Compositions comprising cyclic polyribonucleotides and uses thereof

Abstract

The present invention relates generally to pharmaceutical compositions and formulations of cyclic polyribonucleotides and uses thereof.

Description

Cross-references

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 599,547, filed December 15, 2017, and U.S. Provisional Application No. 62 / 676,688, filed May 25, 2018, each of which is incorporated herein by reference in its entirety. Background Art

[0002] Certain cyclic polyribonucleotides (PRNs) are ubiquitous in human tissues and cells, including those of healthy individuals. Summary of the Invention

[0003] In one aspect, the present invention includes a pharmaceutical composition comprising a cyclic polyribonucleotide comprising at least one structural element selected from the group consisting of: a) an encryptogen; b) a staggered element; c) a regulatory element; d) a replication element; f) a quasi-double-stranded secondary structure; and g) an expressed sequence; and at least one functional characteristic selected from the group consisting of: a) a translation efficiency greater than that of a linear counterpart; b) stoichiometric translation efficiencies of a variety of translation products; c) immunogenicity less than that of a counterpart lacking the encryptogen; d) a half-life greater than that of a linear counterpart; and e) persistence during cell division.

[0004] In certain embodiments, cyclic polyribonucleotide has translation ability.In one such embodiment, quasi-helical structure comprises at least one double-stranded RNA segment and at least one non-double-stranded segment.In another such embodiment, quasi-helical structure comprises the first sequence and the second sequence that are connected with tumor-necrosis factor-receptor sequence for example rich A sequence.

[0005] In some embodiments, the cyclic polyribonucleotide comprises an encryptogen. In some embodiments, the encryptogen comprises at least one modified ribonucleotide, such as pseudouridine, N(6)methyladenosine (m6A). In some embodiments, the encryptogen comprises a protein binding site, such as a ribonucleotide binding protein. In some embodiments, the encryptogen comprises an immune protein binding site, for example, to evade an immune response, such as a CTL response.

[0006] In some embodiments, the cyclic polyribonucleotide comprises at least one modified ribonucleotide.

[0007] In some embodiments, the immunogenicity of the cyclic polyribonucleotide is at least 2-fold lower than that of a counterpart lacking an encryption precursor, as assessed by expression or signaling or activation of at least one of RIG-1, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, IFN-β.

[0008] In some embodiments, the circular polyribonucleotide further comprises a riboswitch.

[0009] In some embodiments, the cyclic polyribonucleotide further comprises an aptazyme.

[0010] In some embodiments, the cyclic polyribonucleotide comprises a translation initiation sequence, such as a GUG or CUG start codon, such as for expression under stress conditions.

[0011] In certain embodiments, the cyclic polyribonucleotide comprises at least one expressed sequence, for example, and coded polypeptide.In this type of embodiment, expressed sequence encoding peptide or polynucleotide.In certain embodiments, the cyclic polyribonucleotide comprises a plurality of identical or different expressed sequences.

[0012] In some embodiments, the circular polyribonucleotide comprises staggered elements, such as 2A.

[0013] In certain embodiments, cyclic polyribonucleotide comprises regulatory nucleic acid, for example non-coding RNA.In certain embodiments, cyclic polyribonucleotide comprises regulatory element, for example, changes the regulatory element of the expression of expressed sequence.

[0014] In some embodiments, the circular polyribonucleotide ranges in size from about 20 bases to about 20 kb.

[0015] In some embodiments, circular polyribonucleotides are synthesized by circularization of linear polynucleotides.

[0016] In some embodiments, the cyclic polyribonucleotide is substantially resistant to degradation by, for example, exonucleases.

[0017] In some embodiments, the cyclic 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 termination element; f) an internal ribosome entry site; g) susceptibility to exonuclease degradation, and h) binding to a cap-binding protein.

[0018] In one aspect, the invention includes a method of producing a composition comprising cyclic polyribonucleotides as described herein.

[0019] In one aspect, the present invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a cyclic polyribonucleotide comprising one or more expression sequences, wherein the cyclic polyribonucleotide is capable of rolling circle translation.

[0020] In certain embodiments, each in one or more expressed sequences is separated from the expressed sequence of succession by the staggered elements in the cyclic polyribonucleotide, wherein the rolling circle translation of one or more expressed sequences generates at least two polypeptide molecules, and for example staggered elements makes ribosome stagnate (stall) or stop, thereby extending polypeptide is fallen off from ribosome.In certain embodiments, staggered elements stop two rounds of translation of (a) single expressed sequence or (b) one or more rounds of translation of two or more expressed sequences to generate single polypeptide.For example, staggered elements can stop two rounds or more rounds of translation of two or more expressed sequences to generate single polypeptide, for example, staggered elements stops ribosome and / or allows extending polypeptide to fall off from ribosome after around cyclic polyribonucleotide loop.

[0021] In some embodiments, the interlaced element is a sequence that is separated from one or more expressed sequences.

[0022] In some embodiments, the interlaced elements comprise a portion of an expressed sequence of one or more expressed sequences.

[0023] On the one hand, the present invention includes a kind of pharmaceutical composition, this pharmaceutical composition comprises pharmaceutically acceptable carrier or excipient and cyclic polyribonucleotide, this cyclic polyribonucleotide comprises one or more expressed sequences and can carry out rolling circle translation, wherein the cyclic polyribonucleotide is configured to make at least 10% in the total polypeptide (mole / mole) generated during the rolling circle translation of the cyclic polyribonucleotide, 20%, 30%, 40%, 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% be discrete polypeptides, and wherein each in the discrete polypeptide is all by single round translation of one or more expressed sequences or is less than single round translation and generates.

[0024] In certain embodiments; The cyclic polyribonucleotide is configured to make at least 10% in the total polypeptide (mole / mole) that generates during the rolling circle translation of the cyclic polyribonucleotide, 20%, 30%, 40%, 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% be discrete polypeptides; And wherein in the in vitro translation system, test discrete product with respect to the amount ratio of total polypeptide.

[0025] In some embodiments, the in vitro translation system comprises rabbit reticulocyte lysate.

[0026] In some embodiments, the stagger element is located downstream or 3' to at least one of the one or more expressed sequences, wherein the stagger element is configured to stall ribosomes during rolling circle translation of the circular polyribonucleotide.

