from the expression sequence of the circular polyribonucleotide in vivo in a cell of the subject for a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days.

Description

This application claims the benefit of United States Provisional Application No. 262 / 599,547, filed December 15, 2017, and United States Provisional Application No. 262 / 676,688, filed May 25, 2018, each one of which is incorporated herein by reference in its entirety. BACKGROUND Certain circular polyribonucleotides are present ubiquitously in tissues and cells of human origin, including tissues and cells from healthy individuals. COMPENDIUM In one aspect, the invention includes a pharmaceutical composition comprising a circular polyribonucleotide comprising at least one structural element selected from a) an encryptogen; b) a stepping element; c) a regulatory element; d) a replication element; f) quasi-double-stranded secondary structure; and g) expression sequence; and at least one functional characteristic selected from: a) higher translation efficiency than a linear homologue; b) a stoichiometric translation efficiency of multiple translation products; c) less immunogenicity than a homologue lacking an encryptogen; d) increased half-life relative to a linear homologue; and e) persistence during cell division. In some embodiments, the circular polyribonucleotide is translation competent. In such an embodiment, the quasi-helical structure comprises at least one double-stranded RNA segment with at least one non-double-stranded segment. In another such embodiment, the quasi-helical structure comprises a first sequence and a second sequence linked to a repetitive sequence, for example, an A-rich sequence. In some embodiments, the circular polyribonucleotide comprises an encryptogen. In some embodiments, the encryptogen comprises at least one modified ribonucleotide, eg, pseudo-uridine, N(6)methyladenosine (m6A). In some embodiments, the encryptogen comprises a protein binding site, eg, ribonucleotide binding protein. In some embodiments, the encryptogen comprises an immunoprotein binding site, eg, to escape immune responses, eg, CTL responses. In some embodiments, the circular polyribonucleotide comprises at least one modified ribonucleotide. In some embodiments, the circular polyribonucleotide has at least 2x less immunogenicity than a homologue lacking the encryptogen, eg, as assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR -8, MDA5, LGP-2, OAS, OASL, PKR, IFN-beta. In some embodiments, the circular polyribonucleotide further comprises a riboswitch. In some embodiments, the circular polyribonucleotide further comprises an aptazime. In some embodiments, the circular polyribonucleotide comprises a translation start sequence, eg, GUG, CUG start codon, eg, expression under stress conditions. In some embodiments, the circular polyribonucleotide comprises at least one expression sequence, eg, encoding a polypeptide. In one such embodiment, the expression sequence encodes a peptide or polynucleotide. In some embodiments, the circular polyribonucleotide comprises a plurality of expression sequences, either the same or different. In some embodiments, the circular polyribonucleotide comprises a staggered element, eg, 2A. In some embodiments, the circular polyribonucleotide comprises a regulatory nucleic acid, eg, an antisense RNA. In some embodiments, the circular polyribonucleotide comprises a regulatory element, eg, that alters expression of an expression sequence. In some embodiments, the circular polyribonucleotide ranges in size from about 20 bases to about 20 kb. In some embodiments, the circular polyribonucleotide is synthesized by circularization of a linear polynucleotide. In some embodiments, the circular polyribonucleotide is substantially resistant to degradation, eg, by exonucleases. In some embodiments, the circular polyribonucleotide lacks at least one of: a) a 5'-UTR; b) a 3'-UTR; c) a poly-A sequence; d) a 5' cap; e) a terminating element; f) an internal ribosome entry site; g) susceptibility to degradation by exonucleases and h) binding to a cap-binding protein. In one aspect, the invention includes a method of producing the composition comprising a circular polyribonucleotide described herein. In one aspect, the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide comprising one or more expression sequences, wherein the circular polyribonucleotide is competent for rolling circle translation. ncionn / nznz / E / Y In some embodiments, each of the one or more expression sequences is separated from a subsequent expression sequence by means of a staggered element in the circular polyribonucleotide, wherein rolling circle translation of the one or more expression sequences generates at least two polypeptide molecules, eg, the staggering element disrupts or stops the ribosome, such that the elongating polypeptide is detached from the ribosome. In some embodiments, the staggering element prevents the 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. For example, the stagger element can prevent the generation of a single polypeptide from two or more rounds of translation of two or more expression sequences, for example, the stagger element arrests the ribosome and / or allows the elongating polypeptide to detach from the ribosome after a loop around the circular polyribonucleotide. In some embodiments, the staging element is a sequence separate from the one or more expression sequences. In some embodiments, the stagger element comprises an expression sequence portion of the one or more expression sequences. In one aspect, the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide comprising one or more expression sequences and is competent for rolling circle translation, wherein the circular polyribonucleotide is so configured that at least 10%, 20%, 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least At least 96%, at least 97%, at least 98%, at least 99%, or 100% of the total polypeptides (molar / molar) generated during rolling circle translation of the circular polyribonucleotide are isolated polypeptides and in wherein each of the isolated polypeptides is generated from a single round of translation or less than a single round of translation of the one or more expression sequences. In some embodiments, the circular polyribonucleotide is configured such that at least 10%, 20%, 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, at less than 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the total polypeptides (molar / molar) generated during the rolling circle translation of the circular polyribonucleotide are isolated polypeptides and wherein the ratio of isolated products to total polypeptides is assessed in an in vitro translation system. In some embodiments, the in vitro translation system comprises rabbit reticulocyte lysate. ncionn / nznz / E / Y In some embodiments, the stagger element is located downstream of or 3' to at least one or more expression sequences, wherein the stagger element is configured to disrupt a ribosome during rolling circle translation of the circular polyribonucleotide. In one aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide comprising one or more expression sequences and a staggered element downstream of or 3' to at least one of the one or more expression sequences. or more expression sequences. In some embodiments, the staggering element is configured to disrupt a ribosome during rolling circle translation of the circular polyribonucleotide. In some embodiments, the staggering element encodes a peptide sequence selected from the group consisting of a 2A sequence and a 2A-like sequence. In some embodiments, the stagger element encodes a sequence with a C-terminal sequence that is GP. In some embodiments, the staggered element encodes a sequence with a C-terminal consensus sequence that is D(V / l)ExNPG P, where x= any amino acid. In some embodiments, the stagger element encodes at least one of GDVESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, GDVEQNPGP, IESNPGP, GDIELNPGP, HDIETNPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEEFGPNP . In some embodiments, the stagger element is located downstream of or 3' to each of the one or more expression sequences. In some embodiments, the first expression sequence stagger element in the circular polyribonucleotide is located upstream of (5' to) a first expression sequence translation initiation sequence following the first expression sequence. expression in the circular polyribonucleotide and wherein a distance between the stagger element and the first translation initiation sequence enables continuous translation of the first expression sequence and the expression sequence that follows. In some embodiments, the stagger element comprises a termination element of a first expression sequence in the circular polyribonucleotide having a distance upstream of (5' to) a translation initiation sequence of an expression sequence a continuation of the first expression sequence in the circular polyribonucleotide and wherein the distance allows for continued translation of the first expression sequence and its subsequent expression sequence. ncionn / nznz / E / Y In some embodiments, a first stagger element is located upstream of (5' to) a first translation initiation sequence of a first expression sequence in the circular polyribonucleotide, wherein the circular polyribonucleotide is continuously translated. , wherein a corresponding circular polyribonucleotide comprises a second staggering element upstream of a second translation initiation sequence of a second expression sequence in the corresponding circular polyribonucleotide that is not continuously translated and wherein the second staggering element in the corresponding circular polyribonucleotide is located at a greater distance from the second translation initiation sequence, eg, at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, than a distance between the first staggered element and the first translation initiation in the circular polyribonucleotide. In some embodiments, the staggering element comprises a first termination element upstream of (5' to) a first translation initiation sequence of a first expression sequence on the circular polyribonucleotide, wherein the circular polyribonucleotide is translated. continuously and a corresponding circular polyribonucleotide comprises a staggering element comprising a second termination element upstream of a second expression sequence in the corresponding circular polyribonucleotide that is not continuously translated and where the second termination element in the polyribonucleotide corresponding circular is at a greater distance from the second translation start sequence, eg at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, than a distance between the first termination element and the first translation initiation on the circular polyribonucleotide. In some embodiments, the distance between the first stagger element and the first translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt , 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or more. In some embodiments, the distance between the second stagger element and the second translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt , 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or more than the distance between the first stagger element and the first translation start. In some embodiments, the circular polyribonucleotide comprises more than one expression sequence. In some embodiments, the circular polyribonucleotide has a translational efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 2 times, at least 3 times times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times larger than a linear counterpart. In some embodiments, the circular polyribonucleotide has a translation efficiency of at least 5-fold greater than a linear counterpart. In some embodiments, the circular polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the one or more expression sequences comprise a Kozak initiation sequence. In some embodiments, the one or more expression sequences encode a peptide. In some embodiments, the circular polyribonucleotide comprises a regulatory nucleic acid, eg, an antisense RNA. In some embodiments, the circular polyribonucleotide comprises a regulatory element, eg, that alters expression of an expression sequence. In one aspect, the invention provides a circular polyribonucleotide of any of the pharmaceutical compositions provided herein. In one aspect, the invention includes a method of producing the pharmaceutical composition provided herein, comprising combining the circular polyribonucleotide described herein and the pharmaceutically acceptable carrier or excipient described herein. In one aspect, the invention includes a method of administering the composition comprising a circular polyribonucleotide described herein. In one aspect, the invention includes a method for protein expression, comprising translating at least one region of the circular polyribonucleotide provided herein. In some embodiments, translation of at least one region of the circular polyribonucleotide occurs in vitro. In some embodiments, translation of the at least one region of the circular polyribonucleotide occurs in vivo. In one aspect, the invention includes a polynucleotide, eg, a DNA vector, that encodes the circular polyribonucleotide provided herein. In one aspect, the invention includes a method of producing the circular polyribonucleotide as provided herein. In some embodiments, the method comprises bridging-mediated circularization of a linear polyribonucleotide. In some embodiments, circularization, eg, bridging-mediated circularization, has an efficiency of at least 2%, at least 5%, at least ncionn / nznz / E / Y 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least one 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least one 42%, at least 44%, at least 46%, at least 48%, or at least 50%. In some embodiments, bridging-mediated circularization has an efficiency of about 40% to about 50% or greater than 50%. Definitions The present invention will be described with respect to particular embodiments and with reference to certain figures, but the invention is not limited thereto, but only by the claims. The terms set forth below in this document are to be understood in their common sense, unless otherwise indicated. The expressions obtainable by, producible by or the like are used to indicate that a claim or embodiment refers to a compound, composition, product, etc. itself, that is, that the compound, composition, product, etc. can be obtained or produced by a method that is described for the production of the compound, composition, product, etc., but that the compound, composition, product, etc. it can also be obtained or produced by methods other than the one described. The terms obtained by, produced by, or the like indicate that the compound, composition, or product is obtained or produced by a specific mentioned method. It is to be understood that the terms obtainable by, producible by and the like also disclose the terms obtained by, produced by and the like as a preferred embodiment of obtainable by, producible by and the like. It is to be understood that the expressions compound, composition, product, etc. to treat, modulate, etc. refer to a compound, a composition, a product, etc. per se, which are suitable for the stated purposes of processing, modulation, etc. The expressions compound, composition, product, etc. to treat, modulate, etc. further discloses that, as a preferred embodiment, said compound, composition, product, etc. it is for use in treatment, modulation, etc. The phrase compound, composition, product, etc. for use in... or use of a compound, composition, product, etc. in the manufacture of a medicine, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for...indicates that said compounds, compositions, products, etc. they are to be used in therapeutic methods that can be practiced in the human or animal body. They are considered as an equivalent disclosure of embodiments and claims pertaining to methods of treatment, etc. Therefore, if an embodiment or claim refers to a compound for use in the treatment of a human or animal suspected of having a disease, this should also be considered a disclosure of a use of a compound in the manufacture ncionn / nznz / E / Y of a medicament for treating a human or animal suspected of suffering from a disease or a method of treatment by administering a compound to a human or animal suspected of suffering from a disease. It is to be understood that the phrase compound, composition, product, etc. to treat, modulate, etc. refers to a compound, composition, product, etc. itself that it is suitable for the stated purposes of processing, modulation, etc. The expression "pharmaceutical composition" is also intended to disclose that the circular polyribonucleotide comprised in a pharmaceutical composition can be used for the treatment of the human or animal body by therapy. Therefore, a circular polyribonucleotide for use in therapy is intended to be equivalent to the expression. Circular polyribonucleotides, compositions comprising said circular polyribonucleotides, methods of using said circular polyribonucleotides, etc. as described herein are based, in part, on examples that illustrate how circular polyribonucleotide effectors can be used that comprise different elements, for example, a replication element, an expression sequence, a staging element, and a encryptogen (see, for example, Example 1) or, for example, an expression sequence, a staging element, and a regulatory element (see, for example, Examples 30 and 38), to achieve different technical effects (for example, increased efficiency of translation relative to a linear homologue in Examples 1 and 38 and increased half-life relative to a linear homologue in Example 38). It is based on, among others, these examples that the description hereinafter contemplates various variations of the specific findings and combinations considered in the examples. As used herein, the term circRNA or the terms circular polyribonucleotide or circular RNA are used interchangeably and can refer to a polyribonucleotide that forms a circular structure through covalent or non-covalent bonding. As used herein, the term "encryptogen" can refer to a circular polyribonucleotide nucleic acid sequence or structure that helps reduce, evade, and / or avoid detection by an immune cell and / or reduces the induction of a response. immunity against circular polyribonucleotide. As used herein, the term "expression sequence" can refer to a nucleic acid sequence that encodes a product, eg, a peptide or polypeptide or a regulatory nucleic acid. An exemplary expression sequence encoding a peptide or polypeptide may comprise a plurality of nucleotide triads, each of which may encode an amino acid and is known as a codon. ncionn / nznz / Ε / γ As used herein, the term "immunoprotein binding site" can refer to a nucleotide sequence that binds to an immunoprotein. In some embodiments, the immunoprotein-binding site helps to mask that the circular polyribonucleotide is exogenous, for example, the immunoprotein-binding site may bind to a protein (eg, a competitive inhibitor) that prevents the circular polyribonucleotide from being recognized. by and binds to an immunoprotein, thereby reducing or preventing an immune response against the circular polyribonucleotide. As used herein, the term "immunoprotein" can refer to any protein or peptide that is associated with an immune response, eg, such as against an immunogen, eg, circular polyribonucleotide. Non-limiting examples of immunoproteins include T cell receptors (TCRs), antibodies (immunoglobulins), major histocompatibility complex (MHC) proteins, complement proteins, and RNA binding proteins. As used herein, the term "modified ribonucleotide" can refer to a nucleotide with at least one sugar, nucleobase, or internucleoside bond modification. As used herein, the phrase "quasi-helical structure" can refer to a higher order structure of the circular polyribonucleotide, wherein at least a portion of the circular polyribonucleotide folds into a helical structure. As used herein, the phrase quasi-double-stranded secondary structure can refer to a higher order structure of the circular polyribonucleotide, wherein at least a portion of the circular polyribonucleotide creates an internal double helix. As used herein, the term "regulatory element" can refer to a moiety, such as a nucleic acid sequence, that modifies the expression of an expression sequence in the circular polyribonucleotide. As used herein, the term "repetitive nucleotide sequence" can refer to a repetitive nucleic acid sequence in a stretch of DNA or RNA or throughout a genome. In some embodiments, the repetitive nucleotide sequence includes poly CA or poly TG (UG) sequences. In some embodiments, the repetitive nucleotide sequence includes repeat sequences from the Alu family of introns. As used herein, the term "replication element" can refer to a sequence and / or motif useful for replication or that initiates transcription of the circular polyribonucleotide. As used herein, the term "staggering element" can refer to a nucleotide sequence that induces ribosomal pausing during translation. In some embodiments, the staggering element is a conserved non-ncionn / nznz / E / Y amino acid sequence with a strong alpha-helical bias, followed by the consensus sequence D(V / l)ExNPG P, where x = any amino acid. In some embodiments, the staging element can include a chemical moiety, such as glycerol, a non-nucleic acid linker moiety, a chemical modification, a modified nucleic acid, or any combination thereof. As used herein, the term "substantially resistant" can refer to one that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , 96%, 97%, 98% or 99% resistance compared to a reference. As used herein, the term "stoichiometric translation" can refer to a substantially equivalent production of translated expression products from the circular polyribonucleotide. For example, for a circular polyribonucleotide that has two expression sequences, stoichiometric translation of the circular polyribonucleotide can mean that the expression products of the two expression sequences can have substantially equivalent amounts, for example, the difference in amount between the two sequences. expression (eg, molar difference) can be about 0 or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%. As used herein, the term "translation initiation sequence" can refer to a nucleic acid sequence that initiates translation of an expression sequence on the circular polyribonucleotide. As used herein, the term "termination element" can refer to a moiety, such as a nucleic acid sequence, that terminates the translation of the expression sequence into the circular polyribonucleotide. As used herein, the term "translational efficiency" can refer to 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 by a given amount of transcript encoding the protein or peptide, eg, in a given period of time, eg in a translation system. given, for example, an in vitro translation system, such as a rabbit reticulocyte lysate or an in vivo translation system, such as a eukaryotic cell or a prokaryotic cell. As used herein, the term "circularization efficiency" can refer to a measure of the resulting circular polyribonucleotide against its starting material. As used herein, the term "immunogenic" can refer to a potential to induce an immune response against a substance. In some ncionn / nznz / E / Y embodiments, an immune response may be induced when an organism's immune system or a certain type of immune cells is exposed to an immunogenic substance. The term "non-immunogenic" can refer to the lack or absence of an immune response above a detectable threshold for a substance. In some embodiments, an immune response is not detected when an organism's immune system or a given type of immune cells is exposed to a non-immunogenic substance. In some embodiments, a non-immunogenic circular polyribonucleotide provided herein does not induce an immune response above a predetermined threshold when measured by an immunogenicity assay. For example, when an immunogenicity assay is used to measure antibodies generated against a circular polyribonucleotide or inflammatory markers, a non-immunogenic circular polyribonucleotide provided herein may result in the production of antibodies or markers at a level less than a threshold. predetermined. The predetermined threshold can be, for example, at most 1.5 times, 2 times, 3 times, 4 times or 5 times the level of antibodies or markers generated by a control reference. INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, embodiments, which are illustrated herein, are shown in the drawings. However, it is to be understood that the invention is not limited to the precise arrangement and instrumentation of the embodiments shown in the drawings. Figure 1 shows different exemplary circularization methods. Figure 2 shows a schematic of an exemplary in vitro production process of a circular RNA containing a start codon, an ORF (open reading frame) encoding GFP, a staggered element (2A), an encryptogen, and an IRES. (internal ribosome entry site). Figure 3 shows a schematic of an exemplary in vivo production process for circular RNA. Figure 4 shows the design of an exemplary circular RNA comprising a start codon, an ORF encoding GFP, a staggered element (2A) and an encryptogen. nc i οηη / ηζηζ / Ε / γ Figures 5A and 5B are schematics demonstrating in vivo stoichiometric protein expression of two different circular RNAs. Figure 6 shows a schematic of a control circular RNA that has an intron and expresses GFP. Figure 7 shows a schematic of an exemplary circular RNA having a synthetic riboswitch (in red) that regulates GFP expression from the circular RNA in the presence or absence of ligands for the riboswitch. Figure 8 is a schematic demonstrating in vivo protein expression in a mouse model from the exemplary circular RNAs. Figure 9 is a schematic demonstrating the in vivo biodistribution of an exemplary circular RNA in a mouse model. Figure 10 is a schematic demonstrating in vivo protein expression in a mouse model from an exemplary circular RNA carrying an encryptogen (intron). Figure 11 shows a schematic of an exemplary circular RNA having a double-stranded RNA segment that can be subjected to dot blot analysis to determine its structural information. Figure 12 shows a schematic of an exemplary circular RNA having a quasi-helical structure (HDVmin), which can be subjected to SHAPE analysis to determine its structural information. Figure 13 shows a schematic of an exemplary circular RNA having a functional quasi-helical (HDVmin) structure demonstrating HDAg binding activity. Figure 14 is a schematic demonstrating the transcription, self-cleavage, and ligation of an exemplary self-replicating circular RNA. Figure 15 shows a schematic of an exemplary circular RNA that is expressed in vivo and has improved stability in vivo. Figure 16 shows a schematic of an exemplary circular RNA that is conserved through mitosis and persists in daughter cells. A BrdU pulse is used to label the divided cells. Figure 17 is a denaturing PAGE gel image demonstrating the in vitro production of different exemplary circular RNAs. Figure 18 is a graph summarizing the circularization efficiencies of different exemplary circular RNAs. Figure 19 is a denaturing PAGE gel image demonstrating the reduced susceptibility to degradation of an exemplary circular RNA compared to its linear counterpart. ncionn / nznz / E / Y Figure 20 is a denaturing PAGE gel image demonstrating exemplary circular RNA after an exemplary purification process. Figure 21 is a Western blot image demonstrating the expression of the Flag protein (~15 kDa) by an exemplary circular RNA lacking IRES, cap, and 5' and 3' UTR. Figure 22 is a Western blot image demonstrating rolling circle translation of an exemplary circular RNA. Figure 23 shows Western blot images demonstrating the production of isolated proteins or long continuous peptides from different exemplary circular RNAs with or without an exemplary stagger element. Figure 24A is a Western blot image showing the comparison of protein expression between different exemplary circular RNAs with a stagger element or a termination element (stop codon). Figure 24B is a graph summarizing the signal intensity of Western blot analysis of protein products translated from the two exemplary circular RNAs. Figure 25 is a graph summarizing the luciferase activity of the translation products of an exemplary circular RNA and its linear counterpart, compared to a vehicle control RNA. Figure 26 is a graph summarizing the amounts of RNA at different collection time points in a time course experiment evaluating the half-life of an exemplary circular RNA. Figure 27A is a graph showing qRT-PCR (quantitative reverse transcriptase polymerase chain reaction) analysis of linear and circular RNA levels 24 hours after delivery to cells using primers that captured both linear and circular RNA. . Figure 27B is a graph showing qRT-PCR analysis of linear and circular RNA levels using a primer specific for circular RNA. Figure 28 is an image showing a blot of Cellulars from circular RNA and linear RNA probed for EGF protein and a beta-tubulin loading control. Figure 29 is a graph showing qRT-PCR analysis of immunity-related genes from 293T cells transfected with circular RNA or linear RNA. Figure 30 is a graph showing luciferase activity of protein expressed from circular RNA by rolling circle translation. Figure 31 is a graph showing the luciferase activity of protein expressed from circular RNA or linear RNA. ncionn / nznz / E / Y Figure 32 is a graph showing luciferase activity of protein expressed from linear RNA or circular RNA by rolling circle translation. Figure 33 is a graph showing luciferase activity of protein expressed from circular RNA by IRES translation initiation. Figure 34 is a graph showing luciferase activity of protein expressed from circular RNA by IRES initiation and rolling circle translation. Figure 35 is an image showing a protein blot of expression products from circular RNA or linear RNA. Figure 36 is an image showing a protein blot of expression products from circular RNA or linear RNA. Figure 37 shows the predicted structure with a quasi-double-stranded structure of an exemplary circular RNA. Figure 38 shows the predicted structure with a quasi-helical structure of an exemplary circular RNA. Figure 39 shows the predicted structure with a quasi-helical structure ligated with a repetitive sequence of an exemplary circular RNA. Figure 40 demonstrates experimental data that RNase H degradation of an exemplary circular RNA produced nucleic acid degradation products consistent with a circular RNA and not a concatameric one. Figure 41 shows an electrophoresis image of the DNA length differences that were generated for the creation of a wide variety of RNA lengths. Figure 42 shows experimental data confirming RNA circularization using RNase H treatment and qPCR analysis against circular junctions of a wide variety of lengths. Figure 43 shows the generation of exemplary circular RNAs with a miRNA binding site. Figure 44 shows the generation of exemplary circular RNA by self-splicing. Figure 45 shows the generation of exemplary circular RNAs with a protein binding site. Figure 46 shows experimental data demonstrating the greater stability of circular RNA in a dividing cell, compared to linear controls. Figure 47 shows experimental data demonstrating protein expression from exemplary circular RNAs with a plurality of expression sequences and rolling circle translation of exemplary circular RNAs with multiple expression sequences. ncionn / nznz / E / Y Figure 48 shows experimental data demonstrating the reduced toxicity to transfected cells of an exemplary circular RNA compared to the linear control. Figure 49 shows that exemplary circular RNA was translated at a higher level compared to linear RNA under stress conditions. Figure 50 shows the generation of circular RNAs with a riboswitch. Figures 51A, 51B and 51C show that the modified circular RNAs were translated in cells. Figures 52A-52C show that the modified circular RNAs had reduced immunogenicity compared to unmodified circular RNAs for cells, as assessed by the expression of MDA5, OAS and IFN-beta in the transfected cells. Figure 53 shows that after injection into mice, circular RNA was detected at higher levels than linear RNA in mouse livers at 3, 4, and 7 days post-injection. Figures 54A and 54B show that after injection of circular RNA or linear RNA expressing Gaussian luciferase into mice, Gaussian luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23 days after injection. the administration of the circular RNAs, while their activity was only detected in plasma at 1 and 2 days after the administration of the modified linear RNA. Figure 55 shows that after RNA injection, circular RNA but not linear RNA was detected in liver and spleen at 16 days after RNA administration. Figure 56 shows that after RNA injection, linear RNA but not circular RNA showed immunogenicity as assessed by RIG-I, MDA-5, IFN-B and OAS. DETAILED DESCRIPTION The present invention relates generally to pharmaceutical compositions and preparations of circular polyribonucleotides and uses thereof. circular polyribonucleotides In some aspects, the invention described herein encompasses compositions and methods for using and producing circular polyribonucleotides and circular polyribonucleotide delivery. In some embodiments, the circular polyribonucleotide is non-immunogenic in a mammal, eg, a human. In some embodiments, the circular polyribonucleotide is capable of or replicates in an aquaculture animal cell (fish, crab, shrimp, oyster, etc.), a mammalian cell, eg, a companion animal cell. or zoo (cats, dogs, lizards, birds, lions, tigers and bears, etc.), a cell of a farm or work animal (horses, cows, pigs, chickens, etc.), a human cell, cells cultured, primary cells or cell lines, stem cells, ncionn / nznz / E / Y progenitor cells, differentiated cells, germ cells, cancer cells (eg, tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, cells embryonic, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, the invention includes a cell comprising the circular polyribonucleotide described herein, wherein the cell is a cell from an aquaculture animal (fish, crab, shrimp, oyster, etc.), a mammalian cell, for example, a cell of a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears, etc.), a cell of a farm or work 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 (for example, tumorigenic, metastatic), a non-tumorigenic cell (normal cells), a fetal cell, an embryonic cell, an adult cell, a mitotic cell, a non-mitotic cell, or any combination thereof. In some embodiments, the cell is modified to comprise the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide includes expression sequences or products. In some embodiments, the circular polyribonucleotide has a half-life of at least that of a linear homologue, eg, linear expression sequence or linear circular polyribonucleotide. In some embodiments, the circular polyribonucleotide has a half-life that is increased relative to 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 of at least about 1 hr to about 30 days or at least about 2 hr, 6 hr, 12 hr, 18 hr, 24 hr, 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, 60 days or more, or any time in between. In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a cell of no more than about 10 min to about 7 days or no more than about 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 8 am, 9 am, 10 am, 11 am, 12 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 midnight, 36h, 48h, 60h, 72h, 4 days, 5 days, 6 days, 7 days or any time in between. 