[0027] On the one hand, the invention provides a kind of pharmaceutical composition, this pharmaceutical composition comprises pharmaceutically acceptable carrier or excipient and cyclic polyribonucleotide, and this cyclic polyribonucleotide comprises one or more expressed sequences and in one or more expressed sequences at least one downstream or 3 ' staggered element.In certain embodiments, staggered element is configured to make ribosome stagnate during the rolling circle translation of cyclic polyribonucleotide.

[0028] In some embodiments, the interlaced elements encode a peptide sequence selected from the group consisting of a 2A sequence and a 2A-like sequence.

[0029] In some embodiments, the staggered elements encode a sequence having a C-terminal sequence of GP.

[0030] In some embodiments, the staggered elements encode a sequence having a C-terminal consensus sequence of D(V / I)ExNPG P, where x = any amino acid.

[0031] In some embodiments, the interleaved elements encode at least one of GDVESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, GDVEQNPGP, IESNPGP, GDIELNPGP, HDIETNPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP.

[0032] In some embodiments, the staggered element is located downstream or 3' to each of the one or more expressed sequences.

[0033] In certain embodiments, the staggered elements of the first expressed sequence in the cyclic polyribonucleotide are upstream (5') of the first translation initiation sequence of the expressed sequence that follows the first expressed sequence in the cyclic polyribonucleotide, and wherein the distance between staggered elements and the first translation initiation sequence enables the first expressed sequence and subsequent expressed sequence to be translated continuously. In certain embodiments, the staggered elements comprise the termination element of the first expressed sequence on the cyclic polyribonucleotide, and there is a certain distance between the upstream (5') of the translation initiation sequence of the first expressed sequence subsequent expressed sequence on this staggered element and the cyclic polyribonucleotide, and wherein the distance enables the first expressed sequence and subsequent expressed sequence thereof to be translated continuously.

[0034] In some embodiments, the first staggered element is located upstream (5') of the first translation initiation sequence of a first expressed sequence in the circular polyribonucleotide, wherein the circular polyribonucleotide is continuously translated, wherein the corresponding circular polyribonucleotide comprising the second staggered element upstream of the second translation initiation sequence of a second expressed sequence in the corresponding circular polyribonucleotide is not continuously translated 5', and wherein the distance between the second staggered element in the corresponding circular polyribonucleotide and the second translation initiation sequence is greater, for example, at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times the distance between the first staggered element and the first translation initiation sequence in the circular polyribonucleotide. In some embodiments, the staggered element comprises a first termination element upstream (5') of a first translation initiation sequence of a first expressed sequence in the circular polyribonucleotide, wherein the circular polyribonucleotide is continuously translated, and the corresponding circular polyribonucleotide comprising the staggered element (comprising the second termination element) upstream of a second translation initiation sequence of a second expressed sequence in the corresponding circular polyribonucleotide is not continuously translated, and wherein the distance between the second termination element in the corresponding circular polyribonucleotide and the second translation initiation sequence is larger, for example, at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times the distance between the first termination element and the first translation initiation in the circular polyribonucleotide.

[0035] In some embodiments, the distance between the first staggered element and the first translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or more. In some embodiments, the distance between the second staggered element and the second translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or greater than the distance between the first staggered element and the first translation start.

[0036] In some embodiments, the circular polyribonucleotide comprises more than one expression sequence.

[0037] In some embodiments, the circular polyribonucleotide has a translation efficiency that is 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, 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 higher than its linear counterpart.

[0038] In some embodiments, the circular polyribonucleotide has a translation efficiency at least 5-fold higher than its linear counterpart.

[0039] In some embodiments, the circular polyribonucleotide lacks an internal ribosome entry site.

[0040] In some embodiments, one or more expressed sequences comprises a Kozak initiation sequence.

[0041] In some embodiments, one or more expressed sequences encode a peptide.

[0042] In certain embodiments, cyclic polyribonucleotide comprises regulatory nucleic acid, for example non-coding RNA.In certain embodiments, cyclic polyribonucleotide comprises regulatory element, for example, changes the regulatory element of the expression of expressed sequence.

[0043] In one aspect, the invention provides a cyclic polyribonucleotide of any of the pharmaceutical compositions provided herein.

[0044] In one aspect, the present invention includes a method of producing a pharmaceutical composition provided herein, comprising combining a cyclic polyribonucleotide as described herein with a pharmaceutically acceptable carrier or excipient as described herein.

[0045] In one aspect, the invention includes a method of administering a composition comprising cyclic polyribonucleotides as described herein.

[0046] In one aspect, the present invention includes a method for protein expression comprising translating at least a region of a cyclic polyribonucleotide provided herein.

[0047] In certain embodiments, the translation in at least one region of the cyclic polyribonucleotide is carried out in vitro.In certain embodiments, the translation in at least one region of the cyclic polyribonucleotide is carried out in vivo.

[0048] In one aspect, the present invention includes a polynucleotide, such as a DNA vector, encoding a circular polyribonucleotide provided herein.

[0049] In one aspect, the present invention includes a method of producing a cyclic polyribonucleotide provided herein.

[0050] In some embodiments, the method comprises splint ligation-mediated circularization of linear polyribonucleotides.

[0051] In some embodiments, the cyclization, e.g., splint-mediated cyclization, has an efficiency of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, or at least 50%. In some embodiments, the splint-mediated cyclization has an efficiency of about 40% to about 50% or greater. definition

[0052] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims.Unless otherwise indicated, the terms set out below should generally be understood in their common sense.

[0053] The term "obtainable by," "producible by," or other similar phrases are used to indicate that a claim or embodiment refers to the compound, composition, product, etc. itself, that is, the compound, composition, product, etc. can be obtained or produced by the method described for making the compound, composition, product, etc., but the compound, composition, product, etc. can also be obtained or produced by methods other than the described method. The term "obtained by," "produced by," or the like indicates that the compound, composition, product is obtained or produced by the specific method recited. It should be understood that the terms "obtainable by," "producible by," and the like also disclose the terms "obtained by," "produced by," and the like as preferred embodiments of "obtainable by," "producible by," and the like.

[0054] The phrase "compounds, compositions, products, etc. for use in treatment, regulation, etc." should be understood to mean compounds, compositions, products, etc. that are suitable for the indicated purpose of treatment, regulation, etc. The phrase "compounds, compositions, products, etc. for use in treatment, regulation, etc." further discloses that, as a preferred embodiment, such compounds, compositions, products, etc. are used for treatment, regulation, etc.

[0055] The words "compounds, compositions, products, etc. for use in..." or "use of compounds, compositions, products, etc. for the manufacture of a medicament, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for..." indicate that these compounds, compositions, products, etc. are to be used in a method of treatment that can be practiced on humans or animals. They are considered to be equivalent disclosures to the examples and claims relating to methods of treatment, etc. If an example or claim thus refers to a "compound for use in treating a human or animal suspected of having a disease", this is also considered to disclose "the use of the compound in the manufacture of a medicament for treating a human or animal suspected of having a disease" or "a method of treatment by administering the compound to a human or animal suspected of having a disease". The words "compounds, compositions, products, etc. for treatment, regulation, etc." should be understood to mean compounds, compositions, products, etc. that are themselves suitable for the indicated purpose of treatment, regulation, etc.

[0056] Term " pharmaceutical composition " is intended to disclose equally that the cyclic polyribonucleotide that is included in the pharmaceutical composition can be used for by therapy treatment human body or animal body.Therefore, this means to be equal to " cyclic polyribonucleotide for the treatment of ".

[0057] Circular polyribonucleotide as herein described, the compositions comprising this type of cyclic polyribonucleotide, the method using this type of cyclic polyribonucleotide etc. are partly based on following example, these examples have been illustrated and comprise different elements, for example replication element, expressed sequence, staggered element and encryption original (for example, referring to example 1) or for example expressed sequence, staggered element and regulating and controlling element (for example, referring to example 30 and example 38) cyclic polyribonucleotide effector how can be used for realizing different technical effects (for example, in example 1 and example 38, be higher than the translation efficiency of linear counterpart and the half-life that linear counterpart increases in example 38).Based on especially these examples, description hereinafter has considered the various modifications of the specific discovery considered in example and combination.

[0058] As used herein, the terms "circRNA" or "cyclic polyribonucleotide" or "circular RNA" are used interchangeably and may refer to a polyribonucleotide that forms a circular structure through covalent or non-covalent bonds.

[0059] As used herein, term " encryption original " can refer to nucleotide sequence or the structure of cyclic polyribonucleotide, and this nucleotide sequence or structure help to reduce, escape and / or avoid being detected by immunocyte and / or reduce the induction of the immune response at cyclic polyribonucleotide.

[0060] As used herein, the term "expressed sequence" may refer to a nucleic acid sequence encoding a product, such as a peptide or polypeptide or a regulatory nucleic acid. An exemplary expressed sequence encoding a peptide or polypeptide may comprise a plurality of nucleotide triplets, each of which may encode an amino acid and is referred to as a "codon."

[0061] As used herein, the term "immune protein binding site" can refer to a nucleotide sequence that is combined with an immune protein. In certain embodiments, the immune protein binding site helps to mask the cyclic polyribonucleotide into exogenous, for example, the immune protein binding site can be combined by protein (such as a competitive inhibitor), thereby preventing the cyclic polyribonucleotide from being identified and combined by the immune protein, and then reducing or avoiding the immune response for the cyclic polyribonucleotide. As used herein, the term "immune protein" can refer to any protein or peptide related to the immune response (such as for example, as for immunogens, such as cyclic polyribonucleotides). The non-limiting examples of immune protein include T cell receptor (TCR), antibodies (immunoglobulins), major histocompatibility complex (MHC) proteins, complement proteins and RNA binding proteins.

[0062] As used herein, the term "ribonucleotide" may refer to a nucleotide having at least one modification to the sugar, nucleobase, or internucleoside linkage.

[0063] As used herein, the phrase "quasi-helical structure" may refer to the higher-order structure of a cyclic polyribonucleotide, wherein at least a portion of the cyclic polyribonucleotide is folded into a helical structure.

[0064] As used herein, the phrase "quasi-double-stranded secondary structure" may refer to the higher-order structure of a cyclic polyribonucleotide, wherein at least a portion of the cyclic polyribonucleotide creates an internal double strand.

[0065] As used herein, the term "regulatory element" may refer to a moiety, such as a nucleic acid sequence, that modifies the expression of an expressed sequence within a circular polyribonucleotide.

[0066] As used herein, the term "repetitive nucleotide sequence" may refer to a repetitive nucleic acid sequence within a DNA or RNA segment or within an entire genome. In some embodiments, the repetitive nucleotide sequence comprises a poly-CA sequence or a poly-TG (UG) sequence. In some embodiments, the repetitive nucleotide sequence comprises a repetitive sequence in the intron Alu family.

[0067] As used herein, the term "replication element" may refer to a sequence and / or motif that can be used to replicate or initiate transcription of a circular polyribonucleotide.

[0068] As used herein, the term "staggered element" can refer to a moiety, such as a nucleotide sequence, that induces ribosome pausing during translation. In some embodiments, the staggered element is a non-conserved sequence of amino acids with a strong α-helical propensity followed by the consensus sequence -D(V / I)ExNPG P, where x = any amino acid. In some embodiments, the staggered element can include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof.

[0069] As used herein, the term "substantially resistant to" can refer to a substance that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% resistant compared to a reference.

[0070] As used herein, term " stoichiometric translation " can refer to the substantially equal generation of the expression product that obtains from cyclic polyribonucleotide translation.For example, for the cyclic polyribonucleotide with two expressed sequences, the stoichiometric translation of cyclic polyribonucleotide can represent that the expression product of two expressed sequences can have substantially equal amount, and for example the amount difference (for example molar difference) between two expressed sequences can be about 0, or be less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%.

[0071] As used herein, the term "translation initiation sequence" may refer to a nucleic acid sequence in a circular polyribonucleotide that initiates translation of an expressed sequence.

[0072] As used herein, the term "termination element" may refer to a portion, such as a nucleic acid sequence, that terminates translation of an expressed sequence in a circular polyribonucleotide.