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 after division. In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a dividing cell of greater than about 10 minutes to about 30 days or at least about 1 ncionn / nznz / E / Y hr, 2 hr, 3 hr, 4 hr, 5am, 6am, 7am, 8am, 9am, 10am, 11am, 12pm, 1pm, 2pm, 3pm, 4pm, 5pm, 6pm, 12pm, 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, 60 days or more, or any time in between. In some embodiments, the circular polyribonucleotide modulates a cellular function, eg, transiently or long-term. In certain embodiments, cell function is stably altered, such as a modulation that persists for at least about 1 hour to about 30 days or at least about 2h, 6h, 12h, 18h, 24h, 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, 60 days or more, or any time in between. In certain embodiments, cellular function is transiently altered, for example, such as a modulation that persists for no longer than about 30 min to about 7 days, or no longer than about 1 hr, 2 hr, 3 hr, 4 hr. , 5 am, 6 am, 7 am, 8 am, 9 am, 10 am, 11 am, 12 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm 22h, 24h, 36h, 48h, 60h, 72h, 4 days, 5 days, 6 days, 7 days or any time in between. In some embodiments, the circular polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 n nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments, the circular polyribonucleotide may be of a size sufficient to accommodate a binding site for a ribosome. One of skill in the art can appreciate that the maximum size of a circular polyribonucleotide can be as large as is within the technical constraints of the production of a circular polyribonucleotide and / or the use of the circular polyribonucleotide. Without being bound by theory, it is possible that multiple RNA segments can be produced from DNA and nc i οηη / ηζηζ / Ε / γίΛΐ anneal their free 5' and 3' ends to produce an RNA strand, which ultimately instance can be circularized, only one 5' end and one 3' free end remains. In some embodiments, the maximum size of a circular polyribonucleotide may be limited by the ability to package and deliver the RNA to a target. In some embodiments, the size of a circular polyribonucleotide is a length sufficient to encode useful polypeptides and thus, lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides may be useful. nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides. In some embodiments, the circular polyribonucleotide comprises one or more elements described elsewhere herein. In some embodiments, the elements may be separated from one another by means of a spacer or linker sequence. In some embodiments, the elements may be separated from one another by 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, approximately 100 nucleotides, approximately 150 nucleotides, approximately 200 nucleotides, approximately 250 nucleotides, approximately 300 nucleotides, approximately 400 nucleotides, approximately 500 nucleotides, approximately 600 nucleotides, approximately 700 nucleotides, approximately 800 nucleotides, approximately 900 nucleotides, approximately 100 nucleotides , up to about 1 kb, at least about 1000 nucleotides, or any number of nucleotides in between. In some embodiments, one or more elements are adjacent to one another, eg, they lack a spacer element. In some embodiments, one or more elements in the circular polyribonucleotide are conformationally flexible. In some embodiments, the conformational flexibility is due to the sequence being substantially free of secondary structure. In some embodiments, the circular polyribonucleotide comprises a secondary or tertiary structure that accommodates one or more desired functions or features described herein, eg, accommodates a binding site for a ribosome, eg, translation, eg, translation into rolling circle. In some embodiments, the circular polyribonucleotide comprises particular sequence features. For example, the circular polyribonucleotide may comprise a particular nucleotide composition. In some such embodiments, the circular ncionn / nznz / E / Y polyribonucleotide may include one or more purine (adenosine or guanosine) rich regions. In some such embodiments, the circular polyribonucleotide may include one or more purine (adenosine or guanosine) rich regions. In some embodiments, the circular polyribonucleotide can include one or more AU-rich regions or elements (AREs). In some embodiments, the circular polyribonucleotide can include one or more adenine-rich regions. In some embodiments, the circular polyribonucleotide can include one or more repeat elements described elsewhere herein. In some embodiments, the circular polyribonucleotide comprises one or more modifications described elsewhere herein. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences and is configured for persistent expression in a cell of a subject in vivo. In some embodiments, the circular polyribonucleotide is configured such that the expression of the one or more expression sequences in the cell at a later time point is equal to or greater than at an earlier time point. In such embodiments, the expression of the one or more expression sequences can either be maintained at a relatively stable level or increase over time. The expression of the expression sequences can be relatively stable over an extended period of time. For example, in some cases, the expression of the one or more expression sequences in the cell over at least 7, 8, 9,10,12,14,16,18, 20, 22, 23 or more days it is not reduced by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some cases, the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% for at least 7, 8, 9, 10, 12, 14, 16,18, 20, 22, 23 or more days. expression sequences Peptides or polypeptides In some embodiments, the circular polyribonucleotide comprises at least one expression sequence that encodes a peptide or polypeptide. Said peptide can include, but is not limited to, a small peptide, peptidomimetic (eg, peptoid), amino acids, and amino acid analogues. The peptide can be linear or branched. Said peptide may have a molecular weight of less than about 5,000 grams per mole, a molecular weight of less than about 2,000 grams per mole, a molecular weight of less than about 1,000 grams per mole, a molecular weight of less than about 500 grams per mole, and salts, esters and other pharmaceutically acceptable forms of said compounds. Said peptide may include, but is not limited to, a neurotransmitter, a hormone, a drug, a toxin, ncionn / nznz / E / Y, a viral or microbial particle, a synthetic molecule, and agonists or antagonists thereof. The polypeptide can be linear or branched. The polypeptide may be from about 5 to about 40,000 amino acids in length, from about 15 to about 35,000 amino acids, from about 20 to about 30,000 amino acids, from about 25 to about 25,000 amino acids, from about 50 to about 20,000 amino acids, from about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1,000 to about 2,500 amino acids, or any range in between. In some embodiments, it may be useful for the polypeptide to be less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or less. Such examples of a peptide or polypeptide include, but are not limited to, a fluorescent label or marker, an antigen, a therapeutic peptide, a synthetic peptide or a peptide analog of a naturally bioactive peptide, an agonist or antagonist peptide, an antimicrobial peptide, a pore-forming peptide, a bicyclic peptide, a targeting or cytotoxic peptide, a degrading or self-destructing peptide, and degrading or self-destructing peptides. Peptides useful in the invention described herein also include antigen-binding peptides, eg, antigen-binding antibody or antibody-like fragments, such as single-chain antibodies, nanobodies (see, eg, Steeland et al. 2016 Nanobodies as therapeutics: big opportunities for small antibodies Drug Discov Today: 21(7):1076-113). Said ncionn / nznz / E / Y antigen-binding peptides may bind to a cytosolic antigen, a nuclear antigen or an intraorganular antigen. In some embodiments, the circular polyribonucleotide comprises one or more RNA expression sequences, each of which may encode a polypeptide. The polypeptide can be produced in substantial amounts. As such, the polypeptide can be any protein molecule that can be produced. A polypeptide can be a polypeptide that can be secreted by a cell or located in the cytoplasmic, nuclear or membrane compartments of a cell. Some polypeptides include, but are not limited to, at least a portion of a viral envelope protein, metabolic regulatory enzymes (eg, that regulate lipid or steroid production), an antigen, a tolerogen, a cytokine, a toxin, enzymes whose absence is associated with disease; and polypeptides that are not active in an animal until cleaved (eg, in the intestine of an animal); and a hormone. In some embodiments, the circular polyribonucleotide includes an expression sequence that encodes a protein, eg, a therapeutic protein. In some embodiments, therapeutic proteins that can be expressed from the circular polyribonucleotide disclosed herein have antioxidant activity, binding, cargo receptor activity, catalytic activity, molecular carrier activity, molecular function regulator, transducer activity molecular, nutrient depot activity, protein marker, structural molecule activity, toxin activity, translational regulator activity, or transporter activity. Some examples of therapeutic proteins may include, but are not limited to, an enzyme replacement protein, a protein for vaccination, antigens (eg, tumor, viral, or bacterial antigens), hormones, cytokines, antibodies, immunotherapy (eg, cancer), cell transdifferentiation / reprogramming factor, transcription factors, chimeric antigen receptor, transposase or nuclease, immune effector (eg, influences susceptibility to an immune response / signal), a death-regulated effector protein (eg, an inducer of apoptosis or necrosis), a non-lytic inhibitor of a tumor (for example, an inhibitor of an oncoprotein), an epigenetic modifying agent, an epigenetic enzyme, a transcription factor, a DNA or protein modifying enzyme, a DNA intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a com competitive for an enzyme, an effector or inhibitor of protein synthesis, a nuclease, a protein fragment or domain, a ligand or a receptor, and a CRISPR system or component thereof. In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include human proteins, ncionn / nznz / Ε / γ for example, a receptor binding protein, a hormone, a growth factor, a growth factor receptor modulator and a regenerative protein (eg, proteins involved in proliferation and differentiation, eg, a therapeutic protein, for wound healing). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include EGF (epithelial growth factor). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include enzymes, for example, oxidoreductase enzymes, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, and desaturases. In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include an intracellular protein or a cytosolic protein. In some embodiments, the circular polyribonucleotide expresses a NanoLuc® luciferase (nLuc). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide disclosed herein include a secretory protein, eg, a secretory enzyme. In some cases, the circular polyribonucleotide expresses a secretory protein which may have a short therapeutic half-life in blood or may be a subcellular localization signal protein or secretory signal peptide protein. In some embodiments, the circular polyribonucleotide expresses a Gaussian luciferase (gLuc). In some cases, the circular polyribonucleotide expresses a non-human protein, for example, a fluorescent protein, an energy transfer acceptor, or a protein tag, such as Flag, Myc, or His. In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide include a GFP. In some embodiments, the circular polyribonucleotide expresses tagged proteins, eg, fusion proteins or engineered proteins that contain a protein tag, eg, chitin-binding protein (CBP), maltose-binding protein (MBP), Fe tag, glutathione-S-transferase (GST), AviTag (GLNDIFEAQKIEWHE), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL); polyglutamate tag (EEEEEE); label E (GAPVPYPDPLEPR); FLAG tag (DYKDDDDK), HA tag (YPYDVPDYA); His tag (HHHHHH); Myc tag (EQKLISEEDL); tag NE(TKENPRSNQEESYDDNES); label S (KETAAAKFERQHMDS); SBP tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP); Softag 1 (SLAELLNAGLGGS); Softag 3 (TQDPSRVG); Spot tag (PDRVRAVSHWSS); Strep tag (Strep II tag: WSHPQFEK); TC label (CCPGCC); Ty tag (EVHTNQDPLD); V5 tag (GKPIPNPLLGLDST); VSV tag (YTDIEMNRLGK); or Xpress tag (DLYDDDDK). ncionn / nznz / E / Y In some embodiments, the circular polyribonucleotide expresses an antibody, eg, an antibody fragment or portion thereof. In some embodiments, the antibody expressed by the circular polyribonucleotide can be of any isotype, such as IgA, IgD, IgE, IgG, IgM. In some embodiments, the circular polyribonucleotide expresses a portion of an antibody, such as a light chain, heavy chain, Fe fragment, CDR (complementarity determining region), Fv fragment, or Fab fragment, or an additional portion of the antibody. same. In some embodiments, the circular polyribonucleotide expresses one or more portions of an antibody. For example, the circular polyribonucleotide may comprise more than one expression sequence, each expressing a portion of an antibody, and the sum of which may constitute the antibody. In some cases, the circular polyribonucleotide comprises an expression sequence encoding the heavy chain of an antibody and another expression sequence encoding the light chain of the antibody. In some instances, when the circular polyribonucleotide is expressed in a cell or cell-free environment, the light chain and heavy chain may undergo suitable post-translational modifications, folding, or other modifications to form a functional antibody. regulatory elements In some embodiments, the circular polyribonucleotide comprises a regulatory element, eg, a sequence that modifies the expression of an expression sequence on the circular polyribonucleotide. A regulatory element can include a sequence that is located adjacent to an expression sequence that encodes an expression product. A regulatory element can be operatively linked to the adjacent sequence. A regulatory element can increase an expressed product amount compared to an expressed product amount when there is no regulatory element. In addition, a regulatory element can increase an amount of expressed products for multiple expression sequences linked in tandem. Thus, a regulatory element can enhance the expression of one or more expression sequences. Multiple regulatory elements are well known to those of ordinary skill in the art. A regulatory element as provided herein can include a selective translation sequence. As used herein, the term "selective translation sequence" can refer to a nucleic acid sequence that selectively initiates or activates translation of an expression sequence on the circular polyribonucleotide, eg, certain riboswitch aptazymes. A regulatory element can also include a selective degradation sequence. As used herein, the term "selective degradation sequence" can refer to a nucleic acid sequence that initiates degradation of the circular ncionn / nznz / E / Y polyribonucleotide or an expression product of the circular polyribonucleotide. Exemplary selective degradation sequence may include riboswitch aptazymes and miRNA binding sites. In some embodiments, the regulatory element is a modulator of translation. A translation modulator can modulate the translation of the expression sequence on the circular polyribonucleotide. A translation modulator can be a translation enhancer or suppressor. In some embodiments, the circular polyribonucleotide includes at least one translation modulator adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide includes a translation modulator adjacent to each expression sequence. In some embodiments, the translation modulator is present on one or both sides of each expression sequence, resulting in separation of expression products, eg, one or more peptides and / or one or more polypeptides. In some embodiments, a translation initiation sequence can function as a regulatory element. In some embodiments, a translation initiation sequence comprises an AUG codon. In some embodiments, a translation initiation sequence comprises any eukaryotic start codon, such as AUG, CUG, GUG, UUG, ACG, AUG, AUU, AAG, AUA, or AGG. In some embodiments, a translation initiation sequence comprises a Kozak sequence. In some embodiments, translation begins at an alternative translation initiation sequence, eg, a translation initiation sequence other than an AUG codon, under selective conditions, eg, stress-induced conditions. As a non-limiting example, translation of the circular polyribonucleotide can begin at an alternative translation start sequence, such as ACG. As another non-limiting example, translation of the circular polyribonucleotide can begin at an alternative translation start sequence, CTG / CUG. As another non-limiting example, translation of the circular polyribonucleotide may begin at an alternative translation start sequence, GTG / GUG. As yet another non-limiting example, the circular polyribonucleotide may begin translation at a non-AUG repeat-associated (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA, eg, CGG, GGGGCC, CAG, CTG. Nucleotides flanking a translation initiation codon, such as, but not limited to, a start codon or alternative start codon, are known to affect the translation efficiency, length, and / or structure of the translation. circular polyribonucleotide. (See, for example, Matsuda and Mauro, PLoS ONE, 2010 5:11; the contents of which are incorporated herein by reference in their entirety.) Masking of any of the nucleotides flanking a translation initiation codon can be used to alter the translation start position ncionn / nznz / E / Y, translation efficiency, length, and / or structure of the polyribonucleotide. circular. In one embodiment, a masking agent near the start codon or alternative start codon can be used to mask or mask the codon to reduce the likelihood of translation initiation at the masked start codon or alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acid (LNA) oligonucleotides and exon-junctional complexes (EJCs). (See, eg, Matsuda and Mauro describing LNA and EJC oligonucleotide masking agents (PLoS ONE, 2010 5:11); the contents of which are incorporated herein by reference in their entirety.) In another embodiment, a masking agent can be used to mask a start codon of the circular polyribonucleotide to increase the likelihood that translation will initiate at an alternative start codon. In some embodiments, translation is initiated under selective conditions, such as, but not limited to, virus-induced selection in the presence of GRSF-1 and the circular polyribonucleotide includes GRSF-1 binding sites, see, for example, http: / / jvi.asm.org / content / 76 / 20 / 10417.fu 11. Translation start sequence In some embodiments, the circular polyribonucleotide encodes a polypeptide and may comprise a translation initiation sequence, eg, a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the circular polyribonucleotide includes the translation initiation sequence, eg, 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, eg, Kozak sequence, is present on one or both sides of each expression sequence, resulting in separation of expression products. In some embodiments, the circular 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 circular polyribonucleotide. In some embodiments, the translation initiation sequence is found in a substantially single-stranded region of the circular polyribonucleotide. The circular 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 at least 30, at least 35, at least 40, at least 50, at least 60, or more than 60 start codons. The ncionn / nznz / E / Y translation can start at the first start codon or it can start downstream of the first start codon. In some embodiments, the circular polyribonucleotide can start at a codon that is not the first start codon, eg, AUG. Translation of the circular polyribonucleotide can be initiated 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 ( see Touriol et al., Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 2010 5:11; the contents of each being incorporated herein by reference in their entirety). In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions, eg, stress-induced conditions. As a non-limiting example, translation of the circular polyribonucleotide can begin at an alternative translation start sequence, such as ACG. As another non-limiting example, translation of the circular polyribonucleotide can begin at an alternative translation start sequence, CTG / CUG. As another non-limiting example, translation of the circular polyribonucleotide can begin at an alternative translation start sequence, GTG / GUG. As yet another non-limiting example, the circular polyribonucleotide may begin translation at a non-AUG repeat-associated (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA, eg, CGG, GGGGCC, CAG, CTG. In some embodiments, translation is initiated by treatment of eukaryotic initiation factor 4A (elF4A) with Rocaglatos (translation is repressed by blocking 43S scavenging, leading to upstream premature translation initiation and reduced expression). of protein from transcripts carrying the RocA-elF4A target sequence, see, for example, www.nature.com / articles / nature17978). GOES In some embodiments, the circular polyribonucleotide described herein comprises an internal ribosome entry site (IRES) element. An IRES element suitable for inclusion in a circular polyribonucleotide comprises an RNA sequence capable of binding to a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt , at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt. In one embodiment, the IRES element is derived from the DNA of an organism including, but not limited to, an ncionn / nznz / E / Y virus, a mammal, and a Drosophila. Said viral DNA may be derived from, but is not limited to, picornavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, the Drosophila DNA from which an IRES element is derived includes, but is not limited to, a Drosophila melanogaster Antennapedia gene. In some embodiments, the IRES element is at least partially derived from a virus, for example, it may be derived from a viral IRES element, such as ABPVIGRpred, AEV, ALPVJGRpred, BQCVJGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_5NCR, CrPVIGR, crTMVJREScp, crTMV_IRESmp75, crTMV_IRESmp228, crTMVJREScp, crTMVJREScp, CSFV, CVB3, DCVJGR, EMCV-R, EoPV_5NTR, ERAV_245-961, ERBVJ62920, EV71 1-748, FeLV-Notch2, FMDV,GBC-V_type,GBC-V_A , gypsy_env, gypsyD5, gypsyD2, HAV_HM175, HCV_type_1a, H¡PV_IGRpred, HIV-1, HoCV1 JGRpred, HRV-2, lAPVJGRpred, idefix, KBV IGRpred, LINE-1_ORF1_-101 Jo_-1, LINE-1_ORF1_-202,jo LINE-1 ORF2 -138 to_-86, LINE-1_ORF1_-44jo_-1, PSIVJGR, PV_type1_Mahoney, PV type3_Leon, REV-A, RhPV_5NCR, RhPV_IGR , SINV1_IGRpred, SV40_661-830, TMEV, TMV UIJRESmp228, TRV_5NTR or TRV_5NTR. In some embodiments, the IRES element is at least partially derived from a cellular IRES, such as AML1 / RUNX1, Antp-D, Antp-DE, Antp-CDE, Apaf-1, Apaf-1, AQP4, AT1 R_var1, AT1 R_var2 , AT1 R_var3, AT1 R_var4, BAG1_p36delta236nt, BAG1_p36, BCL2, B¡P_-222_-3, c-IAP1_285-1399, C-IAP1 _1313-1462, c-jun, c-myc, Cat-1_224, CCND1, DAP5, elF4G, elF4GI-ext, elF4GII, elF4GII-long, ELG1, ELH, FGF1A, FMR1, Gtx-133-141, Gtx-1-166, Gtx-1 -120, Gtx-1-196, hairless, HAP4, HIF1 a , hSNM1, Hsp101, hsp70, hsp70, Hsp90, IGF2_leader2, Kv1.4J.2, L-myc, LamB1_-335_-1, LEF1, MNT_75-267, MNT_36-160, MTG8a, MYB, MYT2_997-1152, n-MYC , NDST1, NDST2, NDST3, NDST4L, NDST4S, NRF_-653_-17, NtHSFI, ODC1, p27kip1, p53J 28-269, PDGF2 / c-sis, Pim1, PITSLRE_p58, Rbm3 ,reaper, Scamper, TFIID, TIF4631, Ubx_1- 966, Ubx_373-961, UNR, Ure2, UtrA, VEGF-A_-133_-1, XIAP_5-464, XIAP_305-466 or YAP1. In some embodiments, the IRES element comprises a synthetic IRES, eg, (GAAA)16, (PPT19)4, KMI1, KMI1, KMI2, KMI2, KMIX, Χ1, or X2. In some embodiments, the circular polyribonucleotide includes at least one IRES that flanks at least one expression sequence (eg, 2, 3, 4, 5, or more). In some embodiments, the IRES flanks both sides of at least one expression sequence (eg, 2, 3, 4, 5, or more). In some embodiments, the circular polyribonucleotide includes one or more IRES sequences on one or both sides of each expression sequence, which results in separation of the one or more peptides and / or the resulting one or more polypeptides. ncionn / nznz / E / Y termination element 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 translated continuously. Exclusion of a termination element can result in rolling circle translation or continued expression of the expression product, eg, peptides or polypeptides, due to the absence of ribosome disruption or detachment. In such an embodiment, rolling circle translation expresses a continuous expression product through each expression sequence. In some different embodiments, a termination element of an expression sequence may be part of a rung element. In some embodiments, one or more expression sequences in the circular polyribonucleotide comprise a termination element. However, rolling circle translation of a subsequent expression sequence (eg, second, third, fourth, fifth, etc.) into the circular polyribonucleotide occurs. In such cases, the expression product can be shed from the ribosome when the ribosome encounters the termination element, eg, a stop codon, and translation is terminated. In some embodiments, translation is terminated while the ribosome, eg, at least one ribosome subunit, 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 comprise two or more successive termination elements. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome is completely uncoupled from the circular polyribonucleotide. In some such embodiments, the production of a subsequent expression sequence (eg, second, third, fourth, fifth, etc.) on the circular polyribonucleotide may require the ribosome to reattach to the circular polyribonucleotide prior to initiation of translation. In general, termination elements include an in-frame nucleotide triplet that signals translation termination, eg, UAA, UGA, UAG. In some embodiments, one or more termination elements on the circular polyribonucleotide are in-frame termination elements, such as, but not limited to, out-of-frame reading frames or -1 and +1-shifted (eg, hidden stop). that they can finish the translation. Frameshift termination elements include nucleotide triplets, TAA, TAG, and TGA, which appear in the second and third reading frames of an expression sequence. Frameshift termination elements may be ncionn / nznz / E / Y important in preventing mRNA misreads, which is normally detrimental to the cell. stepping element In some embodiments, the circular polyribonucleotide includes at least one stagger element adjacent to an expression sequence. In some embodiments, the circular polyribonucleotide includes a staggered element adjacent to each expression sequence. In some embodiments, the staggering element is present on one or both sides of each expression sequence, resulting in separation of expression products, eg, one or more peptides and / or one or more polypeptides. In some embodiments, the staging element is a portion of the one or more expression sequences. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, and each of the one or more expression sequences is separated from a subsequent expression sequence by a stagger element in the circular polyribonucleotide. In some embodiments, the staggering element prevents the 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 staging element is a sequence separate from the one or more expression sequences. In some embodiments, the stagger element comprises an expression sequence portion of the one or more expression sequences. In some embodiments, the circular polyribonucleotide includes a staggered element. To avoid production of a continuous expression product, eg, peptide or polypeptide, while maintaining rolling circle translation, a stagger element can be included to induce ribosomal pausing during translation. In some embodiments, the stagger element is located at the 3' end of at least one of the one or more expression sequences. Staggering element may be configured to disrupt a ribosome during rolling circle translation of the circular polyribonucleotide. The staggered element may include, but is not limited to, a 2A-like sequence or CHYSEL (cis-acting hydrolase element). In some embodiments, the staggering element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP, where X1 is absent or is G or Η, X2 is absent or is D or G, X3 is D or V or I or S or M and X5 is any amino acid. In some embodiments, this sequence comprises a non-conserved amino acid sequence with a strong alpha-helical bias followed by the consensus sequence -D(V / l)ExNPG P, where x = any amino acid. Some non-limiting examples of tiering elements include GDVESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, ncionn / nznz / E / Y GDVEQNPGP, IESNPGP, GDIELNPGP, HDIETNPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP. In some embodiments, the staggering element described herein cleaves an expression product, such as between G and P of the consensus sequence described herein. As a non-limiting example, the circular polyribonucleotide includes at least one staggering element for cleaving the expression product. In some embodiments, the circular polyribonucleotide includes a staggered 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 staggered element that is present on one or both sides of each expression sequence, resulting in translation of one or more individual peptides and / or one or more polypeptides from each expression sequence. expression. In some embodiments, a staggering element comprises one or more modified nucleotides or unnatural nucleotides that induce ribosomal pausing during translation. Unnatural nucleotides can 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 may include any modification to the sugar, nucleobase, internucleoside bond (for example, to a phosphate linker / to a phosphodiester bond / to the phosphodiester backbone), and any combination thereof that can induce pausing. ribosomal during translation. Some of the exemplary modifications provided herein are described elsewhere in this document. In some embodiments, the staggered element is present in the circular polyribonucleotide in other forms. For example, in some circular polyribonucleotides, a stagger element comprises a termination element of a first expression sequence in the circular polyribonucleotide and a nucleotide spacing sequence separating the termination element of a first translation initiation sequence from a expression sequence following the first expression sequence. In some examples, the first stagger element of the first expression sequence is located upstream of (5' to) a first translation initiation sequence of the expression sequence following the first expression sequence in the circular polyribonucleotide. In some cases, the first expression sequence and the expression sequence following the first expression sequence are two separate expression sequences on the circular polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence may allow for continuous translation of the first expression sequence and its subsequent expression sequence. In some embodiments, the first staging element comprises a termination element and separates an expression product of the first expression sequence from an expression product of its subsequent expression sequences, thereby creating isolated expression products. In some cases, the circular polyribonucleotide comprising the first staggering element upstream of the first translation initiation sequence of the sequence below in the circular polyribonucleotide is translated continuously, while a corresponding circular polyribonucleotide comprising a staggering of a second expression sequence that is upstream of a second translation initiation sequence of an expression sequence following the second expression sequence is not translated continuously. In some cases, there is only one expression sequence in the circular polyribonucleotide and the first expression sequence and its subsequent expression sequence are the same expression sequence. In some exemplary circular polyribonucleotides, a stagger element comprises a first termination element of a first expression sequence in the circular polyribonucleotide and a nucleotide spacer sequence separating the termination element from the translation initiation sequence downstream. In some such examples, the first stagger element is located 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 allows for continued translation of the first expression sequence and any expression sequences that follow. In some embodiments, the first stagger element separates the expression product of one round of the first expression sequence from the expression product of the next round of the first expression sequences, thereby creating isolated expression products. In some cases, the circular polyribonucleotide comprising the first staggering element upstream of the first translation initiation sequence of the first expression sequence in the circular polyribonucleotide is translated continuously, whereas a corresponding circular polyribonucleotide comprising a The staggering element upstream of a second translation initiation sequence of a second expression sequence in the corresponding circular polyribonucleotide is not translated continuously. In some cases, the distance between the second stagger element and the second translation initiation sequence is at least 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 sequence in the circular polyribonucleotide.In some cases, the distance between the first ncionn / nznz / Ε / γ step element and the first translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt or more. In some embodiments, the distance between the second stagger element and the second translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt , 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or more than the distance between the first stagger element and the first translation start. In some embodiments, the circular polyribonucleotide comprises more than one expression sequence. Regulatory nucleic acids In some embodiments, the circular polyribonucleotide comprises one or more expression sequences that encode a regulatory nucleic acid, eg, that modifies the expression of an endogenous gene and / or an exogenous gene. In some embodiments, the expression sequence of a circular polyribonucleotide provided herein may comprise a sequence that is in an antisense orientation to a regulatory nucleic acid, such as non-coding RNA, such as, but not limited to, tRNA, cRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scRNA, and RNA and hRNA. In one embodiment, the regulatory nucleic acid targets a gene in the host. Regulatory nucleic acids may include, but are not limited to, a nucleic acid that hybridizes to an endogenous gene (eg, miRNA, siRNA, mRNA, cRNA, RNA, DNA, an antisense RNA, gRNA as described elsewhere herein). document), a nucleic acid that hybridizes to an exogenous nucleic acid, such as viral DNA or RNA, a nucleic acid that hybridizes to RNA, a nucleic acid that interferes with gene transcription, a nucleic acid that interferes with translation RNA, a nucleic acid that stabilizes RNA or destabilizes RNA; such as directing it for degradation and a nucleic acid modulating a DNA or RNA binding factor. In one embodiment, the sequence is a miRNA. In some embodiments, the regulatory nucleic acid targets a sense strand of a host gene. In some embodiments, the regulatory nucleic acid targets an antisense strand of a host gene. In some embodiments, the circular polyribonucleotide comprises a regulatory nucleic acid, such as a guide RNA (gRNA). In some embodiments, the circular polyribonucleotide comprises a guide RNA or encodes the guide RNA. A short gRNA synthetic RNA can be comprised of a framework sequence necessary to bind to the incomplete effector moiety and a user-defined ~20 nucleotide targeting sequence for a genomic target. In practice, guide RNA sequences are generally designed to be between 17-24 nucleotides (eg, 19, 20, or 21 nucleotides) ncionn / nznz / E / Y in length and are complementary to the nucleic acid sequence. Diana. Commercial custom gRNA algorithms and generators are commercially available for use in designing efficient guide RNAs. Gene editing has also been achieved using chimeric single guide RNA (sgRNA), a single molecule of genetically engineered (synthetic) RNA that mimics a naturally occurring crRNA-tracr complex and contains both a tracrRNA (for nuclease binding) and at least one crRNA (to guide the nuclease to the target sequence for editing). Chemically modified sgRNAs have also been shown to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991. The gRNA can recognize specific DNA sequences (eg, sequences adjacent to or in a promoter, silencer, or repressor of a gene). In one embodiment, the gRNA is used as part of a CRISPR system for gene editing. For gene editing purposes, the circular polyribonucleotide can be designed to include one or more guide RNA sequences corresponding to a target DNA sequence; see, for example, Gong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. At least about 16 or 17 nucleotides of gRNA sequence are required per Cas9 for DNA cleavage to occur; for Cpf1, at least about 16 nucleotides of gRNA sequence is needed to achieve detectable DNA cleavage. Certain regulatory nucleic acids can inhibit gene expression through the biological process of RNA interference (¡RNA). iRNA molecules comprise RNA or RNA-like structures that typically contain 15-50 base pairs (such as about 18-25 base pairs) and have an identical (complementary) or substantially identical (substantially complementary) nucleobase sequence. ) to a coding sequence in a target gene expressed in the cell. RNAi molecules include, but are not limited to: small interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), meroduplexes, and dicer substrates (US Pat. Nos. .28,084,599 8,349,809 and 8,513,207). In some embodiments, the circular polyribonucleotide comprises regulatory nucleic acids that are RNA or RNA-like structures, typically between about 5-500 base pairs (depending on the specific RNA structure, eg, 5-30 bp miRNA, 200-500 bp jRNA) and may have an identical (complementary) or nearly identical (substantially complementary) nucleobase sequence to a coding sequence in a target gene expressed in the cell. Long noncoding RNAs (ncRNAs) are defined as non-coding protein transcripts longer than 100 nucleotides. This somewhat arbitrary limit distinguishes the ncionn / nznz / Ε / γ cRNAs of small regulatory RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and other short RNAs. In general, the majority (-78%) of the cRNAs are characterized as tissue-specific. Divergent ncRNAs that are transcribed in the opposite direction to nearby protein-coding genes (they comprise a significant proportion -20% of the total ncRNAs in mammalian genomes) may possibly regulate nearby gene transcription. In one embodiment, the circular polyribonucleotide provided herein comprises a sense strand of a dncRNA. In one embodiment, the circular polyribonucleotide provided herein comprises an antisense strand of a dncRNA. The circular polyribonucleotide can encode a regulatory nucleic acid that is substantially complementary or completely complementary to all or a fragment of an endogenous gene or gene product (eg, mRNA). Regulatory nucleic acids can complement sequences at the intron-exon boundary and between exons or adjacent to exons, to prevent maturation of newly generated nuclear RNA transcripts of specific genes into mRNA for transcription. Regulatory nucleic acids that are complementary to specific genes can hybridize to the mRNA for that gene and prevent its translation. The antisense regulatory nucleic acid can be DNA, RNA, or a derivative or hybrid thereof. In some embodiments, the regulatory nucleic acid comprises a protein binding site that can bind to a protein that is involved in regulating the expression of an endogenous gene or an exogenous gene. The circular polyribonucleotide length can encode a regulatory nucleic acid that hybridizes