[0073] As used herein, the term "translational efficiency" can refer to the rate or amount of protein or peptide produced from ribonucleotide transcription. In some embodiments, translation efficiency can be expressed as the amount of protein or peptide produced from a given amount of transcripts encoding the protein or peptide, e.g., within a given time period, e.g., in a given translation system, e.g., an in vitro translation system (e.g., rabbit reticulocyte lysate) or an in vivo translation system (e.g., eukaryotic or prokaryotic cells).

[0074] As used herein, the term "cyclization efficiency" may refer to a measure of the resulting circular polyribonucleotide relative to its starting material.

[0075] As used herein, the term "immunogenicity" can refer to the potential for inducing an immune response to a certain substance. In certain embodiments, when the immune system of an organism or a certain type of immune cell is exposed to an immunogenic substance, an immune response can be induced. The term "non-immunogenicity" can refer to lacking or not having an immune response on the detectable threshold of the material. In certain embodiments, when the immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic substance, no immune response is detected. In certain embodiments, when measured by an immunogenicity assay, non-immunogenic cyclic polyribonucleotides as provided herein can not induce an immune response that exceeds a predetermined threshold. For example, when using an immunogenicity assay to measure the antibody produced for cyclic polyribonucleotides or inflammatory markers, non-immunogenic polyribonucleotides provided herein can cause the generation of antibody or marker to be lower than the level of a predetermined threshold. The predetermined threshold can be at most 1.5 times, 2 times, 3 times, 4 times or 5 times the antibody or marker level produced by a control reference. Incorporated by Reference

[0076] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The embodiments of the present invention described in detail below will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, exemplary embodiments of the present invention are shown in the accompanying drawings. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0078] Figure 1 Various exemplary cyclization methods are shown.

[0079] Figure 2 Schematic diagram of an exemplary in vitro production process of circular RNA comprising a start codon, an ORF (open reading frame) encoding GFP, an interlaced element (2A), an encipherogen, and an IRES (internal ribosome entry site).

[0080] Figure 3 A schematic diagram showing an exemplary in vivo production process of circular RNA is shown.

[0081] Figure 4 Shown is the design of an exemplary circular RNA comprising a start codon, an ORF encoding GFP, an interlaced element (2A), and an encipherogen.

[0082] Figure 5 A and 5B are schematic diagrams showing the in vivo stoichiometric protein expression of two different circular RNAs.

[0083] Figure 6 A schematic showing a control circular RNA with an intron and expressing GFP.

[0084] Figure 7 A schematic showing an exemplary circular RNA with a synthetic riboswitch (shown in red) that regulates the expression of GFP from the circular RNA in the presence or absence of a riboswitch ligand.

[0085] Figure 8 A schematic showing in vivo protein expression of an exemplary circular RNA in a mouse model.

[0086] Figure 9 A schematic showing in vivo biodistribution of an exemplary circular RNA in a mouse model.

[0087] Figure 10 A schematic showing in vivo protein expression of an exemplary circular RNA with an encryption origin (intron) in a mouse model.

[0088] Figure 11 A schematic showing an exemplary circular RNA with a double-stranded RNA segment that can be subjected to dot blot analysis of its structural information.

[0089] Figure 12 A schematic showing an exemplary circular RNA with a quasi-helical structure (HDVmin) that can be subjected to SHAPE analysis of its structural information.

[0090] Figure 13 A schematic showing an exemplary circular RNA with a functional quasi-helical structure (HDVmin) that exhibits HDAg binding activity.

[0091] Figure 14 A schematic showing transcription, self-cleavage, and ligation of an exemplary self-replicating circular RNA.

[0092] Figure 15 A schematic showing an exemplary circular RNA that is expressed in vivo and has improved in vivo stability.

[0093] Figure 16 A schematic showing an exemplary circular RNA that is retained during mitosis and persists in daughter cells. A BrdU pulse as shown was used to label dividing cells.

[0094] Figure 17is a denaturing PAGE gel image showing in vitro production of different exemplary circular RNAs.

[0095] Figure 18 is a graph summarizing circularization efficiency of different exemplary circular RNAs.

[0096] Figure 19 is a denaturing PAGE gel image showing that exemplary circular RNAs are less susceptible to degradation compared to their linear counterparts.

[0097] Figure 20 is a denaturing PAGE gel image showing exemplary circular RNAs after an exemplary purification process.

[0098] Figure 21 is a Western blot image showing Flag protein (about 15 kDa) expression by exemplary circular RNAs (lacking IRES, cap, 5' and 3' UTR).

[0099] Figure 22 is a Western blot image showing rolling circle translation of exemplary circular RNAs.

[0100] Figure 23 shows a Western blot image showing production of discrete proteins or continuous long peptides from different exemplary circular RNAs with or without exemplary interleave elements.

[0101] Figure 24A is a Western blot image showing comparison of protein expression between different exemplary circular RNAs with exemplary interleave elements or termination elements (stop codon).

[0102] Figure 24B is a graph summarizing signal intensity of Western blot analysis of protein products translated from two exemplary circular RNAs.

[0103] Figure 25 is a graph summarizing luciferase activity of translation products of exemplary circular RNAs and their linear counterparts compared to vehicle control RNA.

[0104] Figure 26 is a graph summarizing RNA amounts at different collection time points in a time course experiment testing half-life of exemplary circular RNAs.

[0105] Figure 27A is a graph showing qRT-PCR analysis of linear and circular RNA levels 24 hours after delivery of primers to cells that capture linear and circular RNAs.

[0106] Figure 27B Figure 2 is a graph showing qRT-PCR analysis of linear and circular RNA levels using primers specific for circular RNA.

[0107] Figure 28 Figure 3 is a blot image showing cell lysates of circular and linear RNA probed for EGF protein and beta-tubulin loading control.

[0108] Figure 29 Figure 4 is a graph showing qRT-PCR analysis of immune-related genes from 293T cells transfected with circular or linear RNA.

[0109] Figure 30 Figure 5 is a graph showing luciferase activity of proteins expressed by circular RNA via rolling circle translation.

[0110] Figure 31 Figure 6 is a graph showing luciferase activity of proteins expressed by circular or linear RNA.

[0111] Figure 32 Figure 7 is a graph showing luciferase activity of proteins expressed by linear or circular RNA via rolling circle translation.

[0112] Figure 33 Figure 8 is a graph showing luciferase activity of proteins expressed by circular RNA via IRES translation initiation.

[0113] Figure 34 Figure 9 is a graph showing luciferase activity of proteins expressed by circular RNA via IRES initiation and rolling circle translation.

[0114] Figure 35 Figure 10 is an image showing a Western blot of expression products of circular or linear RNA.

[0115] Figure 36 Figure 11 is an image showing a Western blot of expression products of circular or linear RNA.

[0116] Figure 37 Figure 12 shows a predicted structure of an exemplary circular RNA having a quasi- duplex structure.

[0117] Figure 38 Figure 13 shows a predicted structure of an exemplary circular RNA having a quasi- helix structure.

[0118] Figure 39 Figure 14 shows a predicted structure of an exemplary circular RNA having a quasi- helix structure linked to a repeat sequence.

[0119] Figure 40Experiments data showing that the nucleic acid degradation products produced by RNase H degradation of exemplary circular RNAs are consistent with the circular RNA (and not linear RNA) are shown.

[0120] Figure 41 Gel images showing different lengths of DNA generated to establish various RNA lengths are shown.

[0121] Figure 42 Experiments data showing that RNA circularization is confirmed using RNase R treatment and qPCR analysis for circular junctions of various lengths are shown.

[0122] Figure 43 Generation of exemplary circular RNA with miRNA binding sites is shown.

[0123] Figure 44 Generation of exemplary circular RNA by self-splicing is shown.

[0124] Figure 45 Generation of exemplary circular RNA with protein binding sites is shown.

[0125] Figure 46 Experiments data showing that circular RNA has higher stability in dividing cells compared to linear controls are shown.

[0126] Figure 47 Experiments data showing protein expression of exemplary circular RNA with multiple expression sequences and rolling circle translation of exemplary circular RNA with multiple expression sequences are shown.

[0127] Figure 48 Experiments data showing that exemplary circular RNA has lower toxicity to transfected cells compared to linear controls are shown.

[0128] Figure 49 Experiments data showing that exemplary circular RNA is translated at higher levels compared to linear RNA under stress conditions are shown.

[0129] Figure 50 Generation of circular RNA with riboswitches is shown.

[0130] Figure 51A , Figure 51B and Figure 51C Modified circular RNA is shown to be translated in cells.

[0131] Figures 52A to 52CIt is shown that the modified circular RNA has reduced immunogenicity to cells compared to unmodified circular RNA as assessed by MDA5, OAS and IFN-beta expression in transfected cells.

[0132] Figure 53 It is shown that the level of circular RNA detected in the liver of mice is higher than linear RNA at day 3, day 4 and day 7 post injection after injection into mice.

[0133] Figure 54A and Figure 54B It is shown that after injection of circular RNA or linear RNA expressing Gaussia luciferase into mice, Gaussia luciferase activity was detected in the plasma at day 1, day 2, day 7, day 11, day 16 and day 23 after administration of the circular RNA, while its activity was only detected in the plasma at day 1 and day 2 after administration of the modified linear RNA.

[0134] Figure 55 It is shown that after injection of RNA, circular RNA but not linear RNA was detected in the liver and spleen at 16 days post administration of the RNA.

[0135] Figure 56 It is shown that after injection of RNA, linear RNA but not circular RNA showed immunogenicity as assessed by RIG-I, MDA-5, IFN-B and OAS. DETAILED DESCRIPTION

[0136] The present application relates generally to pharmaceutical compositions and formulations of circular polyribonucleotides and uses thereof. Circular polyribonucleotides

[0137] In some aspects, the application described herein includes compositions and methods of using and making circular polyribonucleotides and delivering circular polyribonucleotides. In some embodiments, the circular polyribonucleotides are non-immunogenic in mammals, e.g., humans. In some embodiments, the circular polyribonucleotides are capable of replication in or at cells from aquaculture animals (fish, crabs, shrimp, oysters, etc.), mammalian cells (e.g., cells from a pet or zoo animal (cat, dog, lizard, bird, lion, tiger, and bear, etc.), cells from a farm or work animal (horse, cow, pig, chicken, etc.), human cells), cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, the application includes a cell comprising a circular polyribonucleotide described herein, wherein the cell is from aquaculture animals (fish, crabs, shrimp, oysters, etc.), mammalian cells (e.g., cells from a pet or zoo animal (cat, dog, lizard, bird, lion, tiger, and bear, etc.), cells from a farm or work animal (horse, cow, pig, chicken, etc.), human cells), cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, the cell is modified to comprise the circular polyribonucleotide.

[0138] In some embodiments, the circular polyribonucleotide comprises a sequence or expression product.

[0139] In some embodiments, the circular polyribonucleotide has a half-life that is at least the half-life of a linear counterpart (e.g., a linear expression sequence or a linear circular polyribonucleotide). In some embodiments, the circular polyribonucleotide has a half-life that is increased relative to the half-life 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 half-life or persistence of the circular polyribonucleotide in a cell is at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or more, or any time therein. In certain embodiments, the half-life or persistence of the circular polyribonucleotide in a cell is no more than about 10 minutes to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or any time therein. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell that is undergoing cell division. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a post-division cell. In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a cell that is undergoing division that is greater than about 10 minutes to about 30 days, or is at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or more, or any time therein.

[0140] In some embodiments, the circular polyribonucleotides modulate cell function, e.g., transiently or long term. In certain embodiments, the cell function is stably altered, e.g., the modulation persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or more, or any time therein. In certain embodiments, the cell function is transiently altered, e.g., the modulation persists for no more than about 30 minutes to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or any time therein.

[0141] 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 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 can be of sufficient size to accommodate the binding site of a ribosome. One of skill in the art will appreciate that the maximum size of a circular polyribonucleotide can be as large as is limited by the technology for producing and / or using the circular polyribonucleotide. Without being bound by theory, it is possible that multiple segments of RNA can be produced from DNA and anneal with their 5' and 3' overhangs to produce a "string" of RNA that can eventually be circularized when only one 5' overhang and one 3' overhang remain. In some embodiments, the maximum size of a circular polyribonucleotide can be limited by the ability to package the RNA and deliver it to a target. In some embodiments, the size of a circular polyribonucleotide is of a length sufficient to encode a useful polypeptide, and thus a length 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, 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 can be useful.