to a transcript of interest that is between about 5 to 30 nucleotides, between about 10 to 30 nucleotides, or about 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. The degree of identity of the regulatory nucleic acid to the target transcript should be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. The circular polyribonucleotide can encode a micro RNA (miRNA) molecule identical to from about 5 to about 25 contiguous nucleotides of a target gene. In some embodiments, the miRNA sequence targets an mRNA and begins with the AA dinucleotide, comprises a GC content of about 30-70% (about 30-60%, about 40-60%, or about 45%). % 55%) and does not have a high percentage identity to any non-target nucleotide sequence in the genome of the mammal into which it is to be introduced, eg as determined by a standard BLAST search. In some embodiments, the circular polyribonucleotide comprises at least one miRNA, eg, 2, 3, 4, 5, 6, or more. In some embodiments, the ncionn / nznz / E / Y circular polyribonucleotide comprises a sequence encoding a miRNA with at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% nucleotide sequence identity to any one of the nucleotide sequences or a sequence that is complementary to a target sequence. siRNA and shRNA resemble intermediates in the endogenous microRNA (miRNA) gene processing pathway (Bartel, Cell 116:281-297, 2004). In some embodiments, siRNAs can function as miRNAs and vice versa (Zeng et al., Mol Cell 9:1327-1333, 2002; Doench et al., Genes Dev 17:438-442, 2003). MicroRNAs, such as siRNAs, use RISC to downregulate target genes, but unlike siRNAs, most animal miRNAs do not cleave mRNA. In contrast, miRNAs reduce protein production through translational suppression or polyA knockdown and mRNA degradation (Wu et al., Proc Nati Acad Sci USA 103:4034-4039, 2006). Known miRNA binding sites are within the 3' UTR of mRNA; miRNAs resemble target sites with near perfect complementarity to nucleotides 2-8 of the 5' end of the miRNA (Rajewsky, Nat Genet 38 Suppl:S8-13, 2006; Lim et aL, Nature 433:769-773, 2005 ). This region is known as the seed region. Because siRNAs and miRNAs are interchangeable, exogenous siRNAs downregulate mRNAs with seed complementarity to siRNA (Birmingham et al, Nat Methods 3:199-204, 2006. Multiple target sites within a 3' UTR provide a stronger negative regulation (Doench et al., Genes Dev 17:438-442, 2003). Lists of known miRNA sequences can be found in databases maintained by research organizations such as the Wellcome Trust Sanger Institute, Penn Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and the European Molecule Biology Laboratory, among others. Known effective siRNA sequences and analogous binding sites are also well represented in the relevant literature. iRNA molecules are easily designed and produced by technologies known in the art. In addition, there are computational tools that increase the possibility of finding efficient and specific sequence motifs (Lagaña et al., Methods Mol. Bio., 2015,1269:393-412). The circular polyribonucleotide can modulate the expression of the RNA encoded by a gene. Because multiple genes may share some degree of sequence homology with one another, in some embodiments, the circular polyribonucleotide may be designed to target a class of genes with sufficient sequence homology. In some embodiments, the circular polyribonucleotide may contain a sequence that is complementary to sequences that are shared between different target genes or that are unique to a specific target gene. In some embodiments, the circular polyribonucleotide can be designed to target conserved regions of an RNA sequence that have homology among multiple genes, thereby targeting multiple genes in a gene family (for example, different genes). gene isoforms, splice variants, mutant genes, etc.). In some embodiments, the circular polyribonucleotide can be designed to target a sequence that is unique to a specific single gene DNA sequence. In some embodiments, the expression sequence is less than 5,000 bp in length (eg, less than about 5,000 bp, 4,000 bp, 3,000 bp, 2,000 bp, 1,000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp). sc, 400 sc, 300 sc, 200 sc, 100 sc, 50 sc, 40 sc, 30 sc, 20 sc, 10 sc or less). In some embodiments, the expression sequence is, independently or in addition to, greater than 10 bp in length (eg, at least about 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp , 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb , 4.9 kb, 5 kb or more). In some embodiments, the expression sequence comprises one or more of the features described herein, eg, a sequence encoding one or more peptides or proteins, one or more regulatory elements, one or more regulatory nucleic acids, eg , one or more non-coding RNAs, other expression sequences, and any combination thereof. Translation efficiency In some embodiments, the translation efficiency of a circular polyribonucleotide as provided herein is greater than a reference, eg, a linear homologue, a linear expression sequence, or a linear circular polyribonucleotide. In some embodiments, a circular polyribonucleotide as provided herein has a translation efficiency that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250% , 300%, 350%, 400%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2000%, 5000%, 10000%, 100000% or more greater than that of a reference . In some embodiments, a circular polyribonucleotide has a 10% higher translation efficiency than a linear counterpart. In some embodiments, a circular polyribonucleotide has a 300% higher translation efficiency than a linear counterpart. In some embodiments, the circular polyribonucleotide produces stoichiometric ratios of expression products. Rolling circle translation continuously produces expression products at substantially equivalent ratios. In some embodiments, the circular polyribonucleotide has stoichiometric translation efficiency such that expression products are produced at substantially equivalent ncionn / nznz / E / Y ratios. In some embodiments, the circular polyribonucleotide has a stoichiometric translation efficiency of multiple expression products, eg, products of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more sequences of expression. Rolling Circle Translation In some embodiments, once translation of the circular polyribonucleotide is initiated, the ribosome attached to the circular polyribonucleotide does not disengage from the circular polyribonucleotide before completing at least one round of translation of the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide as described herein is competent for rolling circle translation. In some embodiments, during rolling circle translation, once translation of the circular polyribonucleotide is initiated, the ribosome attached to the circular polyribonucleotide does not disengage from the circular polyribonucleotide before completing at least 2 rounds, at least 3 rounds, at least 4 rounds. rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least 9 rounds, at least 10 rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds at least 15 rounds, at least 20 rounds, at least 30 rounds, at least 40 rounds, at least 50 rounds, at least 60 rounds, at least 70 rounds, at least 80 rounds, at least 90 rounds, at least 100 rounds at least 150 rounds, at least 200 rounds, at least 250 rounds, at least 500 rounds, at least 1000 rounds, at least 1500 rounds, at least 2000 rounds, at least 5000 rounds, at least 10000 rounds, at least 105 rounds or at least 106 translation bands of the circular polyribonucleotide. In some embodiments, rolling circle translation of the circular polyribonucleotide results in the generation of a polypeptide product that is translated from more than one round of translation of the circular polyribonucleotide (continuous expression product). In some embodiments, the circular polyribonucleotide comprises a staggered element and rolling circle translation of the circular polyribonucleotide results in the generation of a polypeptide product that is generated from a single round of translation or less than a single round of translation of the circular polyribonucleotide. circular polyribonucleotide (isolated expression product). In some embodiments, the circular polyribonucleotide is configured such that at least 10%, 20%, 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, at less than 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the total polypeptides (molar / molar) generated during the rolling circle translation of the circular polyribonucleotide are isolated polypeptides. In some embodiments, the ratio of amount of isolated products to total polypeptides is assessed in an in vitro translation system. In some embodiments, the in vitro translation system used to assess the amount ratio comprises used from rabbit reticulocytes. In some embodiments, the amount ratio is assessed in an in vivo translation ncionn / nznz / E / Y system, such as a eukaryotic cell or a prokaryotic cell, a cultured cell, or a cell in an organism. Untranslated regions In some embodiments, the circular polyribonucleotide comprises untranslated regions (UTRs). UTRs from a genomic region comprising a gene can 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 cases, a UTR for the first expression sequence is the same as or is continuous with or overlaps with another UTR for a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full length human intron, eg, ZKSCAN1. In some embodiments, the circular polyribonucleotide comprises a UTR with one or more stretches of adenosines and uridines included therebetween. These AU-rich signatures can increase production rates of the expression product. The introduction, removal, or modification of AU-rich UTR elements (AREs) may be useful to modulate the stability or immunogenicity of the circular polyribonucleotide. When specific circular polyribonucleotides are engineered, one or more copies of an ARE can be introduced into the circular polyribonucleotide and the copies of an ARE can modulate translation and / or production of an expression product. Similarly, AREs can be identified and removed or engineered into the circular polyribonucleotide to modulate intracellular stability and thus affect translation and production of the resulting protein. It is to be understood that any UTR from any gene can be incorporated into the respective flanking regions of the circular polyribonucleotide. As a non-limiting example, the UTR or a fragment thereof that can be incorporated is a UTR listed in United States Provisional Applications No. 2 US 61 / 775,509 and US 61 / 829,372 or International Patent Application No. 2PCT / US2014 / 021522; the contents of each of these being incorporated herein by reference in their entirety. In addition, multiple wild-type UTRs from any known gene can be used. It is also within the scope of the present invention to provide artificial UTRs that are not variants of wild-type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they are selected or may be altered in orientation or location. Thus, a 5' or 3' UTR can be inverted, shortened, elongated, made chimeric with one or more other 5' UTR or 3' UTR. As used herein, the term "altered" in reference to a UTR sequence means that the ncionn / nznz / E / Y UTR has been changed in some way relative to a reference sequence. For example, a 3' or 5' UTR can be altered relative to a wild-type or native UTR by a change in orientation or location as taught above or it can be altered by inclusion of additional nucleotides, deletion of nucleotides, the exchange or rearrangement of nucleotides. Any of these changes that produce an altered UTR (either 3' or 5') comprise a variant UTR. In one embodiment, a UTR, such as a double, triple, or quadruple 5' or 3' UTR, may be used. As used herein, a double UTR is one in which two copies of the same UTR are encoded either serially or substantially serially. For example, a beta-globin double 3' UTR can be used, as described in US Patent Publication 20100129877, the contents of which are incorporated herein by reference in their entirety. polyA sequence In some embodiments, the circular polyribonucleotide can include a poly-A 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 (eg, 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, 1,100,1,200,1,300,1,400,1,600 , 1,700,1,800,1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A sequence is from about 10 to about 3,000 nucleotides (for example, 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000 to 3,050 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000). In one embodiment, the poly-A sequence is designed relative to the length of the overall circular polyribonucleotide. This design can be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking regions), or based on the length of the last product expressed from the circular polyribonucleotide. In this context, the poly-A sequence can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% longer than the circular polyribonucleotide or a feature thereof. The poly-A sequence can also be designed as a fraction of the circular polyribonucleotide to which it belongs. In this context, the poly-A sequence can nc i οηη / ηζηζ / Ε / γ be a 10, 20, 30, 40, 50, 60, 70, 80 or 90% or more of the total length of the construct or the total length of the construct minus the poly-A sequence. In addition, engineered binding sites and circular polyribonucleotide conjugation to poly-A binding protein may enhance expression. In one embodiment, the circular polyribonucleotide is designed to include a polyA-G quartet. The G quartet is a hydrogen-bonded cyclic series of four guanine nucleotides that can be formed by G-rich sequences in both DNA and brain. RNA. In one embodiment, the G quartet is incorporated at the end of the poly-A sequence. The resulting circular polyribonucleotide construct is evaluated for its stability, protein production, and / or other parameters, including half-life at various time points. In some embodiments, the polyA-G quartet results in protein production equivalent to at least 75% of that observed using only a 120 nucleotide poly-A sequence. In some embodiments, the circular polyribonucleotide comprises a polyA, lacks a polyA, or has a polyA modified 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, eg, immunogenicity, half-life, efficiency of expression, etc. RNA binding In some embodiments, the circular polyribonucleotide comprises one or more RNA binding sites. MicroRNAs (or miRNAs) are short non-coding RNAs that bind to the 3' UTR of nucleic acid molecules and negatively regulate gene expression by either reducing the stability of the nucleic acid molecule or inhibiting translation. The circular polyribonucleotide may comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Said sequences may correspond to any known microRNA, such as those taught in United States Publication US2005 / 0261218 and United States Publication US2005 / 0059005, the contents of which are incorporated herein by reference in their entirety. A microRNA sequence comprises a seed region, ie, a sequence in the region of positions 2-8 of the mature microRNA, the sequence of which has perfect Watson-Crick complementarity with the miRNA target sequence. A microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA. In some embodiments, a microRNA seed may comprise 7 nucleotides (for example, nucleotides 2-8 of the mature microRNA), where the seed complementarity site in the corresponding miRNA target is flanked by a facing adenine (A). to position 1 of the microRNA. In some embodiments, a microRNA seed may ncionn / nznz / E / Y comprise 6 nucleotides (for example, nucleotides 2-7 of the mature microRNA), where the seed complementarity site in the corresponding miRNA target is flanked. by an adenine (A) facing position 1 of the microRNA. See, for example, Grimson A, Farh K, Johnston WK, Garrett-Engele P, Lim LP, Barrel DP; Mol Cell. 2007 Jul 6;27(1):91-105; each of which is incorporated herein by reference in its entirety. The microRNA seed bases are substantially complementary to the target sequence. By engineering microRNA target sequences into the circular polyribonucleotide, the circular polyribonucleotide can evade or be detected by the host's immune system, has modulated degradation or modulated translation, as long as the microRNA in question is available. This process will reduce the risk of off-target effects upon delivery of the circular polyribonucleotide. The identification of microRNAs, microRNA target regions and their expression patterns and their role in biology have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176 ;Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi: 10.1038 / leu.2011.356);Barrel Cell 2009 136:215-233;Landgraf et al, Cell, 2007 129:1401-1414;each one of which is incorporated herein by reference in its entirety). Conversely, microRNA binding sites can be engineered (ie, removed from) the circular polyribonucleotide to modulate protein expression in specific tissues. Regulation of expression in multiple tissues can be achieved by the introduction or removal of one or more microRNA binding sites. Examples of tissues where microRNAs are known to regulate mRNA and thus protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, m¡R-206, miR-208 ), endothelial cells (m¡R-17-92, miR-126), myeloid cells (m¡R-142-3p, m¡R-142-5p, m¡R-16, miR-21, miR-223 , m¡R-24, m¡R-27), adipose tissue (let-7, m¡R-30c), heart (m¡R-ld, m¡R-149), kidney (miR-192 , m¡R-194, miR-204) and lung epithelial cells (let-7, m¡R-133, m¡R-126). MicroRNA can also regulate complex biological processes, such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176; incorporated herein by reference in its entirety). In the circular polyribonucleotide described herein, binding sites for microRNAs that are involved in such processes can be removed or introduced to engineer expression of the circular polyribonucleotide in biologically relevant cell types or in the context of relevant biological processes. A listing of microRNAs, m¡R sequences, and m¡R binding sites is presented in Table 9 of United States Provisional Application No. s61 / 753,661 filed January 17, 2013, in Table 9 of the Request ncionn / nznz / Ε / γ United States Provisional Application No. 61 / 754,159 filed January 18, 2013 and in Table 7 of United States Provisional Application No. 61 / 758,921 filed January 31, 2013, each of which is incorporated herein by reference in its entirety. In some embodiments, the microRNA binding site includes, for example, m¡R7. The circular polyribonucleotide disclosed herein may comprise a miRNA binding site that hybridizes to any miRNA, such as any of those disclosed in miRNA databases, such as miRBase, deepBase, miRBase, microRNA.org, miRGen 2.0 ; miRNAMap, PMRD, TargetScan, or VIRmiRNA. In some cases, the miRNA binding site can be any site that is complementary to a miRNA whose target sequence is reported in target microRNA gene datasets, such as StarBase, StarScan, Cupid, TargetScan, TarBase, Diana-microT, miRecords, PicTar, PITA, RepTarm RNA22, miRTarBase, miRwalko MBSTAR. By understanding microRNA patterns in different cell types, the circular polyribonucleotide described herein can be engineered for more targeted expression in specific cell types or only under specific biological conditions. By introducing tissue-specific microRNA binding sites, the circular polyribonucleotide can be engineered for optimal protein expression in a tissue or in the context of a biological condition. Examples of using microRNAs to drive tissue- or disease-specific gene expression are listed (Getner and Naldini, Tissue Antigens. 2012, 80:393-403; incorporated herein by reference in its entirety). In addition, microRNA seed sites can be incorporated into the circular polyribonucleotide to modulate expression in certain cells, resulting in biological enhancement. An example of this is the incorporation of miR-142 sites. Incorporation of miR-142 sites into the circular polyribonucleotide described herein can modulate expression in hematopoietic cells, but also reduces or suppresses immune responses to a protein encoded on the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide includes one or more long intergenic noncoding RNA (jncRNA) binding sites. Long noncoding intergenic RNAs (ncRNAs) form the majority of long noncoding RNAs. The jRNAs are non-coding transcripts and, in some embodiments, are greater than about 200 nucleotides in length. In some embodiments, they have an exon-intron-exon structure similar to protein-coding genes, but do not span open reading frames and do not code for proteins. More than 8,000 jRNAs have recently been described and are believed to be the largest RNA subclass, originating from the noncoding transcriptome in humans. Thousands of jRNAs are known and some appear to be key regulators of various cellular processes. ncionn / nznz / E / Y remains difficult to determine the function of individual cRNAIs. The expression of jRNAI is surprisingly tissue-specific compared to genes encoding and which are normally co-expressed with their neighboring genes, although to a similar degree to pairs of neighboring protein-coding genes. In some embodiments, the circular polyribonucleotide includes one or more jRNAs, such as FIRRE, LINC00969, PVT1, LINC01608, JPX, LINC01572, LINC00355, C1 orf132, C3orf35, RP11-734, LINC01608, CC-499B15.5, CASC15, LINC00937, RP11 -191, etc. or other ARNInc or ARNInc, such as those from known ARNInc databases. protein binding In some embodiments, the circular polyribonucleotide includes one or more protein binding sites that allow a protein, eg, a ribosome, to bind to an internal site in the RNA sequence. By engineering protein binding sites, eg, ribosome binding sites, into the circular polyribonucleotide, the circular polyribonucleotide can escape or have reduced detection by the host's immune system, have modulated degradation or regulated translation, masking the circular polyribonucleotide of components of the host's immune system. In some embodiments, the circular polyribonucleotide comprises at least one immunoprotein binding site, eg, to evade immune responses, eg, CTL (cytotoxic T lymphocyte) responses. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and helps mask the circular polyribonucleotide as foreign. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and helps mask the circular polyribonucleotide as foreign or foreign. The translational mechanisms of docking of the ribosome to linear RNA, involve the binding of the ribosome to the 5' capped end of an RNA. From the 5' end, the ribosome migrates to a start codon, after which the first peptide bond is formed. In accordance with the present invention, internal (ie, cap-independent) initiation or translation of the circular polyribonucleotide does not require a free end or a capped end. Instead, a ribosome binds to an internal capless site, whereby the ribosome begins elongation of the polypeptide at a start codon. In some embodiments, the circular polyribonucleotide includes one or more RNA sequences that comprise a ribosome binding site, eg, a start codon. Natural 5' UTRs carry features that play roles in translation initiation. These carry signatures, such as Kozak sequences, which are commonly known to be involved in the processes by which the ribosome initiates translation in many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is ncionn / nznz / E / Y a purine (adenine or guanine) three bases upstream of the initiation codon (AUG), followed by another G The 5' UTR is also known to form secondary structures that are involved in elongation factor binding. In some embodiments, the circular polyribonucleotide encodes a protein-binding sequence that binds to a protein. In some embodiments, the protein-binding sequence directs or localizes the circular polyribonucleotide to a specific target. In some embodiments, the protein-binding sequence specifically binds to an arginine-rich region of a protein. In some embodiments, the protein binding site includes, but is not limited to, a protein binding site, 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, GRN2F1, HNTF1, HNTF1 HNRNPA2B1, HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, LIN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO58, NONO-PM, NOP , 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 to RNA. encryptogenic As described herein, the circular polyribonucleotide comprises an encryptogen for reducing, evading, or avoiding the innate immune response of a cell. In one aspect, circular polyribonucleotides are provided herein which, when administered to cells, result in a reduced host immune response compared to the response elicited by a reference compound, eg, a linear polynucleotide corresponding to the polyribonucleotide circular described or a circular polyribonucleotide lacking an encryptogen. In some embodiments, the circular polyribonucleotide has less immunogenicity than a counterpart lacking an encryptogen. In some embodiments, an encryptogen enhances stability. There is increasing evidence about the regulatory roles played by UTRs with regard to the stability of a nucleic acid molecule and translation. Regulatory features of a UTR can be included in the encryptogen to enhance the stability of the circular polyribonucleotide. nc i οηη / ηζηζ / Ε / γ In some embodiments, 5' or 3' UTRs can constitute encryptogens in a circular polyribonucleotide. For example, removal or modification of AU-rich UTR elements (AREs) may be useful to modulate the stability or immunogenicity of the circular polyribonucleotide. In some embodiments, removal or modification of AU-rich elements (AREs) in the expression sequence, eg, translatable regions, may be useful to modulate the stability or immunogenicity of the circular polyribonucleotide. In some embodiments, an encryptogen comprises a miRNA binding site or a binding site for any other non-coding RNA. For example, the incorporation of miR-142 sites into the circular polyribonucleotide described herein may not only modulate expression in hematopoietic cells, but also reduce or abolish immune responses to a protein encoded in the circular polyribonucleotide. In some embodiments, an encryptogen comprises one or more protein binding sites that allow a protein, eg, an immunoprotein, to bind to the RNA sequence. By engineering protein binding sites into the circular polyribonucleotide, the circular polyribonucleotide can escape or have reduced detection by the host's immune system, have modulated degradation or regulated translation, masking the circular polyribonucleotide from components of the host's immune system . In some embodiments, the circular polyribonucleotide comprises at least one immunoprotein binding site, eg, to evade immune responses, eg, CTL responses. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and helps mask the circular polyribonucleotide as foreign. In some embodiments, an encryptogen comprises one or more modified nucleotides. Exemplary modifications may include any modification to the sugar, nucleobase, internucleoside bond (for example, to a phosphate linker / to a phosphodiester bond / to the phosphodiester backbone), and any combination thereof that may prevent or reduce the immune response against the circular polyribonucleotide. Some of the exemplary modifications provided herein are described in detail below. In some embodiments, the circular polyribonucleotide includes one or more modifications as described elsewhere herein to reduce a host immune response, as compared to the response elicited by a reference compound, eg, a circular polyribonucleotide lacking modifications. In particular, the addition of one or more inosines has been shown to discriminate RNA as endogenous versus viral. See, for example, Yu, Z. et al. (2015) RNA editing by ncionn / nznz / E / Y ADAR1 marks dsRNA as self. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety. In some embodiments, the circular polyribonucleotide includes one or more expression sequences for shRNA or an RNA sequence that can be processed into siRNA and the shRNA or siRNA targets RIG-1 and reduces RIG-1 expression. RIG-1 can sense circular foreign RNA and results in degradation of circular foreign RNA. Therefore, a circular polyribonucleotide carrying sequences for shRNA, siRNA, or any other regulatory nucleic acid targeting RIG-1 may reduce immunity, eg, host cell immunity, against the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide lacks a sequence, element, or structure that assists the circular polyribonucleotide to reduce, evade, or prevent an innate immune response from a cell. In some such embodiments, the circular polyribonucleotide may lack a poly A sequence, a 5' end, a 3' end, phosphate group, hydroxyl group, or any combination thereof. Riboswitches In some embodiments, the circular polyribonucleotide comprises one or more riboswitches. Typically, a riboswitch is considered a part of the circular polyribonucleotide that can bind directly to a small target molecule and whose target binding affects RNA translation, expression product stability, and stability (Tucker B J, Breaker R R ( 2005), Curr Opin Struct Biol 15(3): 342-8). Thus, the circular polyribonucleotide including a riboswitch is indirectly involved in regulating its own activity, depending on the presence or absence of its target molecule. In some embodiments, a riboswitch has a region of aptamer-like affinity for a separate molecule. Therefore, in the broader context of the present invention, any aptamer included in an antisense nucleic acid can be used for the sequestration of large volume molecules. Downstream reporting of the event through (ribo)switch activity can be especially advantageous. In some embodiments, the riboswitch may have an effect on gene expression including, but not limited to, transcriptional termination, inhibition of translation initiation, mRNA self-cleavage, and in eukaryotes, disruption of splicing pathways. The riboswitch may function to control gene expression by attachment or removal of a trigger molecule. Thus, subjecting a circular polyribonucleotide including the riboswitch to conditions that activate, deactivate or block the riboswitch to alter expression. Expression may be altered as a result of, for example, termination of transcription or blockage of ribosome binding to RNA. Binding of a trigger molecule or an analogue thereof may, depending on the nature of the ncionn / nznz / Ε / γ riboswitch, reduce or prevent expression of the RNA molecule or promoter or increase expression of the trigger molecule. RNA. Some examples of riboswitches are described herein. In some embodiments, the riboswitch is a cobalamin (also known as element B12) riboswitch, which binds adenosylcobalamin (the coenzyme derived from vitamin B12) to regulate the biosynthesis and transport of cobalamin and similar metabolites. In some embodiments, the riboswitch is a cyclic di-GMP riboswitch, which binds to cyclic di-GMP to regulate a variety of genes. There are two structurally unrelated classes, cyclic di-GMP-1 and cyclic di-GMP-11. In some embodiments, the riboswitch is an FMN riboswitch (also known as an RFN element), which binds to flavin mononucleotide (FMN) to regulate riboflavin biosynthesis and transport. In some embodiments, the riboswitch is a glmS riboswitch, which cleaves itself when there is a sufficient concentration of glucosamine-6-phosphate. In some embodiments, the riboswitch is a glutamine riboswitch, which binds glutamine to regulate genes involved in glutamine and nitrogen metabolism. They also bind to short peptides of unknown function. Such riboswitches are assigned to two classes, which are structurally related: the glnA RNA motif and the downstream peptide motif. In some embodiments, the riboswitch is a glycine riboswitch, which binds to glycine to regulate glycine metabolism genes. It comprises two adjacent aptamer domains on the same mRNA and is the only known natural RNA that exhibits cooperative binding. In some embodiments, the riboswitch is a lysine riboswitch (also known as an L-box), which binds lysine to regulate lysine biosynthesis, catabolism, and transport. In some embodiments, the riboswitch is a PreQ1 riboswitch, which binds to pre-queuosin to regulate genes involved in the synthesis or transport of this precursor to queuosin. Two completely different classes of PreGI riboswitches are known: PreQ1-1 riboswitches and PreQ1-ll riboswitches. The binding domain of the PreQ1-I riboswitches is unusually small among naturally occurring riboswitches. Found only in certain species of the Streptococcus and Lactococcus genera, PreGI-ll riboswitches have a completely different structure and are larger in size. In some embodiments, the riboswitch is a purine riboswitch, which binds purines to regulate purine metabolism and transport. Different forms of the purine ribo-switch ncionn / nznz / E / Y bind either guanine (a form originally known as the G-box) or adenine. The specificity for guanine or adenine depends entirely on Watson-Crick interactions with a single pyrimidine at the riboswitch at position Y74. In the guanine ribo-switch this residue is a cytosine (ie C74), in the adenine residue it is always a uracil (ie U74). Some homologous types of purine riboswitches bind deoxyguanosine, but have more significant differences than a single nucleotide mutation. In some embodiments, the riboswitch is a SAH riboswitch, which binds to S-adenosylhomocysteine ​​to regulate genes involved in the recycling of this metabolite that occurs when S-adenosylmethionine is used in methylation reactions. In some embodiments, the riboswitch is a SAM riboswitch, which binds S-adenosyl methionine (SAM) to regulate the biosynthesis and transport of methionine and SAM. Three different SAM riboswitches are known: SAM-I (originally called S-box), SAM-II, and the SmK-box riboswitch. SAM-I is found widely in bacteria, but SAM-II is found only in α, β, and a few y proteobacteria. The SmK box riboswitch is found only in the order Lactobacillales. These three varieties of riboswitch have no obvious similarities in sequence or structure. A fourth variety, SAM-IV, appears to have a ligand-binding core similar to that of SAM-I, but in the context of a different backbone. In some embodiments, the riboswitch is a SAM-SAH riboswitch, which binds to both SAM and SAH with similar affinities. Since they are always in a position to regulate genes encoding methionine adenosyltransferase, it has been proposed that only their binding to SAM is physiologically relevant. In some embodiments, the riboswitch is a tetrahydrofolate riboswitch, which binds to tetrahydrofolate to regulate gene synthesis and transport. In some embodiments, the riboswitch is a theophylline-binding riboswitch or a thymine pyrophosphate-binding riboswitch. In some embodiments, the riboswitch is a T. tengcongensis glmS catalytic riboswitch that detects glucosamine-6 ​​phosphate (Klein and Ferre-D'Amare 2006). In some embodiments, the riboswitch is a TPP riboswitch (also known as a THI box), which binds thiamine pyrophosphate (TPP) to regulate thiamine biosynthesis and transport, as well as the transport of similar metabolites. It is the riboswitch found so far in eukaryotes. In some embodiments, the riboswitch is a Muco riboswitch, which binds to the molybdenum cofactor, to regulate genes involved in the biosynthesis and transport of this coenzyme, as well as enzymes that use this or its derivatives as a cofactor. ncionn / nznz / E / Y In some embodiments, the ribo-switch is an adenine-sensing add-A ribo-switch, found in the 5' UTR of the gene encoding Vibrio vulnificus adenine deaminase. aptazime In some embodiments, the circular polyribonucleotide comprises an aptazime. The aptazime is a switch for conditional expression, in which a region of aptamer is used as an allosteric control element and coupled to a region of catalytic RNA (a ribozyme, as described below). In some embodiments, the aptazime is active in cell type specific translation. In some embodiments, aptazime is active in cell state specific translation, eg, virus-infected cells or in the presence of viral nucleic acids or viral proteins. A ribozyme (from a ribonucleic acid enzyme, also called an 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 RNAs, but they have also been shown to catalyze the aminotransferase activity of the ribozyme. More recently, it has been discovered that catalytic RNAs can be "evolved" by in vitro methods [1. Agresti J J, Kelly Β T, Jaschke A, Griffiths A D: Selection of ribozymes that catalyze multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization. Proc Nati Acad Sel USA 2005, 102:16170-16175; 2. Sooter L J, Riedel T, Davidson E A, Levy M, Cox J C, Ellington A D: Toward automated nucleic acid enzyme selection. Biological Chemistry 2001, 382(9):1327-1334.]