[0142] In certain embodiments, the cyclic polyribonucleotide comprises one or more elements described in other places of this paper.In certain embodiments, these elements can be separated from each other by spacer sequence or joint.In certain embodiments, these elements can be by 1 ribonucleotide, 2 nucleotide, about 5 nucleotide, about 10 nucleotide, about 15 nucleotide, about 20 nucleotide, about 30 nucleotide, about 40 nucleotide, about 50 nucleotide, about 60 nucleotide, about 80 nucleotide, about 100 nucleotide, about 150 nucleotide, about 200 nucleotide, about 250 nucleotide, about 300 nucleotide, about 400 nucleotide, about 500 nucleotide, about 600 nucleotide, about 700 nucleotide, about 800 nucleotide, about 900 nucleotide, about 1000 nucleotide, at most about 1kb, at least about 1000 nucleotide, the nucleotide of any amount therebetween is separated from each other.In certain embodiments, one or more elements are continuous each other, for example, lack spacer element.In certain embodiments, one or more elements in the cyclic polyribonucleotide are conformational flexibility. In certain embodiments, conformational flexibility is owing to sequence not containing secondary structure basically.In certain embodiments, cyclic polyribonucleotide comprises secondary or tertiary structure, and this secondary or tertiary structure holds function or the feature of one or more expectations as herein described, for example, holds the binding site of ribosome, for example, translates, for example rolling circle translation.

[0143] In certain embodiments, cyclic polyribonucleotide comprises specific sequence signature.For example, cyclic polyribonucleotide can comprise specific nucleotide composition.In some such embodiments, cyclic polyribonucleotide can comprise one or more purine enrichment districts (adenine or guanosine).In some such embodiments, cyclic polyribonucleotide can comprise one or more purine enrichment districts (adenine or guanosine).In certain embodiments, cyclic polyribonucleotide can comprise one or more AU enrichment districts or element (ARE).In certain embodiments, cyclic polyribonucleotide can comprise one or more adenine enrichment districts.

[0144] In some embodiments, the circular polyribonucleotide may include one or more repeat elements described elsewhere herein.

[0145] In some embodiments, the circular polyribonucleotide comprises one or more modifications described elsewhere herein.

[0146] In some embodiments, the circular polyribonucleotide comprises one or more expression sequences and is configured for sustained expression in a cell in a subject. In some embodiments, the circular polyribonucleotide is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than at an earlier time point. In such embodiments, expression of the one or more expression sequences can be maintained at a relatively stable level or can increase over time. Expression of the expression sequences can be relatively stable over an extended period of time. For example, in some cases, expression of the one or more expression sequences in the cell does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23, or more days. In some cases, 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% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23, or more days. expression sequence peptide or polypeptide

[0147] In some embodiments, the circular polyribonucleotide comprises at least one expression sequence that encodes a peptide or polypeptide. Such peptides include, but are not limited to, small peptides, peptidomimetics (e.g., peptoids), amino acids, and amino acid analogs. Peptides can be linear or branched. Such peptides can 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 such compounds. Such peptides can include, but are not limited to, neurotransmitters, hormones, drugs, toxins, viral or microbial particles, synthetic molecules, and agonists or antagonists thereof.

[0148] The polypeptide can be linear or branched. The length of the polypeptide can be from about 5 to about 40,000 amino acids, 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, from about 200 to about 10,000 amino acids, from about 500 to about 5,000 amino acids, from about 1,000 to about 2,500 amino acids, or any range therebetween. In some embodiments, the polypeptide is 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, or less than about 10,000 amino acids in length. In some embodiments, less than about 0 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less may be useful.

[0149] Some examples of peptides or polypeptides include, but are not limited to, fluorescent tags or markers, antigens, therapeutic peptides, synthetic or analog peptides of natural bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degradation or self-destruction peptides, and a variety of degradation or self-destruction peptides. The peptides described herein that can be used in the present invention also include antigen-binding peptides, such as antigen-binding antibodies or antibody-like fragments, such as single-chain antibodies, nanobodies (see, for example, Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21 (7): 1076-113). Such antigen-binding peptides can bind to cytoplasmic antigens, nuclear antigens, intracellular antigens.

[0150] In some embodiments, the circular polyribonucleotides comprise one or more RNA expression sequences, each of which can encode a polypeptide. The polypeptides can be produced in large quantities. As such, the polypeptides can be any protein molecule that can be produced. The polypeptides can be polypeptides that can be secreted from the cell or localized to the cytoplasm, nucleus, or membrane compartments of the cell. Some polypeptides include, but are not limited to, at least a portion of a viral envelope protein, a metabolic regulatory enzyme (e.g., regulating the production of lipids or steroids), an antigen, a tolerogen, a cytokine, a toxin, an enzyme for which the absence is associated with a disease, and a polypeptide that is not active in the animal until cleaved (e.g., in the animal’s gut), and a hormone.

[0151] In some embodiments, the circular polyribonucleotides include expression sequences that encode proteins, such as therapeutic proteins. In some embodiments, the therapeutic proteins that can be expressed from the circular polyribonucleotides disclosed herein have an antioxidant activity, a binding activity, a cargo receptor activity, a catalytic activity, a molecular carrier activity, a molecular function regulator, a molecular transducer activity, a nutritional reservoir activity, a protein tag, a structural molecule activity, a toxin activity, a transcription regulator activity, a translation regulator activity, or a transporter activity. Some examples of therapeutic proteins can include, but are not limited to, enzyme replacement proteins, proteins for supplementation, protein vaccines, antigens (e.g., tumor antigens, viruses, bacteria), hormones, cytokines, antibodies, immunotherapies (e.g., cancer), cell reprogramming / transdifferentiation factors, transcription factors, chimeric antigen receptors, transposases or nucleases, immune effectors (e.g., affecting susceptibility to immune response / signaling), regulated death effector proteins (e.g., inducers of apoptosis or necrosis), non-lysosomal inhibitors of tumors (e.g., cancer protein inhibitors), epigenetic modifiers, epigenetic enzymes, transcription factors, DNA or protein modification enzymes, DNA intercalators, efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors, enzyme competitive inhibitors, protein synthesis effectors or inhibitors, nucleases, protein fragments or domains, ligands or receptors, and CRISPR systems or components thereof.