. Winkler et al. have shown [Winkler W C, Nahvi A, Roth A, Collins J A, Breaker R R: Control of gene expression by a natural metabolite-responsive ribozyme. Nature 2004, 428:281-286.] that, similar to the riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression. In the context of the present invention, it may be particularly advantageous to place a catalytic RNA or ribozyme on a larger non-coding RNA, such that the ribozyme is present in many copies within the cell for the purpose of chemical transformation of a cell. molecule from a bulky volume. Furthermore, encoding both aptamers and ribozymes on the same antisense RNA can be particularly advantageous. Some non-limiting examples of ribozymes include hammerhead ribozyme, VL ribozyme, leadzyme, hairpin ribozyme. In some embodiments, the aptazime is a ribozyme that can cleave RNA sequences and that can be regulated as a result of ligand / modulator binding. The ribozyme may also be a self-cleaving ribozyme. As such, they combine the properties of ribozymes and aptamers. Aptazymes offer advantages over conventional ncionn / nznz / E / Y aptamers due to their potential for activity in trans, the fact that they act catalytically to inactivate expression, and that inactivation, due to cleavage of their own or heterologous transcript, it is irreversible. In some embodiments, the aptazime is included in an untranslated region of the circular polyribonucleotide and in the absence of ligand / modulator is inactive, allowing expression of the transgene. Expression can be turned off (or downregulated) by addition of the ligand. It should be noted that aptazymes that are downregulated in response to the presence of a particular modulator can be used in control systems, where upregulation of gene expression in response to the modulator is desired. Aptazymes may also allow the development of systems for the autoregulation of circular polyribonucleotide expression. For example, the protein product of the circular polyribonucleotide is the rate-determining enzyme in the synthesis of a small molecule, which could be modified to include an enzyme selected to have increased catalytic activity in the presence of such a molecule, thereby providing a self-regulating feedback loop for its synthesis. Alternatively, the activity of the aptazime can be selected to be sensitive to the accumulation of the protein product of the circular polyribonucleotide or any other cellular macromolecule. In some embodiments, the circular polyribonucleotide can include an aptamer sequence. Some non-limiting examples include a lysozyme-binding RNA aptamer, a Toggle-25t, which is an RNA aptamer that includes 2'fluoropyrimidine nucleotides that binds to thrombins with high specificity and affinity, tRNAT that binds to the lysozyme-binding RNA aptamer, human immunodeficiency virus (HIV TAR) trans-acting response, hemin-binding RNA aptamer, interferon-γ-binding RNA aptamer, vascular endothelial growth factor (VEGF)-binding RNA aptamer, Prostate-specific antigen (PSA)-binding RNA, dopamine-binding RNA aptamer, and RNA-binding aptamer to the non-classical oncogene, heat shock factor 1 (HSF1). circularization In one embodiment, a circular polyribonucleotide can be cyclized or concatemerized. In some embodiments, the linear circular polyribonucleotide can be cyclized in vitro prior to formulation and / or administration. In some embodiments, the circular polyribonucleotide can be cyclized within a cell. extracellular circularization In some embodiments, the circular polyribonucleotide 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 (for example, a linear circular polyribonucleotide), include chemically reactive groups that, when closed together, can form a new ncionn / nznz / Ε / γ bond. covalent 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 at the 3' end of a linear RNA molecule will undergo a nucleophilic attack on the 5' NHS-ester moiety, forming a new 5'73'-amide bond. In one embodiment, a DNA or RNA ligase can be used to enzymatically ligate a 5' phosphorylated nucleic acid molecule (eg, a linear circular polyribonucleotide) to the 3' hydroxyl group of a nucleic acid (eg, a linear nucleic acid). forming a new phosphodiester bond. In an exemplary reaction, a linear circular polyribonucleotide is incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. The ligation reaction can occur in the presence of a linear nucleic acid capable of base pairing with both the 5' and 3' region in juxtaposition to aid the enzymatic ligation reaction. In one embodiment, the linkage is bridging linkage. For example, a bridging ligase, such as SpIintR®, can be used for bridging ligation. For bridging, a single-stranded (bridged) polynucleotide, such as a single-stranded RNA, can be designed to hybridize to both ends of a linear polyribonucleotide such that the two ends can be juxtaposed upon hybridization to the single-stranded bridge. Bridging ligase can thus catalyze the ligation of the two juxtaposed ends of the linear polyribonucleotide, generating a circular polyribonucleotide. In one embodiment, a DNA or RNA ligase may be used in the synthesis of the circular polynucleotides. As a non-limiting example, the ligase may be a circ ligase or circular ligase. In one embodiment, the 5' or 3' end of the circular polyribonucleotide may encode a ribozyme ligase sequence, such that during in vitro transcription, the linear circular polyribonucleotide includes an active ribozyme sequence capable of ligating the 5' end of the circular polyribonucleotide. circular polyribonucleotide linear to the 3' end of the circular polyribonucleotide. The ribozyme ligase can be derived from the group I intron, hepatitis delta virus, hairpin ribozyme or can be selected by SELEX (systematic ligand evolution by exponential enrichment). The ribozyme ligase reaction can take from 1 to 24 hours at temperatures between 0 and 37°C. In one embodiment, a linear circular polyribonucleotide can be cyclized or concatemerized using at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety can react with 5' proximal and / or 3' proximal regions or features of the linear circular polyribonucleotide to cyclize or concatemerize the linear circular polyribonucleotide. In one aspect, the at least one nucleic acid moiety can be located at or attached to or near the 5' end and / or 3' end of the linear circular polyribonucleotide. Contemplated non-nucleic acid moieties may be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety can be a bond, such as a hydrophobic bond, ionic bond, a biodegradable bond, and / or a cleavable bond. As another non-limiting example, the non-nucleic acid moiety is a ligation moiety. As another non-limiting example, the non-nucleic acid moiety can be an oligonucleotide or peptide moiety, such as an aptamer or non-nucleic acid linker as described herein. In one embodiment, a linear circular polyribonucleotide may be cyclized or concatemerized due to a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near, or ligated to the 5' and 3' ends of the linear circular polyribonucleotide. As a non-limiting example, one or more linear circular polyribonucleotides can 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, dipole bonds, conjugation, hyperconjugation, and antibonding. In one embodiment, the linear circular polyribonucleotide may comprise a 5' proximal and a 3' proximal ribozyme RNA sequence. The RNA sequence of the ribozyme can be covalently linked to a peptide when the sequence is exposed to the rest of the ribozyme. In one aspect, peptides covalently linked to the RNA sequence of the ribozyme near the 5' end and the 3' end can associate with each other causing a linear circular polyribonucleotide to cycle or concatemerize. In another aspect, peptides covalently linked to ribozyme RNA near the 5' end and 3' end can cause the primary linear construct or linear mRNA to cycle or concatemerize after being ligated using various methods known in the art. , such as, but not limited to, protein ligation. Non-limiting examples of ribozymes for use in the primary linear constructs or linear RNA of the present invention or a non-exhaustive listing of methods for incorporating and / or covalently ligating peptides are described in US Patent Application No. aUS20030082768, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the linear circular polyribonucleotide can include a nucleic acid 5' triphosphate converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase). As an alternative ncionn / nznz / Ε / γ, the conversion of the 5' triphosphate of the linear circular polyribonucleotide to a 5' monophosphate can occur by a two-step reaction comprising: (a) contacting the 5' nucleotide of the linear circular polyribonucleotide with a phosphatase (eg Antarctic phosphatase, shrimp alkaline phosphatase or calf intestinal phosphatase) to remove all three phosphatases; and (b) contacting the 5' nucleotide after step (a) with a kinase (eg, polynucleotide kinase) that adds a single phosphate. In some embodiments, the 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 approximately 90%, at least approximately 95% or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%. splicing element In some embodiments, the circular polyribonucleotide includes at least one splicing element. In a circular polyribonucleotide as provided herein, a splicing element may be a complete splicing element that can mediate splicing of the circular polyribonucleotide. Alternatively, the splice element may also be a residual splice element from a completed splice event. For example, in some cases, a splicing element of a linear circular polyribonucleotide may mediate a splicing event that results in circularization of the linear polyribonucleotide and thus the resulting circular polyribonucleotide comprises a splicing element. residual from said circularization event mediated by splicing. In some cases, the residual splicing element does not have the ability to mediate splicing. In other cases, the residual splicing element may still mediate the splicing under certain circumstances. In some embodiments, the splice element is located adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide includes a splice element adjacent to each expression sequence. In some embodiments, the splice element is found on one or both sides of each expression sequence, resulting in separation of expression products, eg, one or more peptides and / or one or more polypeptides. ncionn / nznz / E / Y In some embodiments, the circular polyribonucleotide includes an internal splicing element that, when replicated, the joined and spliced ​​ends are joined together. Some examples may include miniature introns (<100 nt) with splicing 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) cis-sequence elements close to back-splice events in the 200 bp preceding (upstream of) or following (downstream of) a back-splicing site with flanking exons . In some embodiments, the circular polyribonucleotide includes at least one repetitive nucleotide sequence described elsewhere herein, such as an internal splicing element. In such embodiments, the repetitive nucleotide sequence may include repeat sequences from the Alu family of introns. In some embodiments, a splicing-related ribosome-binding protein may regulate circular polyribonucleotide biogenesis (eg, Muscleblind and Quaking splicing factors (QKI)). In some embodiments, the circular polyribonucleotide can include canonical splice sites flanking head-to-tail junctions of the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide may include a bulge-helix-bulge motif, comprising a 4 base pair stem flanked by two 3 nucleotide bulges. Cleavage occurs at a site in the bulge region, generating characteristic fragments with a terminal 5' hydroxyl group and 2',3' cyclic phosphate. Circularization occurs by nucleophilic attack of the 5'-OH group on the 2',3' cyclic phosphate of the same molecule, forming a 3',5' phosphodiester bridge. In some embodiments, the circular polyribonucleotide can include a multimeric repetitive RNA sequence that carries an HPR element. The HPR comprises a 2',3' cyclic phosphate and a 5'-OH end. The HPR element self-processes the 5' and 3' ends of the circular polyribonucleotide, thereby ligating the ends together. In some embodiments, the circular polyribonucleotide can include a sequence that mediates self-ligation. In one embodiment, the circular polyribonucleotide can include an HDV sequence (eg, conserved sequence of the HDV replication domain, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUUCUGUAAAGAGG AGACUGCUGGACUCGCCGCCCAAAGUUCGAGCAUGAGCC or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUUCUGUAAAGAGGAGACUGCUG GACUCGCCGCCCGAGCC) so that it binds itself. In one embodiment, the circular polyribonucleotide may include the E-loop sequence (eg, in PSTVd) to be self-ligated. In another embodiment, the circular polyribonucleotide may include an autocircularizing intron, ncionn / nznz / E / Y eg, a 5' and 3' splice junction or a catalytic autocircularizing intron, such as group I introns, group II or group III. Non-limiting examples of group I intron self-splicing sequences may include intron-exon permuted self-splicing sequences from the bacteriophage T4 td gene and Tetrahymena midsequence (IVS) rRNA. Other circularization methods In some embodiments, circular polyribonucleotides can include complementary sequences, including either repetitive or non-repeating nucleic acid sequences within individual introns or along flanking introns. Repetitive nucleic acid sequences are sequences that occur within a segment of the circular polyribonucleotide. In some embodiments, the circular 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 circular polyribonucleotide includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the circular polyribonucleotide, the hybridized segment forming an internal double strand. In some embodiments, the repetitive nucleic acid sequences and the complementary repetitive nucleic acid sequences of two separate circular polyribonucleotides anneal to generate a single circularized polyribonucleotide, with the annealed segments forming internal duplexes. In some embodiments, the complementary sequences are found at the 5' and 3' ends of the linear circular polyribonucleotides. In some embodiments, the complementary sequences include approximately 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, circularization chemical methods can 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), define metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof. . In some embodiments, enzymatic methods of circularization can be used to generate the circular polyribonucleotide. In some embodiments, a ligation enzyme, eg, DNA or RNA ligase, can be used to generate a template of the circular polyribonuclease or a complement, a complementary strand of the circular polyribonuclease or circular polyribonuclease. Circularization of the circular polyribonucleotide can be achieved by methods known in the art, for example, those described in RNA circularization strategies in vivo and ncionn / nznz / Ε / γ in vitro by Petkovic and Muller in Nucleic Acids Res, 2015, 43(4) : 2454-2465 and In vitro circularization of RNA by Muller and Appel, in RNABiol, 2017, 14(8):1018-1027. replication element The circular polyribonucleotide can encode a sequence and / or motifs useful for replication. Replication of a circular polyribonucleotide can occur by generating a complementary circular polyribonucleotide. In some embodiments, the circular polyribonucleotide includes a motif to initiate transcription, where transcription is driven by either the endogenous cellular machinery (DNA-dependent RNA polymerase) or an RNA-dependent RNA polymerase encoded by the circular polyribonucleotide. The product of the rolling circle transcriptional event can be cleaved by a ribozyme to generate either a complementary or propagated circular polyribonucleotide of unit length. Ribozymes may be encoded by the circular polyribonucleotide, its complement, or by a trans RNA sequence. In some embodiments, the encoded ribozymes can include a sequence or motif that regulates (inhibits or promotes) ribozyme activity to control circular RNA propagation. In some embodiments, unit length sequences can be ligated in a circular fashion by a cellular RNA ligase. In some embodiments, the circular polyribonucleotide includes a replication element that aids in self-amplification. Examples of such replication elements include, but are not limited to, HDV replication domains described elsewhere in this document, potato spindle tuber viroid promoter RNA (see, for example, Kolonko 2005 Virology) and ribozymes of Circular replication-competent sense and / or antisense RNAs, such as 5'CGGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGA CGCACGUCCACUCGGAUGGCUAAGGGAGAGCCA-3' antigenonomics or 5'UGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGCAGGA CCGUCC'AUGCCGAUGGenomics. In some embodiments, the circular polyribonucleotide includes at least one staggered element as described herein to aid in replication. A staggering element in the circular polyribonucleotide can excise long replicated transcripts from the circular polyribonucleotide to a specific length that could subsequently be circularized to form a complement to the circular polyribonucleotide. In another embodiment, the circular polyribonucleotide includes at least one ribozyme sequence to cleave replicated long transcripts from the circular polyribonucleotide to a specific length, where another encoded ribozyme cleaves the transcripts in the ribozyme sequence. The circularization forms a complement with the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide is substantially resistant to degradation, eg, by exonucleases. nc i οηη / ηζηζ / Ε / γ In some embodiments, the circular polyribonucleotide is replicated within a cell. In some embodiments, the circular polyribonucleotide replicates within a cell at a rate between about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-99% or anything in between. In some embodiments, the circular polyribonucleotide is replicated within a cell and passed to daughter cells. In some embodiments, a cell passes at least one circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, a cell undergoing meiosis passes the circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. . In some embodiments, a cell undergoing mitosis passes the circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. . In some embodiments, the circular polyribonucleotide is replicated within the host cell. In one embodiment, the circular polyribonucleotide is capable of replication in a mammalian cell, eg, a human cell. Although in some embodiments the circular polyribonucleotide is replicated in the host cell, the circular polyribonucleotide does not integrate into the host genome, eg, into the host's chromosomes. In some embodiments, the circular polyribonucleotide has negligible recombination frequency, eg, with the host's chromosomes. In some embodiments, the circular polyribonucleotide has a recombination frequency, for example, less than about 1.0 cM / Mb, 0.9 cM / Mb, 0.8 cM / Mb, 0.7 cM / Mb, 0.6 cM / Mb, 0.5 cM / Mb, 0.4 cM / Mb, 0.3 cM / Mb, 0.2 cM / Mb, 0.1 cM / Mb or less, eg, with host chromosomes. other sequences In some embodiments, the circular polyribonucleotide further includes another nucleic acid sequence. In some embodiments, the circular polyribonucleotide can comprise other sequences including DNA, RNA, or artificial nucleic acids. Other sequences may include, but are not limited to, genomic DNA, cDNA, or sequences encoding tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other iRNA molecules. In one embodiment, the circular polyribonucleotide includes a siRNA for targeting different loci of the same gene expression product as the circular polyribonucleotide. In one embodiment, the circular polyribonucleotide includes a siRNA for targeting a different gene expression product than the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide lacks a 5' UTR. In some embodiments, the circular polyribonucleotide lacks a 3' UTR. In some embodiments, the circular polyribonucleotide lacks a poly-A sequence. In some ncionn / nznz / E / Y embodiments, the circular polyribonucleotide lacks a termination element. In some embodiments, the circular polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the circular polyribonucleotide lacks susceptibility to degradation by exonucleases. In some embodiments, the fact that the circular polyribonucleotide lacks degradation susceptibility may mean that the circular polyribonucleotide is not degraded by an exonuclease or is only degraded in the presence of a nuclease to a limited extent that is comparable to or similar to that of the circular polyribonucleotide. absence of exonuclease. In some embodiments, the circular polyribonucleotide lacks degradation by exonucleases. In some embodiments, the circular polyribonucleotide has reduced degradation when exposed to exonucleases. 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 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 poly-A 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 ribosome entry site and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a cap and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 5' UTR, a 3' UTR, and an IRES and is competent for protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide comprises 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 a foreign gene, a sequence that encodes an agent therapeutic, a regulatory element (eg, translation modulator, eg, translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids that target endogenous genes (siRNA, cRNA, shRNA) and a sequence encoding a therapeutic mRNA or protein. The other sequence may be from about 2 to about 10,000 nt, from about 2 to about 5,000 nt, from about 10 to about 100 nt, from about 50 to about 150 nt, from ncionn / nznz / E / Y to about 100 to from about 200 nt, from about 150 to about 250 nt, from about 200 to about 300 nt, from about 250 to about 350 nt, from about 300 to about 500 nt, from about 10 to about 1000 nt, from about 50 to about 1000 nt, from about 100 to about 1,000 nt, from about 1,000 to about 2,000 nt, from about 2,000 to about 3,000 nt, from about 3,000 to about 4,000 nt, from about 4,000 to about 5,000 nt, or any range in between. As a result of this circularization, the circular polyribonucleotide may include certain features that distinguish it from linear RNA. For example, circular polyribonucleotide is less susceptible to degradation by exonucleases, compared to linear RNA. As such, the circular polyribonucleotide is more stable than a linear RNA, especially when incubated in the presence of an exonuclease. The increased stability of circular polyribonucleotide compared to linear RNA makes circular polyribonucleotide more useful as a cell transformation reagent for producing polypeptides and can be stored more easily and longer than linear RNA. The stability of the exonuclease-treated circular polyribonucleotide can be assayed using methods standard in the art that determine whether RNA degradation has occurred (eg, by gel electrophoresis). Furthermore, unlike linear RNA, the circular polyribonucleotide is less susceptible to dephosphorylation when the circular polyribonucleotide is incubated with phosphatase, such as calf intestine phosphatase. nucleotide spacer sequences In some embodiments, the circular polyribonucleotide comprises a spacer sequence. In some embodiments, the circular polyribonucleotide comprises at least one spacer sequence. In some embodiments, the circular polyribonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or more spacer sequences. In some embodiments, the circular polyribonucleotide comprises a ratio of spacer sequence to non-spacer sequence of the circular polyribonucleotide, eg, expression sequences, of about 0.05:1, about 0.06:1, ncionn / nznz / E / Y about 0.07:1, about 0.08:1, about 0.09:1, about 0.1:1, about 0.12:1, about 0.125:1, about 0.15:1, about 0.175:1, about 0.2:1, about 0.225:1, about 0.25:1, about 0.3:1, about 0.35:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 0.98:1, about 1:1, about 1.02:1, about 1.05:1, about 1.1:1, about 1.15:1, about 1.2:1, about 1.25:1, about 1.3:1, about 1.35:1, about 1.4:1, about 1.45:1, about 1.5:1, about 1.55:1, about 1.6:1, about 1.65:1, about 1.7:1, about 1.75:1, about 1.8:1, about 1.85:1, about 1.9:1, about 1.95:1, about 1.975:1, about 1.98:1, or about 2:1. In some embodiments, the spacer sequence comprises a ratio of spacer sequence to a non-spacer element downstream (eg, 3' to the spacer sequence) of the circular polyribonucleotide of about 0.5:1, about 0.06:1, about 0.07: 1, about 0.08:1, about 0.09:1, about 0.1:1, about 0.12:1, about 0.125:1, about 0.15:1, about 0.175:1, about 0.2:1, about 0.225:1, about 0.25: 1, about 0.3:1, about 0.35:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75: 1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 0.98:1, about 1:1, about 1.02:1, about 1.05:1, about 1.1:1, about 1.15:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 1.95:1, about 1.975:1, about 1.98:1, about 2.1:1, about 2.2:1, about 2.3:1, about 2.4:1, about 2.5:1, about 2.6:1, about 2.7:1, about 2.8:1, about 2.9:1, about 3:1, about 3.1:1, about 3.2:1, about 3.3:1, about 3.4:1, about 3.5:1, about 3.6:1, about 3.7:1, about 3.8:1, about 3.85:1, about 3.9:1, about 3.95:1, about 3.98:1, or about 4:1. In some embodiments, the spacer sequence comprises a ratio of spacer sequence to a non-spacer element upstream (eg, 5' to the spacer sequence) of the circular polyribonucleotide of about 0.5:1, about 0.06:1, about 0.07: 1, about 0.08:1, ncionn / nznz / E / Y about 0.09:1, about 0.1:1, about 0.12:1, about 0.125:1, about 0.15:1, about 0.175:1, about 0.2:1, about 0.225:1, about 0.25:1, about 0.3:1, about 0.35:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 0.98:1, about 1:1, about 1.02:1, about 1.05:1, about 1.1:1, about 1.15:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 1.95:1, about 1.975:1, about 1.98:1, about 2.1:1, about 2.2:1, about 2.3:1, about 2.4:1, about 2.5:1, about 2.6:1, about 2.7:1, about 2.8:1, about 2.9:1, about 3:1, about 3.1:1, about 3.2:1, about 3.3:1, about 3.4:1, about 3.5:1, about 3.6:1, about 3.7:1, about 3.8:1, about 3.85:1, about 3.9:1, about 3.95:1, about 3.98:1, or about 4:1. In some embodiments, the spacer sequence comprises a sequence of 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 700 ribonucleotides, at least about 800 ribonucleotides, at least about 900 ribonucleotides, or at least about 100 ribonucleotides. ncionn / nznz / E / Y In some embodiments, the spacing sequence can be a nucleic acid sequence or molecule that has a low GC content, eg, less than 65%, 60%, 55%, 50%, 55%, 50%, 45%. , 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24 %, 23%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, along the full length of the spacer or along at least 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the contiguous nucleic acid residues of the spacer. In some embodiments, the spacing sequence may comprise at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 55%, 50%, 45% , 40%, 35%, 30%, 20% or any percentage in between of adenine ribonucleotides. In some embodiments, the spacing sequence comprises at least 5 or more consecutive adenine ribonucleotides. In some embodiments, the spacing sequence comprises at least 6 consecutive adenine ribonucleotides, at least 7 consecutive adenine ribonucleotides, at least 8 consecutive adenine ribonucleotides, at least about 10, at least about 12 consecutive adenine ribonucleotides, at least about 15 consecutive adenine ribonucleotides, at least about 20 consecutive adenine ribonucleotides, at least about 25 consecutive adenine ribonucleotides, at least about 30 consecutive adenine ribonucleotides, at least about 40 consecutive adenine ribonucleotides, at least about 50 consecutive adenine ribonucleotides , at least about 60 consecutive adenine ribonucleotides, at least about 70 consecutive adenine ribonucleotides, at least about 80 consecutive adenine ribonucleotides, at least about 90 ad ribonucleotides consecutive enine, at least about 95 consecutive adenine ribonucleotides, at least about 100 consecutive adenine ribonucleotides, at least about 150 consecutive adenine ribonucleotides, at least about 200 consecutive adenine ribonucleotides, at least about 250 consecutive adenine ribonucleotides, at least about 300 consecutive adenine ribonucleotides, at least about 350 consecutive adenine ribonucleotides, at least about 400 consecutive adenine ribonucleotides, at least about 450 consecutive adenine ribonucleotides, at least about 500 consecutive adenine ribonucleotides, at least about 550 adenine ribonucleotides consecutive, at least about 600 consecutive adenine ribonucleotides, at least about 700 consecutive adenine ribonucleotides, at least about 800 consecutive adenine ribonucleotides, to l at least about 900 consecutive adenine ribonucleotides or at least about 1000 consecutive adenine ribonucleotides. ncionn / nznz / Ε / γ In some embodiments, the spacing sequence is located between one or more elements. In some embodiments, the spacing sequence provides conformational flexibility between elements. In some embodiments, the conformational flexibility is due to the spacer sequence being substantially free of secondary structure. In some embodiments, the spacer sequence is substantially free of secondary structure, such as less than 40kcal / mol, less than -39, -38, -37, -36, -35, -34, 33, -32, -31 ,-30, -29, -28, -27, -26, -25, -24, -23, -22, -20, -19, -18, -17, -16, -15, -14, - 13, -12, 11, -10, -9, -8, -7, -6, -5, -4, -3, -2 or -1 kcal / mol. The spacer can include a nucleic acid, such as DNA or RNA. In some embodiments, the spacer sequence may encode an RNA sequence and preferably, a protein or peptide sequence, including a secretion signal peptide. In some embodiments, the spacing sequence may be non-coding. When the spacer is a non-coding sequence, a translation start sequence may be provided in the coding sequence of an adjacent sequence. In some embodiments, it is envisioned that the first nucleic acid residue of the coding sequence may be the A residue of a translational ininido sequence, such as AUG. When the spacer encodes an RNA or protein or peptide sequence, a translation definition sequence may be provided in the spacer sequence. In some embodiments, the spacer is operably linked to another sequence described herein. Non-nucleic acid linkers The circular polyribonucleotide described herein may also comprise a non-nucleic acid linker. In some embodiments, the circular polyribonucleotide described herein has a non-nucleic acid linker between one or more of the sequences or elements described herein. In one embodiment, one or more sequences or elements described herein are ligated to the linker. The non-nucleic acid linker can be a chemical bond, eg, one or more covalent bonds or non-covalent bonds. In some embodiments, the non-nucleic acid linker is a protein or peptide linker. Said linker can be between 2-30 amino acids or more. The linker includes flexible, rigid, or cleavable linkers described herein. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (GS linker). Flexible linkers can be useful for joining domains that require some degree of movement or interaction and can include small nonpolar (eg Gly) or polar (eg ncionn / nznz / E / Y) amino acids. Being or Thr). Incorporation of Ser or Thr can also maintain linker stability in aqueous solutions by forming hydrogen bonds with water molecules and thus reducing unfavorable interactions between linker and protein moieties. Hard linkers are useful for maintaining a fixed distance between domains and for keeping their functions independent. Rigid linkers may also be useful when a spatial separation of the domains is critical to preserve the stability or bioactivity of one or more components in the fusion. Rigid linkers may have an alpha-helical structure or a Pro, (XP)n-rich sequence, where X designates any amino acid, preferably Ala, Lys or Glu. Cleavable linkers can release free functional domains in vivo. In some embodiments, the linkers can be cleaved under specific conditions, such as the presence of reducing reagents or proteases. Cleavable linkers in vivo can utilize the reversible nature of a disulfide bond. An example includes a thromoin-sensitive sequence (eg, PRS) between the two Cys residues. In vitro thromoin treatment of CPRSCs results in cleavage of the thromoin-sensitive sequence, while the reversible disulfide bond remains intact. Such linkers are known and described, for example, in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369. In vivo cleavage of the linkers in the fusions can also be carried out by proteases that are expressed in vivo in pathologies (eg, cancer or inflammation), in specific cells or tissues, or restricted to certain cellular compartments. The specificity of many proteases offers slower cleavage of the linker in restricted compartments. Examples of linker molecules include a hydrophobic linker, such as a negatively charged sulfonate group; lipids, such as a poly(-CH2-) hydrocaride chain, such as a polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof, non-carbon linkers; carbohydrate binders; phosphodiester linkers or other molecule capable of covalently linking two or more polypeptides. Also included are non-covalent linkers, such as hydrophobic lipid globules to which the polypeptide is linked, for example, via a hydrophobic region of the polypeptide or a hydrophobic extension of the polypeptide, such as a leucine, isoleucine, valine-rich residue array. or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine, or some other hydrophobic moiety. The polypeptide can also be ligated using charge-based chemistry, such that a positively charged moiety of the polypeptide is ligated to a negative charge of another polypeptide or nucleic acid. ncionn / nznz / E / Y stability / half-life In some embodiments, the circular polyribonucleotide provided herein has an increased half-life relative to a reference, eg, a linear polyribonucleotide that has the same nucleotide sequence but is not circularized (linear homologue). In some embodiments, the circular polyribonucleotide is substantially resistant to degradation, eg, by exonucleases. In some embodiments, the circular polyribonucleotide is resistant to self-degradation. In some embodiments, the circular polyribonucleotide lacks an enzymatic cleavage site, eg, a dicer cleavage site. In some embodiments, the circular polyribonucleotide has a half-life of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%. %, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 120%, at least approximately 140% , at least approximately 150%, at least approximately 160%, at least approximately 180%, at least approximately 200%, at least approximately 300%, at least approximately 400%, at least approximately 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or at least about 10000% longer than a reference, for example, a linear counterpart. In some embodiments, the circular polyribonucleotide persists in a cell during cell division. In some embodiments, the circular polyribonucleotide persists in daughter cells after mitosis. In some embodiments, the circular polyribonucleotide is replicated within a cell and passed to daughter cells. In some embodiments, the circular polyribonucleotide comprises a replication element that mediates self-replication of the circular polyribonucleotide. In some embodiments, the replication element mediates the transcription of the circular polyribonucleotide into a linear polyribonucleotide that is complementary to the circular polyribonucleotide (linear complementary). In some embodiments, the linear complementary polyribonucleotide can be circularized in vivo in cells into a complementary circular polyribonucleotide. In some embodiments, the complementary polyribonucleotide can further self-replicate into another circular polyribonucleotide, which has the same or similar nucleotide sequence as the starting circular polyribonucleotide. An exemplary self-replicating element includes the HDC replication domain (as described by Beeharry et al, Virol, 2014, 450-451:165-173). In some embodiments, a cell passes at least one circular polyribonucleotide to daughter cells with ncionn / nznz / E / Y with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%. , 95% or 99%. In some embodiments, a cell undergoing meiosis passes the circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. . In some embodiments, a cell undergoing mitosis passes the circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. . Modifications The circular polyribonucleotide may include one or more substitutions, insertions, and / or additions, deletions, and covalent modifications to reference sequences, in particular, the parent polyribonucleotide, are included within the scope of the present invention. In some embodiments, the circular polyribonucleotide includes one or more post-translational modifications (eg, capping, cleavage, polyadenylation, splicing, poly-A sequencing, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the over one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P & McCIoskey, J. (1999). The FINA Modification Database: 1999 update. Nucí Acids Res 27: 196-197). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-ribonucleoside. thiopseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinometh l-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyluridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouhdine, 2-thio-1- methyl-pseudouridine, 1-methyl1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy¡-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1- methyl-pseudoisocytidine, pyrrolo-cytidine, pyrroloppseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methylpseudoisocytidine, 4-thio- 1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebularin, 5-aza-zebularin, 5-methyl-zebularin, 5-aza-2-thio-zebularin, 2thio-zebularin, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine and 4ncionn / nznz / Ε / γmethoxy¡-1-methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deza-adenine, 7-deza-8-aza-adenine, 7-deza-2-aminopurine, 7-deaza-8-aza-2aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cishydroxy¡sopenten¡l)adenosine, 2-methylthio-N6-(c¡s-hydroxy¡isopenten¡l) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threon¡l carbamoyladenosine , N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine and 2-methoxyadenine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wiosine, wibutosine, 7-deazaguanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl- 6-thio-guanosine and N2,N2-dimethyl-6-thioguanosine. The circular polyribonucleotide may include any useful modification, such as to the sugar, nucleobase, or internucleoside linkage (eg, to a phosphate linkage / to a phosphodiester bond / to a 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 (eg, methyl or ethyl), or halo (eg, chloro or fluoro). In certain embodiments, modifications (eg, one or more modifications) are present at each of the sugar and the internucleoside bond. The modifications may be ribonucleic acid (RNA) modifications in deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA) or hybrids of the same. Additional modifications are described in this document. In some embodiments, the circular polyribonucleotide includes at least one N(6)methyladenosine (m6A) modification to increase translational efficiency. In some embodiments, modification of N(6)methyladenosine (m6A) can reduce the immunogenicity of the circular polyribonucleotide. In some embodiments, the modification can include a cell-induced or chemical 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 in Nat Reviews Mol Cell Biol, 2017, 18:202-210. In some embodiments, chemical modifications to the ribonucleotides of the circular polyribonucleotide can enhance immune evasion. The circular ncionn / nznz / Ε / γ polyribonucleotide can 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 incorporated herein by reference. Modifications include, for example, terminal modifications, eg, 5' end modifications (mono-, di- and tri-) phosphorylation, conjugation, reverse linking, etc.), 3' end modifications (conjugation, DNA nucleotides , reverse bonds, etc.), base modifications (eg, replacement with stabilizing bases, destabilizing bases, or bases that form base pairs with an expanded partner repertoire), base removal (abasic nucleotides), or conjugated bases. Modified ribonucleotide bases can also include 5-methylcytidine and pseudouridine. In some embodiments, the base modifications can modulate the expression, immune response, stability, subcellular localization, to name a few functional effects, of the circular polyribonucleotide. In some embodiments, the modification includes biorthogonal nucleotides, eg, a non-natural base. See, for example, Kimoto et al, Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039 / c7cc06661a, which is incorporated herein by reference. In some embodiments, sugar modifications (eg, at the 2' position or 4j position or sugar replacements of one or more ribonucleotides of the circular polyribonucleotide, in addition to backbone modifications, may include modification or replacement of phosphodiester linkages.Specific examples of the circular polyribonucleotide include, but are not limited to, circular polyribonucleotides that include modified backbones or unnatural internucleoside linkages, such as internucleoside modifications, including the modification or replacement of phosphodiester linkages. have modified backbones include, but are not limited to, those that do not have a phosphorus atom in the backbone.For purposes of this application and as is sometimes cited in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered rse oligonucleosides. In particular embodiments, the circular polyribonucleotide will include ribonucleotides with a phosphorus atom in their internucleoside backbone. Modified circular polyribonucleotide backbones can 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 '-Aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates having normal 3'-5' bonds, 2'-5' bond analogues of these and those having reversed polarity, where adjacent pairs ncionn / nznz / E / Y of nucleoside units are linked from 3'-5' to 5'-3' or from 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. Modified nucleotides, which can be incorporated into the circular polyribonucleotide, can be modified at the internucleoside linkage (eg, phosphate backbone). Herein, in the context of polynucleotide backbone, the terms phosphate and phosphodiester are used interchangeably. The main chain phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. In addition, modified nucleosides and nucleotides can include random replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-bonding oxygens replaced by sulfur. The phosphate linker can also be modified by replacing a linking oxygen with nitrogen (bridged phosphoramides), sulfur (bridged phosphorothioates), and carbon (bridged methylene phosphonates). The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers via the unnatural phosphorothioate backbone linkages. Phosphorothioated DNA and RNA have increased resistance to nucleases and therefore 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 (for example, 5'-0-(1-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine (a- thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine or 5'-0-(1-thiophosphate)-pseudouridine). Other internucleoside linkages that may be employed in accordance with the present invention, including internucleoside linkages that do not contain a phosphorus atom, are described herein. In some embodiments, the circular polyribonucleotide can include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides can be incorporated into the circular polyribonucleotide, such as a bifunctional modification. The cytotoxic nucleoside may include, but is not limited to, adenosine arabinoside, 5-azacitidine, 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-Incionn / nznz / E / Y (tetrahydrofuran-2-yl)pyrimidin-2,4(IH,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'methylidenzytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1 -beta-D-arabinofuranosylcytosine, N4-octadecyl-1 -beta-Darabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy¡-beta- D-arabinopentofuranosyl)cytosine and P-4055 (5'-elaidic acid ester of cytarabine). The circular polyribonucleotide may or may not be uniformly modified throughout the entire length of the molecule. For example, one or more or all types of nucleotides (eg, naturally occurring, purine or pyrimidine nucleotides 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 predetermined sequence region thereof. In some embodiments, the circular polyribonucleotide includes a pseudouridine. In some embodiments, the circular polyribonucleotide includes an inosine, which can assist the immune system in characterizing the circular polyribonucleotide as endogenous against viral RNAs. Inosine incorporation 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 the circular polyribonucleotide (or in a given sequence region thereof) are modified. In some embodiments, the modification can include an m6A, which can increase expression; an inosine, which can attenuate an immune response; pseudouridine, which can increase RNA stability or translational readout (staggered element), an m5C, which can increase stability; and a 2,2,7-trimethylguanosine, which aids in subcellular translocation (eg, nuclear localization). Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (eg, backbone structures) may exist at various positions on the circular polyribonucleotide. One of ordinary skill in the art will appreciate that nucleotide analogs or other modifications can be located at any position on the circular polyribonucleotide such that the function of the circular polyribonucleotide is not substantially reduced. A modification can also be a non-coding region modification. The circular polyribonucleotide may include from about 1% to about 100% modified nucleotides (either based on overall nucleotide content or based on one or more nucleotide types, i.e., one or more of A, G , U or C) or any percentage in between (for example, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to ncionn / nznz / Ε / γ 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from a 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 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%). Structure In some embodiments, the circular polyribonucleotide comprises a higher order structure, eg, a secondary or tertiary structure. In some embodiments, the complementary segments of the circular polyribonucleotide fold themselves into a double-stranded segment, which is held together with pairwise hydrogen bonds, eg, A-U and C-G. In some embodiments, helices, also known as stems, are formed within the molecule, having a double-stranded segment connected to a terminal loop. In some embodiments, the circular polyribonucleotide has at least one segment with a quasi-double-stranded secondary structure. In some embodiments, a segment having a quasi-double-stranded secondary structure has at least 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, the circular polyribonucleotide has one or more segments (eg, 2, 3, 4, 5, 6, or more) that have a quasi-double-stranded secondary structure. In some embodiments, the segments are separated by 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 nucleotides. In some embodiments, one or more sequences of the circular polyribonucleotide include substantially single-stranded versus double-stranded regions. In some embodiments, the ratio of single-stranded to double-stranded may influence the functionality of the circular polyribonucleotide. In some embodiments, one or more sequences of the circular polyribonucleotide are substantially single-stranded. In some embodiments, one or more circular polyribonucleotide sequences that are substantially single-stranded may include a protein or RNA binding site. In some embodiments, circular polyribonucleotide sequences that are substantially single-stranded can be conformationally flexible to allow for increased interactions. In some embodiments, the sequence of the circular polyribonucleotide is purposely engineered to include such secondary structures for binding to or to increase binding to proteins or nucleic acids. ncionn / nznz / E / Y In some embodiments, the circular polyribonucleotide sequences are substantially double-stranded. In some embodiments, one or more circular polyribonucleotide sequences that are substantially double-stranded can include a conformational recognition site, eg, a riboswitch or aptazime. In some embodiments, circular polyribonucleotide sequences that are substantially double-stranded can be conformationally rigid. In some such cases, the conformationally rigid sequence may spherically prevent binding of the circular polyribonucleotide to a protein or nucleic acid. In some embodiments, the sequence of the circular polyribonucleotide is purposefully engineered to include such secondary structures to prevent or reduce protein or nucleic acid binding. There are 16 possible base pairings, although of these, six (AU, GU, GC, UA, UG, CG) can form actual base pairings. The rest are called mismatches and occur at very low frequencies in the helices. In some embodiments, the structure of the circular polyribonucleotide cannot be readily altered without impacting its function and lethal consequences, providing selection for maintaining secondary structure. In some embodiments, the primary structure of the stems (ie, their nucleotide sequence) can continue to vary, while still maintaining helical regions. The nature of the bases is secondary to the higher structure and substitutions are possible as long as they retain the secondary structure. In some embodiments, the circular polyribonucleotide has a quasi-helical structure. In some embodiments, the circular polyribonucleotide has at least one segment with a quasi-helical structure. In some embodiments, a segment having a quasi-helical structure has at least 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 nucleotides. In some embodiments, the circular polyribonucleotide has one or more segments (eg, 2,3,4,5, 6 or more) that have a quasi-helical structure. In some embodiments, the segments are separated by 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 nucleotides. In some embodiments, the circular polyribonucleotide includes at least one U-rich or A-rich sequence or a combination thereof. In some embodiments, the U-rich and / or A-rich sequences are arranged in a way that can produce a quasi-triple helical structure. In some embodiments, the circular polyribonucleotide has a double quasi-helical structure. In some embodiments, the circular polyribonucleotide has one or more segments (eg, 2, 3, 4, 5, 6, or more) that have a double quasihelical structure. In some embodiments, the circular polyribonucleotide includes at least one ncionn / nznz / E / Y of a C-rich and / or G-rich sequence. In some embodiments, the C-rich and / or G-rich sequences are arranged in a so that it can produce a quasi-triple helical structure. In some embodiments, the circular polyribonucleotide has a triplet quasi-helical intramolecular structure that aids in stabilization. In some embodiments, the circular polyribonucleotide has two quasi-helical structures (eg, separated by a phosphodiester bond), such that their terminal base pairs stack and the quasi-helical structures become collinear, resulting in a coaxially stacked substructure. . In some embodiments, the circular polyribonucleotide comprises a tertiary structure with one or more motifs, eg, a pseudoknot, a g-quadruplet, a helix, and coaxial stacking. In some embodiments, the circular polyribonucleotide has at least one binding site, eg, at least one protein binding site, at least one miRNA binding site, at least one scRNA binding site, at least one tRNA binding, at least one rRNA binding site, at least one snRNA binding site, at least one siRNA binding site, at least one piRNA binding site, at least one snRNA binding site, al at least one snRNA binding site, at least one exRNA binding site, at least one sRNA binding site, at least one Y RNA binding site, at least one hnRNA binding site, and / or at least one tRNA motif. Administration The circular polyribonucleotide described herein can also be included in pharmaceutical compositions with a delivery carrier. The pharmaceutical compositions described herein can be formulated, for example, including a carrier, such as a pharmaceutical carrier and / or a polymeric carrier, for example, a liposome and administered by known methods to a subject in need thereof (for example, a human or a non-human agricultural or domestic animal, e.g., cattle, dog, cat, horse, poultry). Such methods include, but are not limited to, transfection (eg, lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other membrane disruption methods (eg, nucleofection), viral delivery (eg, lentivirus, retrovirus, adenovirus, AAV), microinjection, microprojectile bombardment (gene gun), fugene, direct sonic charging, compression of cells, optical transfection, protoplast fusion, impalafection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. Delivery methods are also described, for example, in Gori et al., Delivery and Specificity of CRISPR / Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy. 2015 Jul, 26(7): 443-451. do¡:10.1089 / hum.2015.074; and Zuris et al. Cationic lipid-mediated delivery of ncionn / nznz / E / Y proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct 30;33(1):73-80. The invention further relates to a host or host cell comprising the circular polyribonucleotide described herein. In some embodiments, the host or host cell is a plant, insect, bacterium, fungus, vertebrate, mammal (eg, human), or other organism or cell. In some embodiments, the circular polyribonucleotide is non-immunogenic in the host. In some embodiments, the circular polyribonucleotide elicits a reduced or failed response by the host's immune system compared to the response elicited by a reference compound, for example, a linear polyribonucleotide corresponding to the described circular polyribonucleotide or a circular polyribonucleotide lacking of an encryptogen. Some immune responses include, but are not limited to, humoral immune responses (eg, antigen-specific antibody production) and cell-mediated immune responses (eg, lymphocyte proliferation). In some embodiments, a host or host cell is contacted with (eg, supplied or administered) the circular polyribonucleotide. In some embodiments, the host is a mammal, such as a human. The amount of circular polyribonucleotide, expression product, or both in the host can be measured at any time after administration. In certain embodiments, a time course of growth of the host in culture is determined. In the event that growth is increased or decreased in the presence of the circular polyribonucleotide, it is identified that either the circular polyribonucleotide or the expression product or both are effective in increasing or decreasing the growth of the host. production methods In some embodiments, the circular polyribonucleotide includes a deoxyribonucleic acid sequence that is naturally occurring and can be produced using recombinant technology (methods are described in detail below; for example, delivered in vitro using plasmid DNA) or chemical synthesis. . It is within the scope of the invention that a DNA molecule used to produce an RNA circle may comprise 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 (eg, chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, ncionn / nznz / E / Y chemical treatment of a nucleic acid molecule. to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, amplification and / or mutagenesis of selected regions of a nucleic acid sequence by polymerase chain reaction (PCR), synthesis of oligonucleotide mixtures and mixed group ligation to construct a mixture of nucleic acid molecules and combinations thereof. The circular polyribonucleotide can be prepared according to any available technique including, but not limited to, chemical synthesis and enzymatic synthesis. In some embodiments, a linear primary construct or linear mRNA can be cyclized or concatemerized to create a circular polyribonucleotide described herein. The cycling or concatemerization mechanism can occur by methods such as, but not limited to, chemical, enzymatic, bridging, or ribozyme-catalyzed methods. The newly formed 5'73' bond may be an intramolecular bond or an intermolecular bond. Methods for producing the circular polyribonucleotides described herein are described, for example, in Khudyakov and Fields, Artificial DNA: Methods and Applications, ORO Press (2002); in Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); and Egli and Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012). Various methods for synthesizing circular polyribonucleotides have also been described in the art (see, for example, US Patent No. 2US6210931, US Patent No. 2US5773244, US Patent No. 2US5766903, United States Patent No. 2US5712128, United States Patent No. 2US5426180, United States Publication No. 2US20100137407, International Publication No. 2WO1992001813, and International Publication No. 2WO2010084371, the contents of each being incorporated into the this document by reference in its entirety). In some embodiments, circular polyribonucleotides can be cleaned after production to remove production impurities, eg, free ribonucleic acids, linear or fragmented RNA, DNA, proteins, etc. In some embodiments, the circular polyribonucleotides can be purified by any known method commonly used in the art. Examples of non-limiting purification methods include column chromatography, gel cleavage, size exclusion, etc. pharmaceutical compositions The present invention includes compositions in combination with one or more pharmaceutically acceptable excipients. The pharmaceutical compositions may optionally comprise one or more additional active substances, for example ncionn / nznz / E / Y and / or prophylactically active therapeutic substances. The pharmaceutical compositions of the present invention may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents can be found, for example, in Remington: The Science and Practice of Pharmacy 21.- ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Although the descriptions of pharmaceutical compositions provided herein refer primarily to pharmaceutical compositions that are suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, for example , to non-human animals, eg, non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to various animals is well known and the veterinary pharmacologist ordinarily skilled in the art can design and / or effect such modification with merely routine experimentation. , if there were. Subjects for 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 poultry, including commercially relevant poultry, such as poultry, chickens, ducks, geese, and / or turkeys. Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereinafter developed in the art of pharmacology. In general, such methods of preparation include the step of bringing the active ingredient into association with an excipient and / or one or more additional accessory ingredients and then, if necessary and / or desirable, dividing, shaping and / or packaging the product. expression methods The present invention includes a method for protein expression, comprising translating at least one region of the circular polyribonucleotide provided herein. In some embodiments, the methods for protein expression comprise translation of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% , at least 70%, at least 80%, at least 90%, or at least 95% of the total length of the circular polyribonucleotide in polypeptides. In some embodiments, methods for protein expression comprise translation of the circular polyribonucleotide into polypeptides of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, ncionn / nznz / E / And at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids or at least 1000 amino acids. In some embodiments, methods for protein expression comprise translation of the circular polyribonucleotide into polypeptides of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, or about 1000 amino acids. In some embodiments, the methods comprise translation of the circular polyribonucleotide into continuous polypeptides as provided herein, isolated polypeptides as provided herein, or both. In some embodiments, translation of the at least one region of the circular polyribonucleotide occurs in vitro, such as in rabbit reticulocyte lysate. In some embodiments, translation of the at least one region of the circular polyribonucleotide occurs in vivo, for example, after transfection of a eukaryotic cell or transformation of a prokaryotic cell, such as a bacterium. In some aspects, the present disclosure provides methods of in vivo expression of one or more expression sequences in a subject comprising: administering a circular polyribonucleotide to a cell of the subject, wherein the circular polyribonucleotide comprises the one or more expression sequences; and expressing the one or more circular polyribonucleotide expression sequences in the cell. In some embodiments, the circular polyribonucleotide is configured such that the expression of the one or more expression sequences in the cell at a later time point is equal to or greater than at an earlier time point. In some embodiments, the circular polyribonucleotide is configured such that expression of the one or more expression sequences in the cell over a period of time of at least 7, 8, 9, 10, 12, 14, 16 , 18, 20, 22, 23 or more days is not reduced by more than approximately 40%. In some embodiments, the circular polyribonucleotide is configured such that expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than about 40% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days. In some embodiments, administration of the circular polyribonucleotide is carried out ncionn / nznz / E / Y using any method of administration described herein. In some embodiments, the circular polyribonucleotide is administered to the subject by intravenous injection. In some embodiments, administration of the circular polyribonucleotide includes, but is not limited to, prenatal administration, neonatal administration, postnatal administration, oral, by injection (eg, intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous, and intramuscular), by ophthalmic administration, and by intranasal administration. In some embodiments, methods for protein expression comprise post-translational modification, folding, or other modification of the translation product. In some embodiments, methods for protein expression comprise post-translational modification in vivo, eg, by cellular machinery. All references and publications cited herein are hereby incorporated by reference. The above-described embodiments can be combined to achieve the aforementioned functional characteristics. This is also illustrated by the examples below, which set forth exemplary combinations and the functional characteristics achieved. Table 1 provides an exemplary overview showing how the different elements described above can be combined and the functional characteristics observed. Table 1. Exemplary elements in EXAMPLES ncionn / nznz / E / Y Elements (eg, staggered codon, encryptogen, IRES, etc.) start, circular polyribonucleotide Effect element Replication element Expression sequence Stepping element Regulatory element Encrypto gene Secondary structure quasi-double-stranded aria Exemplary function Start of transcription Product coding Ribosomal pause; translate! Rolling circle modifier of expression Modulation of immune response Exam lo 3 X X X Greater translation efficiency than a linear homologue Exam lo 4 X X X Stoichiometric efficiency of translation of multiple translation products Example 5 Example 9 Example 44 Example 47 X X Less immunogenicity than homologue lacking an encryptogen Example x lo 13 Example 14 X X X Increased half-life vs. linear homologue Example 15 X X X Persistence during cell division Example 18 Example 29 X X Increased half-life vs. linear homologue Example 30 X X X Increased half-life vs. linear homologue Example 38 Example 39 X X X Higher translation efficiency than linear homologue Example 10 Example 12 Example 40 Example 41 X Example 48 X Persistence during cell division Example 49 X X Greater translation efficiency than a linear homologue Example 6 Example 52 X X ncionn / nznz / E / Y Exam lo 53 X X Less immunogenicity than homologue lacking an encryptogen Exam X X Greater 54 translation efficiency than 55 linear homologue; Increased half-life vs. a linear counterpart; Less immunogenicity than the homologue lacking an encryptogen ncionn / nznz / E / Y EXAMPLES The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies or techniques known to those skilled in the art may be used as an alternative. Example 1: Production of circular RNA in vitro This example demonstrates the in vitro production of a circular RNA. A circular RNA is designed with a start codon (SEQ ID NO: 1), one or more ORFs (SEQ ID NO: 2), one or more staggering elements (SEQ ID NO: 3), one or more encryptogens (SEQ ID NO: 4) and an IRES (SEQ ID NO: 5), shown in Figure 2. Circularization allows rolling circle translation, multiple open reading frames (ORFs) with alternating stagger elements for isolated ORF expression and controlled stoichiometry of the protein, one or more encryptogens to attenuate or mitigate RNA immunogenicity, and an optional IRES that directs RNA to ribosomal entry without poly-A sequence. In this example, the circular ARN is generated as follows. Unmodified linear RNA is synthesized by in vitro transcription using T7 RNA polymerase from a DNA segment having ZKSCAN1 5' and 3' introns and an ORF encoding GFP ligated to 2A sequences. The RNA transcript is purified with an RNA purification system (QIAGEN), treated with alkaline phosphatase (ThermoFisher Scientific, EF0652) following the manufacturer's instructions, and further purified with the RNA purification system. The circular RNA bridging ligation is generated by treatment of the transcribed linear RNA and a bridging DNA using T4 DNA ligase (New England Bio, Inc., M0202M) and the circular RNA is isolated after enrichment with RNase R treatment. RNA quality is assessed by agarose gel or by automated electrophoresis (Agilent). Example 2: Production of circular RNA in vivo, cell culture This example demonstrates the in vivo production of circular RNA. GFP (SEQ ID NO: 2) is cloned into an expression vector, eg, pcDNA3.1(+) (Addgene) (SEQ ID NO: 6). This vector is mutagenized to induce the production of circular RNA in cells (SEQ ID NO: 6 and described by Kramer et al 2015), shown in Figure 3. HeLa cells are grown at 37°C, 5% CO 2 in high glucose Dulbecco's Modified Eagle's medium (DMEM) (Life Technologies), supplemented with penicillin-streptomycin and 10% fetal calf serum. One microgram of the above-described expression plasmid is transferred using lipid transfection reagent (Life Technologies) and total RNA is isolated from transfected cells using phenol-based RNA isolation reagent (Life Technologies) according to manufacturer's instructions. for between 1 hour and 20 days after transfection. To measure circular RNA and mRNA levels for GFP, reverse transcription qPCR is performed using random hexamers. Briefly, for RT-qPCR, total RNA from HeLa cells and RNase R digested RNA from the same source are used as templates for RT-PCR. To prepare the cDNAs of the mRNAs for GFP and the circular RNAs for GFP, reverse transcription reactions are carried out with a reverse transcriptase (Super-Script II: RNase H; Invitrogen) and random hexamers according to the manufacturer's instructions. Amplified PCR products are analyzed using 6% PAGE and visualized by ethidium bromide staining. To estimate the enrichment factor, PCR products are quantified by densitometry (ImageQuant; Molecular Dynamics) and total RNA sample concentrations are measured by UV absorbance. Further RNA measurement is carried out with Northern blot analysis. Briefly, a complete cell extract was obtained using a phenol-based reagent (TRIzol) or nuclear and cytoplasmic protein extracts were obtained by fractionating the cells with a commercial kit (CelLytic NuCLEAR Extraction Kit, Sigma). To inhibit RNA polymerase II transcription, cells are treated with flavopiridol (1 mM final concentration; Sigma) for 0-6 h at 37°C. For RNase R treatments, 10 mg of total RNA is treated with 20 U of RNase R (Epicentre) for 1 h at 37°C. Northern blots using oligonucleotide probes are carried out as follows. Oligonucleotide probes and PCR primers are designed using standard primer design tools. Add ncionn / nznz / E / Y promoter sequence Τ7 to the reverse primer to obtain an antisense probe in the in vitro transcription reaction. In vitro transcription is carried out using T7 RNA polymerase with DIG-RNA labeling mix according to the manufacturer's instructions. DNA templates are removed by DNase I digestion and RNA probes are purified by phenol-chloroform extraction and subsequent precipitation. The probes are used at 50ng / ml. Total RNA (2 pg - 10 pg) is denatured using Glyoxal loading stain (Ambion) and resolved on a 1.2% agarose gel in MOPS buffer. The gel is soaked in 1 xTBE for 20 min and transferred to a Hybond-N+ membrane (GE Healthcare) for 1 h (15 V) using a semi-dry transfer system (Bio-Rad). The membranes are dried and crosslinked by UV (at 265 nm) 1 χ at 120,000 pJ cm-2. Prehybridization is performed at 68 °C for 1 h and DIG-labeled in vitro transcribed RNA probes are hybridized overnight. Membranes are washed three times in 2x SSC, 0.1% SDS at 68°C for 30 min, followed by three 30 min washes in 0.2x SSC, 0.1% SDS at 68°C. Immunodetection is carried out with anti-DIG antibodies conjugated directly to alkaline phosphatase. Immunoreactive bands are visualized using alkaline phosphatase chemiluminescent substrate (CDP Star reagent) and an image detection and quantification system (LAS-4000 detection system). Example 3: Preparation of circular RNA and translation in vitro This example demonstrates gene expression and gene product detection from circular RNA. In this example, the circular RNA is designed with a start codon (SEQ ID NO: 1), a GFP ORF (SEQ ID NO: 2), one or more stagger elements (SEQ ID NO: 3), one or more plus encryptogens of human origin (SEQ ID NO: 4) and with or without an IRES (SEQ ID NO: 5), see Figure 4. In this example, circular RNA is generated either in vitro or in cells, as shown. described in example 1 and 2. Circular RNA is incubated for 5 h or overnight in rabbit reticulocyte lysate (Promega, Fitchburg, WI, USA) at 30 °C. The final composition of the reaction mixture includes 70% rabbit reticulocyte lysate, 10 pM methionine and leucine, 20 pM non-methionine and leucine amino acids, and 0.8U / pL RNase inhibitor (Toyobo, Osaka, Japan). Aliquots of the mixture are taken and separated on 10-20% polyacrylamide / sodium dodecyl sulfate (SDS) gradient gels (Atto, Tokyo, Japan). The supernatant is removed and the pellet is dissolved in 2x SDS sample buffer (0.125 M Tris-HCI, pH 6.8, 4% SDS, 30% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue). at 70 °C for 15 min. The hemoglobin protein is removed during this process, while the non-hemoglobin proteins are concentrated. ncionn / nznz / E / Y After centrifugation at 1,400x g for 5 min, the supernatant is analyzed on 10-20% polyacrylamide / SDS gradient gels. A commercially available standard (BioRad) is used as a size marker. After electroblotting onto a polyvinylidene fluoride (PVDF) membrane (Millipore) using a semi-dry method, the blot is visualized using a chemiluminescent kit (Rockland). GFP protein is expected to be visualized in cellular Usados ​​and detected in higher amounts in circular RNA than in linear RNA, as a result of rolling circle translation. Example 4: Stoichiometric Expression of Protein from Circular RNA This example demonstrates the ability of circular RNA for stoichiometric expression of proteins. In this example, a circular RNA is designed to include encryptogens (SEQ ID NO: 4) and an ORF encoding GFP (SEQ ID NO: 2) and an ORF encoding RFP (SEQ ID NO: 8) with staggered elements. (SEQ ID NO: 3) flanking the GFP and RFP ORFs, see Figure 5. Another circular RNA is designed in a similar way, although instead of flanking 2A sequences it will have a stop and start codon in between. the ORFs of GFP and RFP. Circular RNAs are generated either in vitro or in cells as described in example 1 and 2. Circular RNAs are incubated for 5 h or overnight in rabbit reticulocyte lysate (Promega, Fitchburg, WI, USA) at 30 °C. The final composition of the reaction mixture includes 70% rabbit reticulocyte lysate, 10 μΜ methionine and leucine, 20 μΜ non-methionine and leucine amino acids, and 0.8 U / pL RNase inhibitor (Toyobo, Osaka, Japan). Aliquots of the mixture are taken and separated on 10-20% polyacrylamide / sodium dodecyl sulfate (SDS) gradient gels (Atto, Tokyo, Japan). The supernatant is removed and the pellet is dissolved in 2x SDS sample buffer (0.125 M Tris-HCI, pH 6.8, 4% SDS, 30% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue). at 70 °C for 15 min. The hemoglobin protein is removed during this process, while the non-hemoglobin proteins are concentrated. After centrifuging at 1,400*g for 5 min, the supernatant is analyzed on 10-20% polyacrylamide / SDS gradient gels. A commercially available standard (BioRad) is used as a size marker. After electroblotting onto a polyvinylidene fluoride (PVDF) membrane (Millipore) using a semi-dry method, the blot is visualized using a chemiluminescent kit (Rockland). Circular RNA with ORFs for GFP and RFP not separated by a stop and start codon are expected to have equal amounts of either protein, while ncionn / nznz / E / Y cells treated with the circular RNA, including the start and stop codon between the ORFs will have different amounts of either protein. Example 5: Lack of immunogenicity in cell culture This example demonstrates the in vivo assessment of circular RNA immunogenicity after cell infection. In this example, the circular RNAs are designed to include an encryptogen, eg, a ZKSCAN1 intron and a GFP ORF. In addition, control circular RNA is designed to include a GFP ORF with and without introns, see Figure 6. Circular RNA is generated either in vitro or in cells as described in example 1 and 2. HeLa cells are transfected with 500 ng of circular RNAs. Circular RNA transfection includes the following conditions: (1) naked circular RNA in cell culture medium (Lingor et al 2004); (2) electroporation (Muller et al 2015); (3) cationic lipids (SNALP, Vaxfectin) (Chesnoy and Huang, 2000); (3) cationic polymers (PEI, polybrene, DEAE-dextran) (Turbofect); (4) virus-like particles (HPV L1, polyomavirus VP1) (Tonges et al 2006); (5) exosomes (Exo-Fect from SBI); (6) nanostructured calcium phosphate (nanoCaP) (Olton et al 2006); (6) peptide transduction domains (TAT, polyR, SP, pVEC, SynB1, etc.) (Zhang et al 2009); (7) vesicles (VSV-G, TAMEL) (Liu et al 2017); (8) cell compression; (SQZ Biotechnologies) (9) nanoparticles (Neuhaus et al 2016); and / or (10) magnetofection (Mair et al 2009). Transfection methods are carried out in cell culture medium (DMEM 10% FBS) and cells are subsequently cultured for 24-48 h. After 2-48 h post-transfection, the medium is removed and the relative expression of the indicated RNA and the transfected RNA is measured by qRT-PCR. For qRT-PCR analysis, total RNA is isolated from cells using phenol-based RNA isolation solution (TRIzol) and RNA isolation kit (QIAGEN) following the manufacturer's instructions. qRT-PCR analysis is performed in triplicate using a PCR master mix (Brilliant II SYBR Green qRT-PCR master mix) and a PCR cycler (LightCycler 480). mRNA levels for well-known regulators of innate immunity such as RIG-I, MDA5, OAS, OASL, and PKR are quantitated and normalized to actin, GAPDH, or HPRT values. The relative expression of RNA genes indicated for circular RNA transfection is normalized by the level of transfected RNA and compared to the expression level of circular RNA transfected cells that do not contain one or more encryptogens. In addition to qRT-PCR analysis, Western blot analysis and immunohistochemistry, as described above in Example 4, are used to assess the efficiency of GFP expression. ncionn / nznz / E / Y GFP-positive cells containing one or more encryptogens are expected to show an attenuated immunogenic response. In addition, (1) primary murine dendritic cells are transfected; (2) human embryonic kidney 293 cells stably expressing TLR-7,8 or 9 (InvivoGen); (3) monocyte-derived dendritic cells (AlICells) or (4) Raw 264.7 cells with a DNA plasmid including the ZKSCAN1 or td introns producing a circular RNA encoding GFP as described above. After 6-48 hours post-transfection, the cell culture supernatant is collected and cytokine expression is measured using ELISA. When the culture supernatant is collected, the cells are collected for Northern blotting, gene expression matrix, and FACS analysis. For the ELISA, ELISA kits are used for interferon-β (IFN-β), chemokine ligand 5 (C-C motif) (CCL5), IL-12 (BD Biosciences), IFN-α, TNF-α, and IL-8. (Biosource International). ELISAs are carried out according to the manufacturer's recommendations. Expression of the indicated cytokines for circular RNA-transfected cells are compared to the level of control RNA-transfected cells. Cells transfected with circular RNA with an encryptogen are expected to have reduced cytokine expression compared to control transfected cells. For Northern blot analysis, samples are processed and analyzed as described above. The probes are derived from plasmids and are specific for the coding regions of human IFN-alpha, IFN-beta (Open Biosystems), TNF-alpha or GAPDH (ATOO). Cells transfected with circular RNA with an encryptogen are expected to have reduced cytokine expression compared to control transfected cells. For the gene expression matrix, RNA is isolated using a phenol-based solution (TRIzol) and / or an RNA isolation kit (RNeasy, Qiagen). The RNA is amplified and analyzed (eg, lllumina Human HT12v4 chip on an lllumina BeadStation 500GX device). Levels in mock control treated cells are used as reference value for calculation of the multiple of increase. Cells transfected with circular RNA with an encryptogen are expected to have reduced cytokine expression compared to control transfected cells. For FACS analysis, cells are stained with an antibody conjugated directly against CD83 (Research Diagnostica Inc), HLA-DR, CD80 or CD86 and analyzed in a flow cytometer. It is expected that cells transfected with circular RNA with an encryptogen will show reduced expression of these markers compared to control transfected cells. ncionn / nznz / E / Y Example 6: Riboswitches for selective expression This example demonstrates the ability to control protein expression from circular RNA in vivo. For this example, the circular RNAs are designed to include one or more encryptogens (SEQ ID NO: 4), a synthetic riboswitch (SEQ ID NO: 9) that regulates the expression of the ORF encoding GFP (SEQ ID NO: 2) with staggered elements (2A sequences) (SEQ ID NO: 3) flanking the GFP ORF, see Figure 7. Circular RNA is generated either in vitro or in cells as described in example 1 and 2. Theophylline induces riboswitch activation, resulting in inactivation of gene expression (as described by Auslander, et al. 2010). The riboswitch is expected to control GFP expression from circular RNA. In the presence of theophylline, GFP expression is not expected to be observed. HeLa cells are transfected with 500 ng of the described circular RNA encoding GFP under the control of the synthetic theophylline-dependent riboswitch (SEQ ID NO: 9) to assess selective expression. Transfection methods are described in Example 5. After 24 h of culture at 37 °C and 5% CO2, cells are treated with and without theophylline, with concentrations in the range of 1 nM-3 mM. After 24 h of continuous culture, cells are fixed in 4% paraformaldehyde for 15 min at room temperature, blocked and permeabilized for 45 min with 10% FBS in PBS with 0.2% detergent. Samples are then incubated with primary antibodies against GFP (Invitrogen) and secondary antibodies conjugated to Alexa 488 and DAPI (Invitrogen) in PBS with 10% FBS and 0.1% detergent for 2 h at room temperature or overnight at 4 °C. c. Cells are then washed with PBS and subsequently analyzed using a fluorescence microscope for GFP expression. Example 7: Expression in vivo This example demonstrates the ability to express protein from circular RNA in vivo. For this example, circular RNAs are designed including one or more encryptogens (SEQ ID NO: 4) and an ORF encoding GFP (SEQ ID NO: 2) or RFP (SEQ ID NO: 8) or luciferase (SEQ ID NO: 10) with staggered element (SEQ ID NO: 3) flanking the ORF of GFP, RFP or luciferase, see Figure 8. Circular RNA is generated either in vitro or in cells as described in example 1 and 2 . Male 6-8 week old BALB / c mice receive 300 mg / kg (6 mg) of circular RNA (50 uL vol) with ORFs for GFP, RFP, or luciferase, as described herein, or linear RNA as control. , by intradermal (ID), intramuscular (IM), oral (PO), intraperitoneal (IP), or intravenous (IV) administration. Animals receive a single dose or three injections (day 1, day 3, day 5). ncionn / nznz / Ε / γ Blood, heart, lung, spleen, kidney, liver and skin are collected from the injection sites of the control mice without administration and at 2, 4, 8, 24, 48, 72, 96, 120, 168 and 264 h later. of administration (n = 4 mice / time point). Blood samples are collected by jugular venipuncture at the end of the study. Circular RNA quantification is performed in both serum and tissues using branched DNA (rDNA) quantification (Panomics / Affymetrix). A standard curve on each plate of known amounts of RNA (added to untreated tissue samples) is used to quantify RNA in treated tissues. The calculated amount in picograms (pg) is normalized to the amount of tissue weighed in the lysate applied to the plate. Protein expression (RFP or GFP) is assessed by FACS or Western blotting in each tissue, as described in a previous example. A separate group of mice given circular RNA for luciferase are injected with 3 mg of luciferin at 6, 24, 48, 72, and 96 h post-dose and the animals are imaged on an imaging system. in vivo imaging (MS Spectrum, PerkinElmer). At 6 h post-dose, three animals are sacrificed and ex vivo muscle, skin, draining lymph nodes, liver and spleen are dissected and imaged. Mice are expected to express GFP, RFP, or luciferase in the treated tissues. Example 8: Biodistribution in vivo This example demonstrates the ability to monitor and measure the biodistribution of circular RNA in vivo. In this example, mice are treated with the circular RNA encoding luciferase as described in Example 9. Briefly, circular RNAs are designed by including one or more encryptogens (SEQ ID NO: 4) and an ORF encoding luciferase. (SEQ ID NO: 10) with staggered element (SEQ ID NO: 3) flanking the luciferase ORF, see Figure 9. Circular RNA is generated either in vitro or in cells as described in Example 1 and 2. Mice given circular RNA for luciferase are injected with 3 mg luciferin at 6, 24, 48, 72, and 96 h post-dose and the animals are imaged on an in-line imaging system. live (IVIS Spectrum, PerkinElmer). At 6 h post-dose, three animals are sacrificed and ex vivo muscle, skin, draining lymph nodes, liver and spleen are dissected and imaged. Circular RNA quantification is performed in both serum and tissues using branched DNA (rDNA) quantification (Panomics / Affymetrix). A standard curve on each plate of known amounts of RNA (added to untreated tissue samples) is used to quantify RNA in treated tissues. The calculated amount in picograms (pg) is normalized to the amount of tissue weighed in the lysate applied to the plate. ncionn / nznz / E / Y A separate group of 6-8 week old BALB / c mice are given circular RNA for luciferase by IM or ID administration at four dose levels: 10, 2, 0.4 and 0.08 mg (n=6 per group). At 6, 24, 48, 72 and 96 h after administration, animals are injected with 3 mg of Iuciferin and imaged on an in vivo imaging system (IVIS Spectrum, PerkinElmer). At 6 h post-dose, three animals are sacrificed and ex vivo muscle, skin, draining lymph nodes, liver and spleen are dissected and imaged. Luciferase expression in the tissues of the mice was also evaluated in Example 9 and the tissue distribution of this expression is discussed. Mice are expected to show luciferase expression in the treated tissues. Example 9: Lack of immunogenicity in vivo This example demonstrates the in vivo assessment of circular RNA immunogenicity after cell infection. This example describes the quantification and comparison of the immune response after administrations of circular RNA carrying an encryptogen, see Figure 10. In one embodiment, any of the RNAs carrying an encryptogen will have a reduced immunogenic response (eg, reduced compared to administration of control RNA) after one or more administrations of circular RNA compared to control. One measure of immunogenicity for circular RNA is serum cytokine levels. In this example, serum cytokine levels are examined after one or more administrations of circular RNA. Circular RNA from any one of the above examples is administered intradermally (ID), intramuscularly (IM), orally (PO), intraperitoneally (IP), or intravenously (IV) in 6-8 week old BALB / c mice. . Serum is drawn from the different cohorts: mice injected systemically and / or locally with one or more injections of circular RNA carrying an encryptogen and circular RNA without an encryptogen. The collected serum samples are diluted 1-1 Ox in PBS and assayed for mouse IFN-α by enzyme-linked immunosorbent assay (PBL Biomedical Labs, Piscataway, NJ) and TNF-α (R&D, Minneapolis, MN). In addition to serum cytokine levels, measurement of inflammatory markers is another measure of immunogenicity. In this example, spleen tissue from mice treated with vehicle (no circular RNA), linear RNA or circular RNA will be collected 1, 4 and 24 hours after administration. Samples will be analyzed using the following techniques: qRTPCR analysis, Northern blot or FACS analysis. For qRT-PCR analysis, mRNA levels for RIG-I, MDA5, OAS, OASL, TNF-alpha, and PKR are quantitated as described above. ncionn / nznz / E / Y For Northern blot analysis, samples are processed and analyzed for IFN-alpha 13 , IFN-beta (Open Biosystems), TNF-alpha or GAPDH (ATCC) as described above. For FACS analysis, cells are stained with an antibody conjugated directly against CD83 (Research Diagnostics Inc), HLA-DR, CD80, or CD86 and analyzed in a flow cytometer. In one embodiment, the circular RNA with an encryptogen will have reduced levels of cytokines (as measured by ELISA; Northern blot, FACS and / or qRT-PCR) after one or multiple administrations, compared to the control RNA. Example 10: Circular RNA includes at least one double-stranded RNA segment This example demonstrates that circular RNA includes at least one double-stranded RNA segment. In this example, circular RNA is synthesized by one of the methods described above, to include a GFP ORF and an IRES, see Figure 11. Dot blot assays with J2 and K1 monoclonal antibodies will be used to measure the structures of Double-stranded RNAs of at least 40 bp in length. Circular RNA (200 ng) is transferred to a nylon membrane (supercharged Nytran), dried, blocked with 5% defatted dry milk in TBS-T buffer (50mM Tris—HCl, 150mM NaCl, 0.05 Tween-20). %, pH 7.4) and incubate with mAb J2 or K1 specific for dsRNA (English & Scientific Consulting) for 60 min. Membranes are washed six times with TBS-T and then treated with HRP-conjugated donkey anti-mouse lg antibody (Jackson Immunology), then washed six times and spots visualized with Western blot detection reagent. enhanced chemiluminescence (Amersham). A circular RNA is expected to create a quasi-double-stranded internal RNA segment. Example 11: Circular RNA including a quasi-double-stranded secondary structure This example demonstrates that circular RNA includes quasi-double-stranded secondary structure. In this example, circular RNA is synthesized by one of the methods described above, with and without the addition of HDVmin expression (Griffin et al. 2014). This RNA sequence forms a quasi-helical structure, see Figure 12, and is used as a positive control (as shown by Griffin et al. 2014). To assess whether the structure of the circular RNA includes quasi-double-stranded secondary structure, the secondary structure will be determined using selective 2ΌΗ acylation by primer extension (SHAPE). SHAPE assesses the local flexibility of the backbone in RNA with a single nucleotide resolution. The reactivity of the base positions with the SHAPE electrophile is related to the secondary structure: ncionn / nznz / E / Y base-paired positions are weakly reactive, while unpaired positions are more highly reactive. SHAPE is carried out on circular RNA, HDVmin, and containerized linear RNA. SHAPE is carried out with N-methylisatoic anhydride (NMIA) or benzoyl cyanide (BzCN) essentially as reported by Wilkinson et al 2006 and Griffin 2014 et al, respectively. Briefly, for SHAPE with BzCN, 1 ul of 800 mM BzCN in dimethyl sulfoxide (DMSO) is added to a 20 ul reaction mix containing 3 to 6 pmol of RNA in 160 mM Tris, pH 8.0, 1 U / L of RNase inhibitor (eg, RNase Inhibitor Get Over It) and incubated for 1 min at 37°C. Reaction mixes include 1 ul of DMSO without BzCN. After incubation with BzCN, RNAs are phenol-chloroform extracted and purified (eg using RNA Clean & Concentrator-5 kit) following manufacturer's instructions and resuspended in 6ul 10mM Tris, pH 8.0. A one dye system is used to detect BzCN adducts. RNAs are hybridized with a 6-carboxyfluorescein (6FAM) labeled primer. Primer extension is carried out using a reverse transcriptase (SuperScript III Invitrogen) according to the manufacturer's recommendations, with the following modifications in incubation conditions: 5 min at 42°C, 30 min at 55°C, 25 min at 65°C and 15 min at 75°C. Two sequencing runs are generated using either 0.5 mM ddATP or 0.5 mM ddCTP in the primer extension reaction. Primer extension products are ethanol precipitated, washed to remove excess salt and resolved by capillary electrophoresis together with a commercial size standard (eg Liz size standard, Genewiz Fragment Analysis Service). Raw electropherograms are generated using a primary fragment analysis tool (eg, PeakScanner, Applied Biosystems). Then, the peaks at each position on the electropherogram are integrated. For each RNA tested, y-axis scaling is performed to correct for loading error such that the background for each primer extension reaction is normalized to that of a negative control reaction run on RNA that it is not treated with BzCN. A signal attenuation correction is applied to the data for each reaction. The peaks are aligned with a scale created from two sequencing reactions. At each position, the peak area of ​​the negative control is subtracted from the peak area in the BzCN-treated samples; these values ​​are then converted to normalized SHAPE reactivities by dividing the subtracted peak areas by the mean of the 2% to the maximum 10% of the subtracted peak areas. In addition to the SHAPE analysis, NMR will be carried out (Marchanka et al 2015); hydroxyl radical probing (Ding et al 2012); or a combination of DMS and CMTC and ketoxal (Tijerina et al 2007 and Ziehler et al 2001). A circular RNA is expected to have a quasi-double-stranded secondary structure. nc i οηη / ηζηζ / Ε / γ Example 12: Circular RNA includes a functional quasi-helical structure This example demonstrates that circular RNA includes a functional quasi-helical structure. In this example, circular RNA is synthesized by one of the methods described above, with the addition of 395L expression (Defenbaugh et al. 2009). This RNA sequence forms a quasi-helical structure (as shown previously, using the mfold RNA secondary structure folding algorithm and Defenbaugh et al 2009), Figure 13. This structure is essential for complexation with antigen. hepatitis D (agHDA). Therefore, to assess whether the circular RNA structure includes a functional quasi-structure, circular RNA and linear RNA will be incubated with HDAg-160 or HDAg-195 and analyzed for binding using EMSA assays. Binding reactions are performed in 25ul including 10 mM Tris-HCI (pH 7.0), 25 mM KCI, 10 mM NaCI, 0.1 g / L bovine serum albumin (New England Biolabs), 5% glycerol, 0.5 mM DTT, 0.2 U / l RNase inhibitor (Applied Biosystems), and 1 mM phenylmethylsulfonyl fluoride solution. Circular RNA is incubated with HDAg protein (obtained as described by Defenbaugh et al 2009) at concentrations in the range of ΟΙ 10nM. Reaction mixtures are assembled on ice, incubated at 37°C for 1 h, and electrophoresed on 6% native polyacrylamide gels in 0.5 Tris-borate-EDTA at 240 V for 2.5 h. Free and bound RNA levels are determined using nucleic acid stain (eg gelred). Binding will be calculated as the intensity of unbound RNA relative to the intensity of the entire lane minus background. A circular RNA is expected to have a functional quasi-helical structure. Example 13: Self-transcription / replication In this example, circular RNA is synthesized by one of the methods described above, with the addition of expression of the one or more replication domains (as described by Beeharry et al. 2014), the replication-competent antigenomic ribozyme, and a nuclear localization signal. These RNA sequences allow circular RNA to localize to the nucleus, where the host RNA polymerase will bind and transcribe the RNA. This RNA is then self-cleaved using the ribozyme. The RNA is then ligated and self-replicates again, see Figure 14. Circular RNA (1-5 micrograms) will be transfected into HeLa cells using the techniques described above. HeLa cells are grown at 37°C, 5% CO 2 in high glucose Dulbecco's Modified Eagle's medium (DMEM) (Life Technologies), supplemented with penicillin-streptomycin and 10% fetal calf serum. After transfection, HeLa cells are cultured for an additional 4-72 h, then total RNA is isolated from transfected cells using phenol-based isolation reagent (Life Technologies) according to manufacturer's instructions between 1 hour and 20 days later. of transfection and ncionn / nznz / E / Y the total amount of circular RNA encoding HDV domains will be determined and compared to control circular RNA using qPCR as described herein. Example 14: Stability / half-life of circular RNA In this example, circular RNA is synthesized by one of the methods described above. A circular RNA is designed to include encryptogens (SEQ ID NO: 4) and an ORF encoding GFP (SEQ ID NO: 2) with stagger elements (SEQ ID NO: 3) flanking the GFP ORF, see Figure fifteen. Human fibroblasts (eg, IMR-90) are grown to confluence in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C with 5% CO2 in treated plates for tissue culture. When fibroblasts reach confluence, they stop dividing due to contact inhibition (Leontieva et al. 2014). Lipid Transfection Reagent (2pL; Invitrogen) is added to a mixture of 1 pg of circular RNA or linear RNA (described above) and 145 pL of medium with reduced serum (Opti-MEM I Solution) in one well of a treated plate. for 12-well tissue culture. After incubation at room temperature for 15min, ~1 x 10Λ5 cells suspended in DMEM with 10% FBS are added to the circular RNA solution (described above). Cells will be cultured and then harvested on day 1, 2, 3, 4, 5, 10, 20 and 30 after circular RNA transfection. Cells will be isolated for qRT-PCR and another subset for FACS analysis. To measure circular RNA and mRNA levels for GFP, reverse transcription qPCR is performed using random hexamers, as described in Example 2. Cells will be FACS analyzed using antibodies to GFP, as described herein. document. Circular RNA is expected to persist in cells for at least several days and to retain functional GFP protein expression. Example 15: Preservation of circular RNA in daughter cells In this example, circular RNA is synthesized by one of the methods described above. A circular RNA is designed to include encryptogens (SEQ ID NO: 4) and an ORF encoding GFP (SEQ ID NO: 2) with stagger elements (SEQ ID NO: 3) flanking the GFP ORF, see Figure 16. Human fibroblasts (eg, IMR-90) are grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C with 5% CO2 in plates treated for tissue culture. Cells are passaged regularly to maintain exponential growth. Lipid Transfection Reagent (2 pL; Invitrogen) is added to a mixture of 1 pg of circular RNA or linear RNA (described above) and 145 pL of medium with reduced serum (Opti-MEM I Solution) in a ncionn / well. nznz / E / Y a 12-well tissue culture treated plate. After incubation at room temperature for 15 min, 1 x 10Λ5 HeLa cells suspended in DMEM with 10% FBS are added to the circular RNA solution (described above). After incubation for 24 h at 37 °C and 5% CO2, cells are pulsed with BrdU (eg Sigma-Aldrich). The duration of BrdU labeling is optimized for each cell type according to its specific doubling time, for example, IMR-90 human fibroblasts have a doubling time of 27 h and are pulsed for 8-9 h as described by Elabd et al. 2013. Cells will be harvested on day 1, 2, 3, 4, 5 and 10 after the BrdU pulse. A subset of the cells will be isolated for qRT-PCR and another subset for FACS analysis. To measure the levels of circular RNA and mRNA for GFP, reverse transcription qPCR is performed using random hexamers, as described in Example 2. Cells will be analyzed with FACS using antibodies to BrdU and GFP, as described in This document. Circular RNA is expected to persist in daughter cells and daughter cells to express GFP protein. Example 16: Circularization of circular RNA This example demonstrates the in vitro production of circular RNA using bridging. A naturally occurring circular RNA can be engineered to include one or more desirable properties and can be produced using recombinant DNA technology. As shown in the following example, bridging ligation circularizes linear RNA. circIRNA was designed to encode triple FLAG-tagged EGF without a stop codon (264 nt). It has a Kozak sequence (SEQ ID NO: 11) at the start codon for translation initiation. CirRNA2 has identical sequences to circular RNA1, except that it has a termination element (triple stop codon) (273 nt, SEQ ID NO: 12). Circular RNA3 was designed to encode triple FLAG-tagged EGF flanked by a staggered element (2A sequence, SEQ ID NO: 13), without a termination (stop codon) element (330 nt). circRNA4 has identical sequences to circular RNA3, except that it has a termination element (triple stop codon) (339 nt). In this example, circular RNA was generated as set forth below. DNA templates for in vitro transcription were amplified from gBlocks gene fragments with corresponding sequences (IDT) with a forward primer carrying the T7 promoter and 2-O-methylated nucleotide with a reverse primer. The amplified DNA templates were gel purified with a DNA gel purification kit (Qiagen). 250-500 ng of purified DNA template was subjected to in vitro transcription. Linear 5'-monophosphorylated transcripts were generated in vitro using T7 RNA polymerase from each DNA template having the corresponding nc i οηη / ηζηζ / Ε / γ sequences in the presence of 7.5mM GMP, 1.5mM GTP, 7.5 UTP mM, CTP 7.5mM and ATP 7.5mM. Approximately 40pg of linear RNA was generated in each reaction. After incubation, each reaction was treated with DNase to remove the DNA template. In vitro transcribed RNA was ethanol precipitated in the presence of 2.5IVI ammonium acetate to remove unincorporated monomers. Transcribed linear RNA was circularized using T4 RNA ligase 2 on a 20 nt bridging DNA oligomer (SEQ ID NO: 14) as a template. The bridging DNA was designed to hybridize to 10 nt from either the 5' or 3' end of the linear RNA. After hybridization with the bridging DNA (3μΜ), 1μΜ linear RNA was incubated with O.SU / μΙ T4 RNA ligase 2 at 37eC for 4 h. The mixture without T4 RNA ligase 2 was used as a negative control. Circularization of linear RNA was monitored by separating RNA on 6% denaturing PAGE. The slower migrating RNA bands correspond to circular RNA rather than linear RNA on denaturing polyacrylamide gels due to their circular structure. As seen in Figure 17, the addition of ligase (lanes +) to the RNA pools generated new bands that appeared above the linear RNA bands that were present in the pools lacking ligase (lanes -). Slower migrating bands appeared in all RNA pools, indicating that bridging (eg, circularization) occurred successfully with multiple constructs, compared to the negative control. Example 17: Efficiency of RNA circularization This example demonstrates the efficiencies of circularization of RNA bridging ligation. A non-naturally occurring circular RNA modified to include one or more desirable properties can be produced using bridging-mediated circularization. As shown in the example below, bridging ligation circularized linear RNA with greater efficiency than controls. In this case circRNAI, circRNA2, circRNA3 and circRNA4 were also used as described in Example 1. circRNAS was designed to encode FLAG-tagged EGF flanked by a 2A sequence and followed by FLAG-tagged nanoluciferase (873 nt, SEQ ID NOT: 17). circRNA6 has an identical sequence to circular RNA 5, except that it included a termination element (triple stop codon) between the EGF and nanoluciferase genes and a termination element (triple stop codon) at the end of the nano sequence. luciferase (762 nt, SEQ ID NO: 18). In this example, to measure RNA circularization efficiency, 6 different sizes of linear RNA (264 nt, 273 nt, 330 nt, 339 nt, 873 nt, and 762 nt) were generated and circularized as described in Example 1. Circular RNAs were resolved by 6% denaturing PAGE and the corresponding RNA bands were excised on the gel for ncionn / nznz / E / Y linear or circular RNA for purification. The excised RNA gel bands were ground up and the RNA eluted with 800 μΙ of 300 mM NaCI overnight. Gel debris was removed by spin filters and RNA was ethanol precipitated in the presence of 0.3M sodium acetate. The circularization efficiency was calculated as follows. The amount of eluted circular RNA was divided by the amount of total eluted RNA (circular + linear RNA) and the result was plotted as a graph in Figure 18. Ligation of linear RNAs using T4 RNA ligase 2 produced circular RNA with higher efficiency rates than control. Trend data indicated that larger buildings circulated at higher rates. Example 18: Circular RNA that has no susceptibility to degradation This example demonstrates the susceptibility of circular RNA to RNase R degradation compared to linear RNA. Circular RNA is more resistant to nuclease degradation than linear RNA due to the absence of 5' and 3' ends. As shown in the example below, circular RNA was less susceptible to degradation than its linear RNA counterpart. circ5RNA was generated and circularized as described in Example 2 for use in the assay described herein. To assess circularization of circ5RNA, 20ng / pl linear or circular circ5RNA was incubated with 2U / pl RNAse R, a 3' to 5' exonuclease that digests linear RNAs but does not digest linear or circular RNA structures, at 37°C for 30min. After incubation, the reaction mixture was analyzed by 6% denaturing PAGE. Bands of linear RNA present in the lanes lacking exonuclease were absent in the circRNA5 lane (see Figure 19), indicating that circRNA5 showed increased resistance to exonuclease treatment compared to the linear RNA control. Example 19: Isolation and purification of circular RNA This example demonstrates the purification of circular RNA. In certain embodiments, circular RNAs, as described in the examples above, can be isolated and purified prior to expression of the encoded protein products. This example describes isolation using UREA gel separation. As shown in the following example, circular RNA was isolated and purified. circRNAI, circRNA2, circRNA3, circRNA4, circRNA5 and circRNA6 were isolated as described herein as described in Example 2. In this example, linear and circular RNAs were generated as described. To purify the circular RNAs, the ligation mixes were resolved on 6% ncionn / nznz / E / Y denaturing PAGE and RNA bands corresponding to each of the circular RNAs were excised. The excised RNA gel fragments were ground and the RNA eluted with 800 μΙ NaCI 300 mlVI overnight. Gel debris was removed by spin filters and RNA was ethanol precipitated in the presence of 0.3M sodium acetate. Eluted circular RNA was analyzed by 6% denaturing PAGE, see Figure 20. Individual bands were visualized by PAGE for circular RNAs that had variable sizes. Example 20: Detection of protein expression This example demonstrates protein expression in vitro from circular RNA. Protein expression is the process of generating a specific protein from mRNA. This process includes the transcription of DNA into messenger RNA (mRNA), followed by translation of the mRNA into polypeptide chains, which are ultimately folded into functional proteins and can be targeted to specific subcellular or extracellular locations. As shown in the following example, a protein was expressed in vitro from a circular RNA sequence. Circular RNA was designed to encode triple FLAG-tagged EGF by a 2A sequence without a termination element (stop codon) (330 nt, SEQ ID NO: 19). Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25μΙ. The final composition of the reaction mixture contained 70% rabbit reticulocytes used, 20μΜ amino acids, 0.SU / μΙ RNase inhibitor and 1 μg of linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32μΙ) and water (300μΙ) to the reaction mixture (16μΙ) and centrifuging at 20,817xg for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 30μΙ of 2x SDS sample buffer and incubated at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit (see Figure 21) and the band of the Western blot was measured by ImageJ. Fluorescence was detected, indicating that expression product was present. Therefore, circular RNA was shown to drive the expression of a protein. Example 21: Independent expression of IRES This example demonstrates circular RNA driven expression in the absence of an IRES. ncionn / nznz / Ε / γ An IRES or internal ribosome entry site is an RNA element that allows initiation of translation in a cap-dependent manner. Circular RNA was shown to drive the expression of Flag protein in the absence of an IRES. Circular RNA was designed to encode triple FLAG-tagged EGF by a 2A sequence without a termination element (stop codon) (330 nt, SEQ ID NO: 19). Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25μΙ. The final composition of the reaction mixture included 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.SU / μΙ RNase inhibitor, and 1 pg linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32pl) and water (300pl) to the reaction mixture (16pl) and centrifuging at 20,817xg for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 30µl of 2x SDS sample buffer and incubated at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an enhanced chemiluminescence (ECL) kit (see Figure 21) and the band of the Western blot was measured by ImageJ. The expression product was detected in the circular RNA reaction mix even in the absence of an IRES. Example 22: Independent expression of cap This example demonstrates that circular RNA is capable of driving expression in the absence of a cap. A cap is a specially altered nucleotide at the 5' end of the mRNA. The 5' cap is useful for the stability, as well as translation initiation, of a linear mRNA. Circular RNA drove the expression of the product in the absence of a cap. Circular RNA was designed to encode triple FLAG-tagged EGF by a 2A sequence without a termination element (stop codon) (330 nt, SEQ ID NO: 19). Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25µl. The final composition of the reaction mixture included 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.SU / μΙ RNase inhibitor, and 1 pg linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32pl) and water (300pl) to the reaction mixture (16pl) and centrifuging at 20,817xg for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 30µl of 2x SDS sample buffer at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. ncionn / nznz / Ε / γ After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit (see Figure 21) and the band of the Western blot was measured by ImageJ. The expression product was detected in the circular RNA reaction mixture even in the absence of a cap. Example 23: Expression without a 5' UTR This example demonstrates protein expression in vitro from circular RNA lacking 5' untranslated regions. The 5' untranslated region (5j UTR) is the region directly upstream of a start codon that assists in protein translation downstream of an RNA transcript. As shown in the following example, a 5' untranslated region in the circular RNA sequence was not required for protein expression in vitro. Circular RNA was designed to encode triple FLAG-tagged EGF by a 2A sequence without a termination element (stop codon) (330 nt, SEQ ID NO: 19). Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25μΙ. The final composition of the reaction mixture included 70% rabbit reticulocyte lysate, 20 μΜ amino acids, O.SU / μΙ RNase inhibitor, and 1 pg linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32μΙ) and water (300μΙ) to the reaction mixture (16μΙ) and centrifuging at 20,817xg for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 30μΙ of 2x SDS sample buffer and incubated at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit (see Figure 21) and the band of the Western blot was measured by ImageJ. The expression product was detected in the circular RNA reaction mixture even in the absence of a 5' UTR. Example 24: Expression without a 3' UTR This example demonstrates protein expression in vitro from circular RNA lacking a 3' UTR. The 3' untranslated region (3j UTR) is the region directly downstream of a translation stop codon and includes regulatory regions that can post-transcriptionally influence gene expression. The 3' untranslated region may also play a role in gene expression influencing the localization, stability, ncionn / nznz / E / Y export and efficiency of translation of an mRNA.In addition, the structural features of the 3' UTR, as well as the use of alternative polyadenylation, may play a role in gene expression . As shown in the following example, a 3' UTR in the circular RNA sequence was not required for protein expression in vitro. Circular RNA was designed to encode triple FLAG-tagged EGF by a 2A sequence without a termination element (stop codon) (330 nt, SEQ ID NO: 19). Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25μΙ. The final composition of the reaction mixture included 70% rabbit reticulocyte lysate, 20μΜ amino acids, 0.8U / pl RNase inhibitor, and 1 pg linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32μΙ) and water (300μΙ) to the reaction mixture (16μΙ) and centrifuging at 20,817xg for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 30μΙ of 2x SDS sample buffer and incubated at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit (see Figure 21) and the band of the Western blot was measured by ImageJ. The expression product was detected in the circular RNA reaction mixture even in the absence of a 3' UTR. Example 25: Expression without a stop codon This example demonstrates the generation of a polypeptide product after rolling circle translation from circular RNA lacking a stop codon. Proteins are based on polypeptides, which are made up of unique amino acid sequences. Each amino acid is encoded in mRNA by triplets of nucleotides called codons. During protein translation, each codon in the mRNA corresponds to an amino acid addition in a growing polypeptide chain. The termination element or stop codons signal the termination of this process by binding to release factors, which cause dissociation of the ribosomal subunits, releasing the amino acid chain. As shown in the following example, a circular RNA lacking a stop codon generated a long polypeptide product made up of repeating polypeptide units by rolling circle translation. ncionn / nznz / E / Y 100 The circular RNA was designed to encode triple FLAG-tagged EGF without a termination element (stop codon) (264 nt, SEQ ID NO: 20) and included a Kozak sequence in the start codon to promote translation initiation. . Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25μΙ. The final composition of the reaction mixture included 70% rabbit reticulocytes used, 20μΜ amino acids, 0.SU / μΙ RNase inhibitor and 1pg linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32μΙ) and water (300μΙ) to the reaction mixture (16μΙ) and centrifuging at 20,817xg for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 30μΙ of 2x SDS sample buffer and incubated at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit (see Figure 22) and the band of the Western blot was measured by ImageJ. The expression product was detected in the circular RNA reaction mixture even in the absence of a stop codon. Example 26: Expression of isolated proteins without a termination element (stop codon) This example demonstrates the generation of an isolated translated protein product from circular RNA lacking a termination element (stop codons). Ladder elements, such as 2A peptides, can include short amino acid sequences, ~20 aa, which make it possible to produce equimolar levels of multiple genes from a single mRNA. The staggering element may function by causing the ribosome to skip the synthesis of a peptide bond at the C-terminus of the 2A element, resulting in a gap between the end of the 2A sequence and the next peptide downstream. The gap occurs between glycine and proline residues found at the C-terminus and the upstream cistron has a few additional residues added to the end, while the downstream cistron begins with a proline. As shown in the example below, circular RNA lacking a termination element (stop codon) generated a long polypeptide polymer (Figure 23, left panel: non-staggered - circular RNA lane) and inclusion of a sequence de 2A at the 3' end of the coding region resulted in the production of isolated protein with a size comparable to that generated by the equivalent linear RNA construct (Figure 23, left panel: staggered - circular RNA lane). ncionn / nznz / E / Y 101 The circular RNA was designed to encode triple FLAG-tagged EGF without a termination element (stop codon) (264 nt, SEQ ID NO: 20) and without a stagger element. A second circular RNA was designed to encode triple FLAG-tagged EGF by a 2A sequence without a termination element (stop codon) (330 nt, SEQ ID NO: 19). Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25μΙ. The final composition of the reaction mix included 70% rabbit reticulocytes used, 20 μΜ amino acids, O.SU / μΙ RNase inhibitor, and 1 μg of linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32μΙ) and water (300μΙ) to the reaction mixture (16μΙ) and centrifuging at 20,817xg for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 30μΙ of 2x SDS sample buffer and incubated at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit (see Figure 23) and the band of the Western blot was measured by ImageJ. Isolated expression products were detected, indicating that circular RNA comprising a stagger element drove the expression of the individual proteins, even in the absence of a termination element (stop codons). Example 27: Rolling circle translation This example demonstrates high in vitro biosynthesis of a protein from circular RNA using a staggering element. Non-naturally occurring circular RNA was engineered to include a stagger element to compare protein expression with that of circular RNA lacking a stagger element. As shown in the example below, a staggered element overexpressed the protein compared to an otherwise identical circular RNA lacking such a sequence. Circular RNA was designed to encode triple FLAG-tagged EGF, with a termination element (eg, three stop codons in tandem) (273 nt, SEQ ID NO: 21). A second circular RNA was designed to encode triple FLAG-tagged EGF by a 2A sequence without a termination element (stop codon) (330 nt, SEQ ID NO: 19). Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25μΙ. The final composition of the reaction mixture contained 70% rabbit reticulocytes used, 20 µΜ amino acids, O.SU / µΙ RNase inhibitor and 1 pg linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32μΙ) and water (300μΙ) to the reaction mixture (16μΙ) and centrifuging at 20,817xg ncionn / nznz / Ε / γ. 102 for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 30μΙ of 2x SDS sample buffer and incubated at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit (see Figure 24) and the band of the Western blot was measured by ImageJ. Isolated expression products were detected, indicating that circular RNA comprising a stagger element drove the expression of the individual proteins, even in the absence of a termination element (stop codons). Example 28: Expression of a biologically active protein in vitro This example demonstrates the in vitro biosynthesis of a biologically active protein from circular RNA. A non-naturally occurring RNA was engineered to express a biologically active therapeutic protein. As shown in the following example, a biologically active protein was expressed from the circular RNA in the reticulocyte lysate. Circular RNA was designed to encode FLAG-tagged EGF flanked by a 2A sequence and followed by FLAG-tagged nano-luciferase (873 nt, SEQ ID NO: 17). Linear or circular RNA was incubated for 5 h in rabbit reticulocyte lysate at 30°C in a volume of 25μΙ. The final composition of the reaction mixture contained 70% rabbit reticulocytes used, 20μΜ amino acids, 0.8U / pl RNase inhibitor. Luciferase activity in the translation mix was monitored using a luciferase assay system according to the manufacturer's protocol (Promega). As shown in Figure 25, much higher fluorescence was detected with both circular RNA and linear RNA than with vehicle control RNA, indicating that the expression product was present. Therefore, circular RNA was shown to express a biologically active protein. Example 29: Circular RNA with Longer Half-Life Than Linear RNA Homolog This example demonstrates circular RNA engineered to have a longer half-life, compared to linear RNA. Circular RNA encoding a therapeutic protein provided recipient cells with the ability to produce higher levels of the encoded protein due to prolonged biological half-life, for example, compared to linear RNA. As shown in the following example, a circular RNA has a longer half-life than its linear RNA counterpart in reticulocyte lysate. ncionn / nznz / Ε / γ 103 A circular RNA was designed to encode FLAG-tagged EGF flanked by a 2A sequence and followed by FLAG-tagged nano-luciferase (873 nt, SEQ ID NO: 17). In this example, a time course experiment was performed to monitor RNA stability. 100ng of linear or circular RNA was incubated with rabbit reticulocyte lysate and samples were collected at 1h, 5h, 18h and 30h. Total RNA was isolated from lysate using a phenol-based reagent (Invitrogen) and cDNA was generated by reverse transcription. Analysis by qRT-PCR was carried out using dy-based quantitative PCR reaction mix (BioRad). As shown in Figure 26, higher concentrations of circular RNA were detected at later time points than linear RNA. Therefore, the circular RNA was more stable or had an increased half-life compared to its linear counterpart. Example 30: Circular RNA Demonstrated Longer Half-Life Than Linear RNA in Cells This example demonstrates that circular RNA delivered to cells has an increased half-life in cells compared to linear RNA. A non-naturally occurring RNA was engineered to express a biologically active therapeutic protein. As shown in the example below, circular RNA was present at higher levels compared to its linear RNA counterpart, demonstrating a longer half-life for circular RNA. In this example, circular RNA and linear RNA were designed to encode a Kozak, EGF, flanked by a 2A, para, or non-stop sequence (SEQ ID NO: 11,19, 20, 21). To monitor the half-life of the RNA in the cells, 0.1x10 6 cells were plated in each well of a 12-well plate. After 1 day, 1 pg of linear or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). Twenty four hours after transfection, total RNA was isolated from cells using a phenol-based extraction reagent (Invitrogen). Total RNA (500 ng) was reverse transcribed to generate cDNA. Analysis by qRT-PCR was carried out using dye-based quantitative PCR mix (BioRad). The primer sequences are as follows: Primers for linear or circular RNA, D: ACGACGGTGTGTGCATGTAT, I: TTCCCACCACTTCAGGTCTC; primers for circular RNA, D: TACGCCTGCAACTGTGTTGT, I: TCGATGATCTTGTCGTCGTC. Circular RNA was successfully transfected into 293T cells, as was its linear counterpart. After 24 hours, the remaining circular and linear RNA was measured using qPCR. As shown in Figures 27A and B, circular RNA was shown to have a longer half-life in cells compared to linear RNA. ncionn / nznz / E / Y 104 Example 31: Synthetic circular RNAs were translated in cells and synthetic circular RNA was translated by rolling circle translation This example demonstrates the translation of synthetic circular RNA in cells. As shown in the example below, circular RNA and linear RNA were designed to encode a Kozak sequence, 3xFLAG-EGF without a termination element (SEQ ID NO: 11). Circular RNA was translated into polymeric EGF, whereas linear RNA was not, demonstrating that cells carried out rolling circle translation of a synthetic circular RNA. In this example, 0.1x10 6 cells were plated in each well of a 12-well plate to monitor the efficiency of linear or circular RNA translation in cells. After 1 day, 1 pg of linear or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). Twenty four hours after transfection, cells were harvested by adding 200µΙ RIPA buffer to each well. Next, 10pg of cell lysate proteins were analyzed on a 10-20% polyacrylamide / SDS gradient gel. After electroblotting onto a nitrocellulose membrane using a dry blot method, the blot was incubated with an anti-FLAG and anti-mouse IgG antibody with peroxidase. As a loading control, anti-beta tubulin antibody was used. The blot was visualized with an enhanced chemiluminescence (ECL) kit. The intensity of the Western blot bands was measured by ImageJ. Circular RNA was successfully transfected into 293T cells, as was its linear counterpart. However, Figure 28 shows that 24 hours after transfection, EGF protein was detected in cells transfected with circular RNA, whereas it was not detected in cells transfected with linear RNA. Thus, circular RNA was transduced into cells by rolling circle translation compared to linear RNA. Example 32: Synthetic Circular RNA Demonstrated Reduced Expression of Immunogenic Genes in Cells This example demonstrates that engineered circular RNA has reduced immunogenicity compared to linear RNA. Circular RNA encoding a therapeutic protein provided reduced induction of immunogenicity-related genes (RIG-I, MDA5, PKA, and IFN-beta) in recipient cells, compared to linear RNA. RIG-I can recognize short 5' capless triphosphate single-stranded or double-stranded RNA, while MDA5 can recognize longer dsRNAs. RIG-I and MDA5 may both be involved in the activation of MAVS and the triggering of antiviral responses. PKR can be activated by dsRNA and induced by interferons, such as IFN-beta. As shown in the example below, circular RNA was shown to have reduced activation of immunity-related genes nc i οηη / ηζηζ / Ε / γ in 293T cells than an analogous linear RNA, assessed by RIG-I expression. , MDA5, PKR and IFN-beta by qPCR. Circular RNA and linear RNA were designed to encode either (1) a Kozak 3xFLAG-EGF sequence without a termination element (SEQ ID NO: 11); (2) a Kozak sequence, 3xFLAG-EGF, flanked by a termination element (stop codon) (SEQ ID NO: 21); (3) a Kozak sequence, 3xFLAG-EGF, flanked by a 2A sequence (SEQ ID NO: 19); or (4) a Kozak sequence, 3xFLAG-EGF flanked by a 2A sequence followed by a termination element (stop codon) (SEQ ID NO: 20). In this example, the level of innate immune response genes in cells was monitored by seeding 0.1x10 6 cells in each well of a 12-well plate. After 1 day, 1 pg of linear or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). Twenty four hours after transfection, total RNA was isolated from cells using a phenol-based extraction reagent (Invitrogen). Total RNA (500 ng) was reverse transcribed to generate cDNA. Analysis by qRT-PCR was carried out using dye-based quantitative PCR mix (BioRad). Primer sequences used: Primers for GAPDH, D: ncionn / nznz / E / Y AGGGCI GC I I I IAACICIGGI, I: CCCCACI IGAI I I IGGAGGGA; RIG-I, D TGTGGGCAATGTCATCAAAA, I: GAAGCACTTGCTACCTCTTGC; MDA5, D GGCACCATGGGAAGTGATT, I ATTTGGTAAGGCCTGAGCTG; PKR, D TCGCTGGTATCACTCGTCTG, I: G ATTCTGAAGACCGCCAGAG; IFN-beta, D CTCTCCTGTTGTGCTTCTCC, I: GTCAAAGTTCATCCTGTCCTTG. As shown in Figure 29, levels of immunity-related genes by qRT-PCR of 293T cells transfected with circular RNA showed reduced RIG-I, MDA5, PKR, and IFN-beta, compared to cells transfected with RNA. linear. Thus, the induction of immunogenic related genes in recipient cells was reduced in cells transfected with circular RNA, compared to cells transfected with linear RNA. Example 33: Increased Expression from Synthetic Circular RNA by Rolling Circle Translation in Cells This example demonstrates the upregulation of expression by rolling circle translation of synthetic circular RNA in cells. Circular RNAs were designed to include an IRES with a nanoluciferase gene or an EGF negative control gene without a termination element (stop codon). Cells were transfected with EGF negative control (SEQ ID NO: 22); nLUC stop (SEQ ID NO: 23): EMCV IRES, staggering sequence (2A sequence), 3x FLAG-tagged nLUC sequences, staggering sequence (2A sequence) and a stop codon; or nLUC stagger (SEQ ID NO: 24): EMCV IRES, stagger sequence (sequence 106 from 2A), 3x FLAG-tagged nLUC sequences and staggered sequence (sequence from 2A). As shown in Figure 30, both circular RNAs produced translation product that had functional luciferase activity. In this example, the translation of circular RNA in the cells was monitored. Specifically, 0.1x106 cells were seeded in each well of a 12-well plate. After 1 day, 300 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). After 24hrs, cells were harvested by adding 100µΙ RIPA buffer. Nanoluciferase activity was measured in Usados ​​using a luciferase assay system according to its manufacturer's protocol (Promega). As shown in Figure 30, both circular RNAs expressed protein in cells. However, circular RNA with a staggering element, e.g., 2A sequence, lacking a termination element (stop codon), produced higher levels of protein product having functional luciferase activity than circular RNA with a termination element (stop codon). Example 34: Synthetic circular RNA translated in cells This example demonstrates the translation of synthetic circular RNA in cells. Furthermore, this example demonstrates that circular RNA produced more expression product than its linear counterpart. Circular RNA was successfully transfected into 293T cells, as was its linear counterpart. Cells were transfected with circular RNA encoding EGF as a negative control (SEQ ID NO: 22): EMCV IRES, staggered sequence (2A sequence), 3x FLAG-tagged EGF sequences, staggered sequence (2A sequence) ; Linear or circular nLUC (SEQ ID NO: 23): EMCV IRES, staggering sequence (2A sequence), 3x FLAG-tagged nLuc sequences, a staggering sequence (2A sequence) and stop codon. As shown in Figure 31, the circular RNA was translated into nanoluciferase in cells. Translation of the linear or circular RNA in cells was monitored. Specifically, 0.1x106 cells were seeded in each well of a 12-well plate. After 1 day, 300 ng of linear or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). After 24hrs, cells were harvested by adding 100µΙ RIPA buffer. Nanoluciferase activity in lysates was measured using a luciferase assay system according to its manufacturer's protocol (Promega). As shown in Figure 31, the circular RNA translation product was detected in cells. In particular, the circular RNA had higher levels of luciferase activity or more protein produced compared to its linear RNA counterpart. ncionn / nznz / E / Y 107 Example 35: Rolling circle translation from synthetic circular RNA produced functional protein product in cells This example demonstrates rolling circle translation of functional protein product from synthetic circular RNA lacking a termination element (stop codon), eg, having a stagger element lacking a termination element (stop codon). stop) in cells. Furthermore, this example demonstrates that circular RNA with a staggered element expressed more functional protein product than its linear counterpart. Circular RNA was successfully transfected into 293T cells, as was its linear counterpart. Cells were transfected with EGF circular RNA negative control (SEQ ID NO: 22); Linear and circular nLUC (SEQ ID NO: 24): EMCV IRES, staggered sequence (2A sequence), 3x FLAG-tagged nLuc sequences a staggered sequence (2A sequence). As shown in Figure 32, the circular RNA was translated into nanoluciferase in cells. Translation of the linear or circular RNA in cells was monitored. Specifically, 0.1x106 cells were seeded in each well of a 12-well plate. After 1 day, 300 ng of linear or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). After 24hrs, cells were harvested by adding 100µΙ RIPA buffer. Nanoluciferase activity in lysates was measured using a luciferase assay system according to its manufacturer's protocol (Promega). As shown in Figure 32, the circular RNA translation product was detected in cells. In particular, circular RNA without a termination element (stop codon) produced higher levels of protein product that has functional luciferase activity than its linear RNA counterpart. Example 36: Synthetic Circular RNA Translated by IRES Initiation in Cells This example demonstrates the initiation of translation with synthetic circular RNA with an IRES in cells. Circular RNAs were designed to include a Kozak sequence or an IRES with a nanoluciferase gene or an EGF negative control gene. Cells were transfected with EGF negative control (SEQ ID NO: 22), nLUC Kozak (SEQ ID NO: 25): Kozak sequence, 1x FLAG-tagged EGF sequence, a staggered sequence (T2A sequence), nLUC labeled with 1x FLAG, staggered sequence (P2A sequence) and a stop codon; o nLUC IRES (SEQ ID NO: 23): EMCV IRES, staggering sequence (2A sequence), 3x FLAG-tagged nLUC sequences, staggering sequence (2A sequence) and a stop codon. As shown in Figure 33, circular RNA with an IRES demonstrated higher levels of luciferase activity, corresponding to higher levels of protein, compared to circular RNA with a Kozak sequence. nc i οηη / ηζηζ / Ε / γ 108 In this example, the translation of circular RNA in the cells was monitored. Specifically, 0.1x106 cells were seeded in each well of a 12-well plate. After 1 day, 300 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). After 24hrs, cells were harvested by adding 100µΙ RIPA buffer. Nanoluciferase activity in lysates was measured using a luciferase assay system according to its manufacturer's protocol (Promega). As shown in Figure 33, circular RNA initiated protein expression with an IRES and produced higher levels of protein product having functional luciferase activity than circular RNA initiation of protein expression with Kozak sequence. Example 37: Rolling circle translation of synthetic circular RNA in cells This example demonstrates increased protein production by rolling circle translation of synthetic circular RNA in cells that initiated protein production with an IRES. Circular RNAs were designed to include a Kozak sequence or an IRES with a nanoluciferase gene or an EGF negative control with or without a termination element (stop codon). Cells were transfected with EGF negative control (SEQ ID NO: 22); nLUC IRES stop (SEQ ID NO: 23): EMCV IRES, staggering sequence (2A sequence), 3x FLAG-tagged nLUC sequences, staggering sequence (2A sequence) and a stop codon; or nLUC IRES staggering (SEQ ID NO: 24): EMCV IRES, staggering sequence (2A sequence), 3x FLAG-tagged nLUC sequences, and staggering sequence (2A sequence). As shown in Figure 34, both circular RNAs produced expression product demonstrated by rolling circle translation and circular RNA without a termination element and an IRES (eg, without a Kozak sequence) initiated and produced higher levels of product. protein with functional luciferase activity than circular RNA with a termination element and IRES (ie, with a Kozak sequence), demonstrating rolling circle translation. In this example, the translation of circular RNA in the cells was monitored. Specifically, 0.1x106 cells were seeded in each well of a 12-well plate. After 1 day, 300 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). After 24hrs, cells were harvested by adding 100µΙ RIPA buffer. Nanoluciferase activity was measured in Usados ​​using a luciferase assay system according to its manufacturer's protocol (Promega). As shown in Figure 34, the circular RNA was translated into protein in cells by a rolling circle method provided from both circular RNAs. However, the circular RNA lacked a termination element (stop codon). However, rolling circle translation of circular RNA initiated increased protein production with an IRES and produced more protein product that had functional ncionn / nznz / Ε / γ activity. 109 luciferase compared to a circular RNA with a Kozak translation initiation termination element. Example 38: Increased Protein Expressed from Circular RNA This example demonstrates the translation of synthetic circular RNA in cells. Furthermore, this example demonstrates that circular RNA produced more expression product of the correct molecular weight than its linear counterpart. Linear and circular RNAs were designed to include a nanoluciferase gene with a termination element (stop codon). Cells were transfected with vehicle: transfection reagent only; linear nLUC (SEQ ID NO: 23): EMCV IRES, staging element (2A sequence), 3x FLAG-tagged nLuc sequences, a staging element (2A sequence) and termination element (stop codon); o Circular nLUC (SEQ ID NO: 23): EMCV IRES, staging element (2A sequence), 3x FLAG-tagged nLuc sequences, one staging element (2A sequence) and one termination element (stop codon ). As shown in Figure 35, circular RNA produced higher levels of protein that had the correct molecular weight compared to linear RNA. After 24hrs, cells were harvested by adding 10ΟμΙ RIPA buffer. After centrifugation at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit and the band of the Western blot was measured by ImageJ. As shown in Figure 35, the circular RNA was translated into protein in the cells. In particular, the circular RNA produced higher levels of protein that had the correct molecular weight compared to its linear RNA counterpart. Example 39: Rolling Circle Translation of Synthetic Circular RNA Produced Protein Products Isolated in Cells This example demonstrates that isolated protein products were translated by rolling circle translation from synthetic circular RNA lacking a termination element (stop codon), eg, having a stagger element instead of a termination element. (stop codon) in cells. Furthermore, this example demonstrates that circular RNA with a staggered element expressed more protein product having the correct molecular weight than its linear counterpart. Circular RNAs were designed to include a nanoluciferase gene with a stagger element instead of a termination element (stop codon). Cells were transfected with vehicle: transfection reagent only; Linear nLUC (SEQ ID NO: 24): IRES ncionn / nznz / E / Y EMCV 110, staggered element (2A sequence), 3x FLAG-tagged nLuc sequences and a staggered element (2A sequence); o Circular nLUC (SEQ ID NO: 24): EMCV IRES, stagger element (2A sequence), nLuc sequences marked with 3x FLAG and a stagger element (2A sequence). As shown in Figure 36, circular RNA produced higher levels of protein that had the correct molecular weight compared to linear RNA. After 24hrs, cells were harvested by adding 10ΟμΙ RIPA buffer. After centrifugation at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. After being electroblotted onto a nitrocellulose membrane using a dry blot method, the blot was incubated with anti-FLAG and anti-mouse IgG antibody with peroxidase. The blot was visualized with an ECL kit and the band of the Western blot was measured by ImageJ. As shown in Figure 36, the circular RNA translation product was detected in cells. In particular, circular RNA without a termination element (stop codon) produced higher levels of isolated protein product having the correct molecular weight than its linear RNA counterpart. Example 40: Preparation of circular RNA with a quasi-double-stranded helical structure This example demonstrates that circular RNA possessed quasi-double-stranded and helical structure. Non-naturally occurring circular RNA was engineered to adopt a quasi-double-stranded helical structure. A similar structure was shown to be involved in the condensation of a naturally occurring circular RNA that possessed a uniquely long half-life in vivo (Griffin et al 2014, J Virol. 2014 Jul;88(13):7402-11. doi: 10.1128 / JVI.00443-14, Guedj et al, Hepatology. 2014 Dec;60(6):1902-10. doi: 10.1002 / hep.27357). In this example, circular RNA was designed to encode an EMCV IRES, 3XFLAG-tagged Nluc as ORF, and staggered sequence (EMCV 2A 3XFLAG Nluc 2A without stop). To assess RNA secondary structure, the RNA structure thermodynamic prediction tool (RNAfoid) (Vienna RNA) was used. Additionally, RNA tertiary structure was analyzed using an RNA modeling algorithm. As shown in Figures 37 and 38, circular RNA is modeled to adopt a quasi-double-stranded helical structure. Example 41: Preparation of circular RNA with a quasi-helical structure ligated with a repetitive sequence This example demonstrates that circular RNA can be designed to have a quasi-helical structure linked with a repetitive sequence. nciann / nznz / E / Y 111 Non-naturally occurring circular RNA was engineered to adopt a quasi-helical structure linked with a repetitive sequence. A similar structure was shown to be involved in the condensation of a naturally occurring circular RNA that possessed a uniquely long half-life in vivo (Griffin et al 2014, Guedj et al 2014). In this example, the circular RNA was designed to encode an EMCV IRES, Nluc, and a spacer including a repetitive sequence (SEQ ID NO: 26). To assess the tertiary structure of RNA, an RNA modeling algorithm was used. As shown in Figure 39, circular RNA is modeled to adopt a quasi-helical structure. Example 42: Circularized RNA is circular and not concatameric This example demonstrates that degradation of circular RNA by RNase H produced nucleic acid degradation products, consistent with a circular RNA and not a concatameric RNA. The RNA, when incubated with a ligase, can either not react or form an intra- or intermolecular bond, generating a circular RNA (without free ends) or a concatameric RNA, respectively. Treatment of each type of RNA with a complementary DNA primer and RNase H, a non-specific endonuclease that recognizes DNA / RNA duplexes, is expected to produce a unique number of degradation products of specific sizes, depending on the RNA material of departure. As shown in the following example, a ligated RNA was shown to be circular and not concatamer based on the number and size of RNAs produced by RNase H degradation. Circular RNA and linear RNA containing EMCV T2A 3XFLAG-Nluc P2A were generated. To assess the circularization status of the RNA (1299 nt), 0.05 pmol / μΙ linear or circular RNA was incubated with 0.25U / pl RNAse H, an endoribonuclease that digests DNA / RNA duplexes, and 0.3 pmol / μΙ oligomer against 1037-1046 nt of RNA (CACCGCTCAGGACAATCCTT, SEQ ID NO: 55) at 37°C for 20min. After incubation, the reaction mixture was analyzed by 6% denaturing PAGE. For the linear RNA used described above, it is expected that after DNA primer ligation and subsequent RNase H cleavage, two cleavage products of 1041 nt and 258 nt are produced. A concatemer is expected to produce three cleavage products of 258.1041 and 1299 nt. A circular one is expected to produce a single cleavage product of 1299 nt. The number of bands in the linear RNA lane incubated with RNase endonuclease produced two bands of 1041 nt and 258 nt as expected, while a single ncionn / nznz / E / Y was produced. 112 band of 1299 nt in the circular RNA lane (see Figure 40), indicating that the circular RNA was, in fact, circular and not concatameric. Example 43: Preparation of large circRNAs This example demonstrates the generation of circular polyribonucleotide in the range of about 20 bases to about 6.2Kb. Genetically engineered non-naturally occurring circular RNA was produced to include one or more desirable properties, in a range of sizes depending on the desired function. As shown in the example below, linear RNA up to 6200 nt was circularized. In this case the plasmid pCDNA3.1 / CAT (6.2kb) was used. Primers were designed to anneal to pCDNA3.1 / CAT at regular intervals to generate DNA oligonucleotides corresponding to 500 nt, 1000 nt, 2000 nt, 4000 nt, 5000 nt and 6200 nt. In vitro transcription of the indicated DNA oligonucleotides was carried out to generate linear RNA of the corresponding sizes. Circular RNAs were generated from these RNA oligonucleotides using bridging DNA. To measure the efficiency of RNA circularization, 6 different sizes of linear RNA (500 nt, 1000 nt, 2000 nt, 4000 nt, 5000 nt and 6200 nt) were generated. They were circularized using a DNA bridge and T4 DNA ligase 2. As a control, a reaction without T4 RNA ligase was carried out. Half of the circularized sample was treated with RNase R to remove linear RNA. To monitor circulation efficiency, each sample was analyzed using qPCR. As shown in Figure 41, circular RNA was generated from a large variety of DNA of different lengths. As shown in Figure 42, circularization of the RNA was confirmed using RNase R treatment and qPCR analysis against circular junctions. This example demonstrates the production of circular RNA for a variety of lengths. Example 44: Circular RNA Engineered with a Protein Binding Site This example demonstrates the generation of a circular RNA with a protein binding site. In this example, a circular RNA is designed to include CVB3 IRES (SEQ ID NO: 56) and an ORF encoding Gaussian luciferase (Gluc) (SEQ ID NO: 57) followed by at least one protein binding site. . For a specific example, a HuR binding sequence (SEQ ID NO: 52) from the Sindbis virus 3' UTR is used to assess the immunogenicity effect of protein binding to circular RNA. The HuR binding sequence comprises two elements, URE (U-rich element; SEQ ID NO: 50) and CSE (Conserved Sequence element; SEQ ID NO: 51). In circular RNA without HuR binding sequence or with URE it is used as a control. Part of the autocatalytic intron and exon sequences of ncionn / nznz / E / Y are located 113 Anabaena before the CVB3 IRES (SEQ ID NO: 56). Circular RNAs are generated in vitro as described. As shown in Figure 45, circular RNA was generated to contain a HuR binding site. To monitor the effect of protein-RNA binding on circular RNA immunogenicity, cells are seeded in each well of a 96-well plate. After 1 day, 500 ng of circular RNA is transfected into each well using a lipid-based transfection reagent (Invitrogen). Transfection efficiency / RNA stability / immunogenicity are monitored daily, up to 72 h. Medium is collected to monitor Gluc activity. Cell lysate is prepared to measure the RNA level with a kit that allows the measurement of relative gene expression by real-time RT-PCR (Invitrogen). Translation efficiency is monitored by measuring Gluc activity with the Gaussia Luciferase Ultrarapid Assay Kit according to the manufacturer's instructions (Pierce). For qRT-PCR analysis, cDNA is generated with the cell lysate preparation kit according to the manufacturer's instructions (Invitrogen). qRTPCR analysis is performed in triplicate using a PCR master mix (Brilliant II SYBR Green qRT-PCR master mix) and a PCR cycler (LightCycler 480). Circular RNA stability is measured by primers against Nluc. mRNA levels for well known regulators of innate immunity such as RIG-I, MDA5, OAS, OASL and PKR are quantitated and normalized to actin values. Example 45: Preparation of circular RNA with regulatory nucleic acid sites This example demonstrates the in vitro production of circular RNA with a regulatory RNA binding site. Different cell types possess unique nucleic acid regulatory machinery to target specific RNA sequences. Encoding these specific sequences in a circular RNA could confer unique properties in different cell types. As shown in the example below, circular RNA was engineered to encode a microRNA binding site. In this example, the circular RNA included a sequence encoding a TS EMCV IRES, a mir692 microRNA binding site (GAGGUGCUCAAAGAGAU), and two spacer elements flanking the IRES-ORF. Circular RNA was generated in vitro. Unmodified linear RNA was transcribed in vitro from a DNA template that includes all of the motifs listed above, plus the T7 RNA polymerase promoter to drive transcription. The RNA transcript was purified with an RNA cleanup kit (New England Biolabs, T2050), treated with an RNA 5'-phosphohydrolase (RppH) (New England Biolabs, M0356) following the manufacturer's instructions, and ncionn / nznz purified. / HEY 114 again with an RNA purification column. RppH-treated RNA was circularized using DNA bridge (GGCTATTCCCAATAGCCGTT) and T4 RNA ligase 2 (New England Biolabs, M0239). Circular RNA was purified by Urea-PAGE (Figure 43), eluted in buffer (0.5 M sodium acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated, and resuspended in RNase-free water. As shown in Figure 43, circular RNA with a miRNA binding site was generated. Example 46: Self-splicing of circular RNA This example demonstrates the ability to produce circular RNA by self-splicing. For this example, the circular RNAs included a CVB3 IRES, an ORF encoding Gaussian luciferase (GLuc), and two spacer elements flanking the IRES-ORF. Circular RNA was generated in vitro. Unmodified linear RNA was transcribed in vitro from a DNA template that includes all of the motifs listed above. In vitro transcription reactions included 1 pg T7 RNA polymerase promoter template DNA, 10X T7 reaction buffer, 7.5 mM ATP, 7.5 mM CTP, 7.5 mM GTP, 7.5 mM DTP, 10 mM DTT, 40U inhibitor RNase and T7 enzyme. Transcription was carried out at 37°C for 4 h. The RNA transcript was DNase-treated with 1 U of DNase I at 37°C for 15 min. To promote circularization by self-splicing, additional GTP was added to a final concentration of 2 mM and incubated at 55 °C for 15 min. The RNA was then column purified and visualized by UREA-PAGE. Figure 44 shows circular RNA generated by self-splicing. Example 47: Circular RNA with a splicing element comprising an encryptogen This example demonstrates a circular RNA engineered to have reduced immunogenicity. For this example, a circular RNA included a CVB3 IRES, an ORF encoding Gaussian luciferase (GLuc), and two spacer elements flanking the IRES-ORF, these two spacer elements comprise a splice element that are part of the autocatalytic exon and intron sequences from Anabaena (SEQ ID NO: 59). Circular RNA is generated in vitro. In this example, the level of innate immune response genes in cells is monitored by seeding the cells in each well of a 12-well plate. After 1 day, 1 pg of linear or circular RNA is transfected into each well using a lipid-based transfection reagent (Invitrogen). Twenty-four hours after transfection, total RNA is isolated from cells using a phenol-based extraction reagent (Invitrogen). Total RNA (500 ncionn / nznz / E / Y 115 ng) is reverse transcribed to generate cDNA. Analysis by qRT-PCR is carried out using dye-based quantitative PCR mix (BioRad). qRT-PCR levels of immunity-related genes from BJ cells transfected with circular RNA comprising a splicing element are expected to show reduced RIG-I, MDA5, PKR, and IFN-beta cells compared to transfected with linear RNA. Therefore, the induction of immunogenic related genes in recipient cells is expected to be reduced in cells transfected with circular RNA compared to cells transfected with linear RNA. Example 48: Persistence of circular RNA during cell division This example demonstrates the persistence of the circular polyribonucleotide during cell division. A non-naturally occurring circular RNA engineered to include one or more desirable properties can persist in cells during cell division without being degraded. As shown in the example below, RNA expressing Gaussian luciferase (GLuc) was monitored over 72 h days in HeLa cells. In this example, a 1307 nt circular RNA included a CVB3 IRES, an ORF encoding Gaussian luciferase (GLuc) and two spacer elements flanking the IRESORF. The persistence of circular RNA was monitored throughout cell division in HeLa cells. 5000 cells / well were transfected in suspension in a 96-well plate with circular RNA. Glow cell imaging was performed on an Avos imaging device (ThermoFisher) and cell counts were performed using a luminescent cell viability assay (Promega) at 0h, 24h, 48h. , 72h and 96h. Gaussian luciferase enzyme activity was monitored daily as a measure of protein expression and gLuc expression was monitored daily in supernatant removed from wells every 24 h using the Gaussian luciferase activity assay (Thermo Scientific , Pierce). 50 µl of 1X Gluc substrate was added to 5 µΙ plasma to perform the Gluc luciferase activity assay. Plates were read just after mixing in a luminometer instrument (Promega). Circular RNA protein expression was detected at higher levels than linear RNA in dividing cells (Figure 46). Cells with circular RNA had higher cell division rates compared to linear RNA at all time points measured. This example demonstrates the increased detection of circular RNA during cell division than its linear RNA counterpart. ncionn / nznz / E / Y 116 Example 49: Rolling circle translation produced a plurality of expression sequences This example demonstrates the ability of circular RNA to express multiple proteins from a single construct. Furthermore, this example demonstrates rolling circle translation of circular RNA encoding multiple ORFs. This example demonstrates the expression of two proteins from a single construct. A circular RNA (mtEMCV T2A 3XFLAG-GFP F2A 3XFLAG-Nluc P2A IS spacer) for rolling circle translation was designed to include EMCV IRES (SEQ ID NO: 58) and an ORF encoding GFP with 3XFLAG marker and an ORF encoding nanoluciferase (Nluc) with 3XFLAG tag. Staggering elements (2A) flanked the GFP and Nluc ORFs. Another circular RNA was designed in a similar way, but included a triple stop codon between the Nluc ORF and the spacer. Part of the Anabaena autocatalytic exon and intron sequences were included before the EMCV IRES. Circular RNAs were generated in vitro as described. Protein expression from circular RNA was monitored either in vitro or in cells. For in vitro analysis, circular RNAs were incubated for 3 h in rabbit reticulocyte lysate (Promega, Fitchburg, WI, USA) at 30 °C. The final composition of the reaction mixture included 70% rabbit reticulocyte lysate, 20 μΜ complete amino acids, and 0.8 U / pL RNase inhibitor (Toyobo, Osaka, Japan). After incubation, hemoglobin protein was removed by adding acetic acid (0.32μΙ) and water (300μΙ) to the reaction mixture (16μΙ) and centrifuging at 20,817xg for 10min at 15°C. The supernatant was removed and the pellet was dissolved in 2x SDS sample buffer and incubated at 70°C for 15 min. After centrifuging at 1400xg for 5 min, the supernatant was analyzed on a 10-20% polyacrylamide / SDS gradient gel. For analysis in cells, cells were seeded in each well of a 12-well plate to monitor the efficiency of circular RNA translation in cells. After 1 day, 500 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). 48 hours after transfection, cells were harvested by adding 200μΙ RIPA buffer to each well. Next, 10pg of cell lysate proteins were analyzed on a 10-20% polyacrylamide / SDS gradient gel. After electroblotting of reticulocyte lysate and cell samples to a nitrocellulose membrane using a dry blot method, the blot was incubated with an anti-FLAG and anti-mouse IgG antibody with peroxidase. As a loading control, anti-beta tubulin antibody was used. The blot was visualized with an enhanced chemiluminescence (ECL) kit. The intensity of the Western blot bands was measured by ImageJ. ncionn / nznz / E / Y 117 As shown in Figure 47, circular RNA encoding GFP and nLuc produced 2 protein products. Translation from circular RNA without the triple stop generated more of both protein products than circular RNA with the triple stop codon. Finally, both circular RNAs with and without the triple stop expressed proteins at ratios of 1 / 3.24 and 1 / 3.37, respectively. Example 50: Circular RNA Shows Reduced Toxicity Compared to Linear RNA This example demonstrates that circular RNA is less toxic than linear RNA. For this example, the circular RNA includes an EMCV IRES, an ORF encoding NanoLuc with a 3XFLAG tag and flanked on both sides by stagger elements (2A) and a termination element (stop codon). Circular RNA was generated in vitro and purified as described herein. The linear RNA used in this example was poly A cap and tail modified RNA or poly A cap and tail unmodified RNA encoding nLuc with the globin UTRs. To monitor RNA toxicity in cells, BJ human fibroblast cells were seeded in each well of a 96-well plate. 50ng of circular or linear RNA modified with polyA cap and tail were transfected after zero, forty-eight and seventy-two hours, using a lipi...