[0152] In some embodiments, exemplary proteins that can be expressed by the cyclic polyribonucleotides disclosed herein include human proteins, such as receptor binding proteins, hormones, growth factors, growth factor receptor regulators, and regeneration proteins (e.g., proteins involved in proliferation and differentiation, such as therapeutic proteins for wound healing). In some embodiments, exemplary proteins that can be expressed by the cyclic polyribonucleotides disclosed herein include EGF (epithelial growth factor). In some embodiments, exemplary proteins that can be expressed by the cyclic polyribonucleotides disclosed herein include enzymes, such as oxidoreductases, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, non-ATP-dependent proteases, and desaturases. In some embodiments, exemplary proteins that can be expressed by the cyclic polyribonucleotides disclosed herein include intracellular proteins or cytoplasmic proteins. In some embodiments, cyclic polyribonucleotides express Luciferase (nLuc). In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotides disclosed herein include secreted proteins, such as secreted enzymes. In some cases, the circular polyribonucleotide expresses a secreted protein that has a short half-life in the blood, or can be a protein with a subcellular localization signal, or is a protein with a secretory signal peptide. In some embodiments, the circular polyribonucleotide expresses Gaussia luciferase (gLuc). In some cases, the circular polyribonucleotide expresses a non-human protein, such as a fluorescent protein, an energy transfer acceptor, or a protein tag like Flag, Myc, or His. In some embodiments, exemplary proteins that can be expressed from the circular polyribonucleotide include GFP. In some embodiments, the circular polyribonucleotide expresses a tag protein, such as a fusion protein containing a protein tag or an engineered protein, such as a chitin binding protein (CBP), maltose binding protein (MBP), Fc tag, glutathione-S-transferase (GST), AviTag (GLNDIFEAQKIEWHE), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL), polyglutamate tag (EEEEEE), E tag (GAPVPYPDPLEPR), FLAG tag (DYKDDDDK), HA tag (YPYDVPDYA), His tag (HHHHHH), Myc tag (EQKLISEEDL), NE tag (TKENPRSNQEESYDDNES), S tag (KETAAAKFERQHMDS), SBP tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), Sof tag 1 (SLAELLNAGLGGS), Sof tag 3 (TQDPSRVG); Spot tag (PDRVRAVSHWSS), Strep tag (Strep tag II: WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDPLD), V5 tag (GKPIPNPLLGLDST), VSV tag (YTDIEMNRLGK), or Xpress tag (DLYDDDDK).

[0153] In certain embodiments, cyclic polyribonucleotides express antibodies, such as antibody fragments or a part thereof. In certain embodiments, the antibody expressed by cyclic polyribonucleotides can be any isotype, such as IgA, IgD, IgE, IgG, IgM. In certain embodiments, cyclic polyribonucleotides express a part for an antibody, such as light chain, heavy chain, Fc fragment, CDR (complementary determining region), Fv fragment or Fab fragment, another part thereof. In certain embodiments, cyclic polyribonucleotides express one or more parts of an antibody. For example, cyclic polyribonucleotides can comprise more than one expression sequence, wherein each of which expresses a part for an antibody, and its summation can constitute an antibody. In some cases, cyclic polyribonucleotides comprise an expression sequence encoding an antibody heavy chain and an expression sequence encoding an antibody light chain. In some cases, when cyclic polyribonucleotides are expressed in a cell or acellular environment, light chain and heavy chain can be subjected to suitable modification, folding or other post-translational modifications to form functional antibodies. Regulatory elements

[0154] In some embodiments, the cyclic polyribonucleotide comprises a regulatory element, eg, a sequence that modifies the expression of an expressed sequence within the cyclic polyribonucleotide.

[0155] The regulatory element can include a sequence adjacent to an expressed sequence encoding an expression product. The regulatory element can be operably linked to an adjacent sequence. Compared to the amount of the product expressed when the regulatory element is not present, the regulatory element can increase the amount of the product expressed. In addition, a regulatory element can increase the amount of the product expressed by a plurality of expressed sequences connected in series. Therefore, a regulatory element can strengthen the expression of one or more expressed sequences. A plurality of regulatory elements are well-known to those of ordinary skill in the art.

[0156] Controlling element provided herein can comprise selective translation sequence.As used herein, term " selective translation sequence " can refer to the nucleotide sequence of the translation that selectively starts or activates expression sequence in cyclic polyribonucleotide, for example some riboswitch aptamer enzyme.Controlling element can also comprise selective degradation sequence.As used herein, term " selective degradation sequence " can refer to the nucleotide sequence of the degraded of the expression product of initial cyclic polyribonucleotide or cyclic polyribonucleotide.Exemplary selective degradation sequence can comprise riboswitch aptamer enzyme and miRNA binding site.

[0157] In some embodiments, the regulatory element is a translational modulator. A translational modulator can modulate translation of an expression sequence in a circular polyribonucleotide. A translational modulator can be a translational enhancer or a translational suppressor. In some embodiments, the circular polyribonucleotide comprises at least one translational modulator adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide comprises a translational modulator proximal to each expression sequence. In some embodiments, a translational modulator is present on one or both sides of each expression sequence, resulting in, for example, the spacing of expression products of one or more peptides and / or one or more polypeptides.

[0158] In some embodiments, a translation initiation sequence can act as a regulatory element. In some embodiments, the translation initiation sequence comprises an AUG codon. In some embodiments, the translation initiation sequence comprises any eukaryotic initiation codon, such as AUG, CUG, GUG, UUG, ACG, AUC, AUU, AAG, AUA, or AGG. In some embodiments, the translation initiation sequence comprises a Kozak sequence. In some embodiments, translation initiates at an alternative translation initiation sequence, such as a translation initiation sequence other than an AUG codon, under selective conditions, such as stress-inducing conditions. As a non-limiting example, translation of a circular polyribonucleotide can initiate at an alternative translation initiation sequence, such as ACG. As another non-limiting example, translation of a circular polyribonucleotide can initiate at an alternative translation initiation sequence, CTG / CUG. As yet another non-limiting example, translation of a circular polyribonucleotide can initiate at an alternative translation initiation sequence, GTG / GUG. As yet another non-limiting example, a circular polyribonucleotide can initiate translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence comprising a short stretch of a repeat RNA, such as CGG, GGGGCC, CAG, CTG.