Claims

CLAIMS 1. A rolling circle translation method of one or more expression sequences comprising expressing the one or more expression sequences from a circular polyribonucleotide by rolling circle translation, wherein the circular polyribonucleotide comprises the one or more expression sequences, a stagger element at a 3' end of at least one of the expression sequences, and lacking a termination element.

2. The method of claim 1, wherein the staggering element is configured to interrupt a ribosome during rolling circle translation of the circular polyribonucleotide.

3. The method of claim 1, wherein the staggering element encodes a sequence with a C-terminal consensus sequence that is D(V / l)ExNPGP, where x = any amino acid.

4. The method of claim 1, wherein the expression of one or more expression sequences generates at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of isolated polypeptides from the total (molar / molar) polypeptides generated during rolling circle translation of the circular polyribonucleotide, and wherein each of the isolated polypeptides is generated from a single expression sequence.

5. The method of claim 1, wherein at least one of the expression sequences encodes a secreted protein.

6. The method of claim 1, wherein the circular polyribonucleotide lacks an internal ribosome entry site.

7. The method of claim 1, wherein the one or more expression sequences comprise a Kozak start sequence.

8. The method of claim 1, wherein the circular polyribonucleotide further comprises at least one structural element selected from: (a) a cryptogene; (b) a regulatory element; (c) a replication element; and (d) quasi-double-stranded secondary structure.

9. The method of claim 1, wherein the circular polyribonucleotide comprises at least one functional feature selected from: (i) greater translation efficiency than a linear homologue; (ii) stoichiometric translation efficiency of multiple translation products; (iii) less immunogenicity than a homologue lacking an encryptogen; (iv) increased half-life compared to a linear homologue; and (v) persistence during cell division.

10. The method of claim 1, wherein the expression of one or more expression sequences generates at least 5 times more expression product than a linear homologue.

11. The method of claim 1, wherein the expression of the one or more expression sequences comprises the expression of the one or more expression sequences in a cell comprising the circular polyribonucleotide.

12. The method of claim 11, wherein the circular polyribonucleotide persists during cell division.

13. The method of claim 11, wherein at least approximately 60% of a quantity of the circular polyribonucleotide persists for at least approximately 7 days in the cell.

14. The method of claim 11, wherein: (a) the expression of the one or more expression sequences in the cell at a later time point is equal to or greater than at an earlier time point; or (b) the expression of the one or more expression sequences in the cell over a period of at least approximately 7 days is not reduced by 40%; or (c) the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 40% for at least approximately 7 days.

15. The method of claim 1, wherein the expression of the one or more expression sequences comprises expressing the expression sequences in a mammalian subject.

16. The method of claim 1, wherein the termination element comprises a stop codon.

17. The method of claim 11, wherein the circular polyribonucleotide further comprises a replication domain configured to mediate self-replication of the circular polyribonucleotide.

18. A pharmaceutical composition comprising a circular polyribonucleotide comprising (a) a circular polyribonucleotide comprising one or more expression sequences and a stagger element at a 3' end of at least one of the one or more expression sequences, wherein the circular polyribonucleotide is competent for rolling circle translation and lacks a termination element; and (b) a pharmaceutically acceptable excipient.

19. A cell comprising a circular polyribonucleotide comprising one or more expression sequences and a stagger element at a 3' end of at least one of the one or more expression sequences, wherein the circular polyribonucleotide is competent for rolling-circle translation and lacks a termination element.

20. A method of in vivo expression of one or more expression sequences in a subject, comprising: administering a circular polyribonucleotide to a cell of the subject wherein the circular polyribonucleotide comprises the one or more expression sequences; and expressing the one or more expression sequences from the circular polyribonucleotide in the cell, wherein the circular polyribonucleotide is configured such that the expression of the one or more expression sequences in the cell at a later time point is equal to or greater than at an earlier time point.

21. A method of in vivo expression of one or more expression sequences in a subject, comprising: administering a circular polyribonucleotide to a cell of the subject wherein the circular polyribonucleotide comprises the one or more expression sequences; and expressing the one or more expression sequences from the circular polyribonucleotide in the cell, wherein the circular polyribonucleotide is configured such that the expression of the one or more expression sequences in the cell over a period of at least 7, 8, 9, 10, 12, 14 or 16 days, is not reduced by more than approximately 40%.

22. A method of in vivo expression of one or more expression sequences in a subject, comprising: administering a circular polyribonucleotide to a cell of the subject wherein the circular polyribonucleotide comprises the one or more expression sequences; and expressing the one or more expression sequences from the circular polyribonucleotide in the cell, wherein the circular polyribonucleotide is configured such that the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than approximately 40% for at least 7, 8, 9, 10, 12, 14, or 16 days.

23. A pharmaceutical composition comprising a circular polyribonucleotide comprising at least one structural element selected from: a) an encryptogen; b) a stagger element; c) a regulatory element; d) a replication element; f) a quasi-double-stranded secondary structure; and g) an expression sequence; and at least one functional feature selected from: a) higher translation efficiency than a linear homolog; b) stoichiometric translation efficiency of multiple translation products; c) less immunogenicity than a homolog lacking an encryptogen; d) increased half-life compared to a linear homolog; and e) persistence during cell division.

24. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide comprising one or more expression sequences, wherein the circular polyribonucleotide is competent for rolling circle translation.

25. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide comprising one or more expression sequences and competent for rolling-circle translation, wherein the circular polyribonucleotide is configured such that at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the total (molar / molar) polypeptides generated during rolling-circle translation of the circular polyribonucleotide are isolated polypeptides, and wherein each of the isolated polypeptides is generated from a single round of translation or less than a single round of translation of the one or more expression sequences.

26. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide comprising one or more expression sequences and a stagger element at the 3' end of at least one of the one or more expression sequences, wherein the stagger element is configured to interrupt a ribosome during rolling-circle translation of the circular polyribonucleotide.

27. A method for producing the composition of any one of claims 18 or 23-26, comprising combining the circular polyribonucleotide of any one of claims 18 or 23-26 and the pharmaceutically acceptable carrier or excipient of any one of claims 18 or 23-26.

28. A treatment method, comprising administering the composition of any one of claims 18 or 23-26.

29. A method for protein expression, comprising translating at least one region of the circular polyribonucleotide of any one of claims 18 or 23-26. ncionn / nznz / E / Y 165 30. The method of claim 29, wherein the translation of at least one region of the circular polyribonucleotide occurs in vitro.

31. The method of claim 29, wherein the translation of at least one region of the circular polyribonucleotide occurs in vivo.

32. A polynucleotide encoding the circular polyribonucleotide of any one of claims 18 or 23-26.

33. A method for producing the circular polyribonucleotide of any one of claims 18 or 23-26.