[0159] Nucleotides flanking the codon initiating translation, such as but not limited to the start codon or an alternative start codon, are known to affect the translation efficiency, length, and / or structure of a circular polyribonucleotide. (See, e.g., Matsuda and Mauro, PLoS ONE [Public Library of Science], 2010 5:11; the contents of which are incorporated by reference herein in their entirety). Masking any nucleotides flanking the codon initiating translation can be used to alter the translation initiation position, translation efficiency, length, and / or structure of a circular polyribonucleotide.

[0160] In one embodiment, a masking agent can be used near the start codon or alternative start codon to mask or hide the codon to reduce the likelihood of initiating translation at the masked start codon or alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acid (LNA) oligonucleotides and exon junction complexes (EJC). (See, e.g., Matsuda and Mauro (PLoS ONE [Public Library of Science], 2010 5:11) describing masking agents LNA oligonucleotides and EJC; the contents of which are incorporated by reference herein in their entirety). In another embodiment, a masking agent can be used to mask the start codon of a circular polyribonucleotide to increase the likelihood that translation will initiate at the alternative start codon.

[0161] In some embodiments, translation initiates under selective conditions, such as but not limited to, selection for viral induction in the presence of GRSF-1, and the circular polyribonucleotide includes a GRSF-1 binding site, see, e.g., / / jvi.asm.org / content / 76 / 20 / 10417.full. Translation initiation sequence

[0162] In some embodiments, the circular polyribonucleotide encodes a polypeptide and can comprise a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence comprises a Kozak or Shine-Dalgarno sequence. In some embodiments, the circular polyribonucleotide comprises a translation initiation sequence, e.g., a Kozak sequence, adjacent to an expression sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, a translation initiation sequence, e.g., a Kozak sequence, is present on one or both sides of each expression sequence, resulting in a spacing of the expression products. In some embodiments, the circular polyribonucleotide comprises 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 substantially within a single-stranded region of the circular polyribonucleotide.

[0163] The circular polyribonucleotide can comprise more than 1 start codon, such as but not limited to at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, or more than 60 start codons. Translation can initiate on the first start codon or can initiate downstream of the first start codon.

[0164] In some embodiments, the circular polyribonucleotide can initiate at a codon other than the first start codon, e.g., AUG. Translation of the circular polyribonucleotide can proceed 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 of which are incorporated by reference herein in their entirety). In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress-induced conditions. As one non-limiting example, translation of the circular polyribonucleotide can begin at an alternative translation initiation sequence, such as ACG. As another non-limiting example, translation of the circular polyribonucleotide can begin at an alternative translation initiation sequence CTG / CUG. As yet another non-limiting example, translation of the circular polyribonucleotide can begin at an alternative translation initiation sequence GTG / GUG. As yet another non-limiting example, the circular polyribonucleotide can begin translation at a Repeat Associated Non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes a short stretch of a repeat RNA, e.g., CGG, GGGGCC, CAG, CTG.

[0165] In some embodiments, translation is initiated by treating eukaryotic initiation factor 4A (eIF4A) with Rocaglates (repress translation by blocking 43S scanning, leading to premature upstream translation initiation and reduced protein expression of transcripts carrying the RocA-eIF4A target sequence, see, e.g., www.nature.com / articles / nature17978). IRES

[0166] In some embodiments, the circular polyribonucleotides described herein comprise an internal ribosome entry site (IRES) element. Suitable IRES elements for inclusion in the circular polyribonucleotides include RNA sequences capable of engaging a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt. In one embodiment, the IRES element is derived from DNA of an organism including, but not limited to, viruses, mammals, and fruit flies. Such viral DNA can be derived from, but is not limited to, a picornavirus complementary DNA (cDNA), an encephalomyocarditis virus (EMCV) cDNA, and a poliovirus cDNA. In one embodiment, the fruit fly DNA from which the IRES element is derived includes, but is not limited to, the Antennapedia gene from Drosophila melanogaster.

[0167] In some embodiments, the IRES element is at least partially derived from a virus, e.g., it can be derived from a viral IRES element such as ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV_IGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_5NCR, CrPV_IGR, crTMV_IREScp, crTMV_IRESmp75, crTMV_IRESmp228, crTMV_IREScp, crTMV_IREScp, CSFV, CVB3, DCV_IGR, EMCV-R, EoPV_5NTR, ERAV_245-961, ERBV_162-920, EV71_1-748, FeLV-Notch2, FMDV_type_C, GBV-A, GBV-B, GBV-C, gypsy_env, gypsyD5, gypsyD2, HAV_HM175, HCV_type_1a, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HRV-2, IAPV_IGRpred, idelfix, KBV_IGRpred, LINE-1_ORF1_-101_to_-1, LINE-1_ORF1_-302_to_-202, LINE-1_ORF2_-138_to_-86, LINE-1_ORF1_-44_to_-1, PSIV_IGR, PV_type1_Mahoney, PV_type3_Leon, REV-A, RhPV_5NCR, RhPV_IGR, SINV1_IGRpred, SV40_661-830, TMEV, TMV_UI_IRESmp228, TRV_5NTR, TrV_IGR, or TSV_IGR.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, AT1R_var1, AT1R_var2, AT1R_var3, AT1R_var4, BAG1_p36delta236nt, BAG1_p36, BCL2, BiP_-222_-3, c-IAP1_285-1399, c-IAP1_1313-1462, c-jun, c-myc, Cat-1_224, CCND1, DAP5, eIF4G, eIF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A, FMR1, Gtx-133-141, Gtx-1-166, Gtx-1-120, Gtx-1-196, hairless, HAP4, HIF1a, hSNM1, Hsp101, hsp70, hsp70, Hsp90, IGF2_leader2, Kv1.4_1.2, 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, NtHSF1, ODC1, p27kip1, p53_128-269, PDGF2 / c-sis, Pim-1, 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, e.g., (GAAA)16, (PPT19)4, KMI1, KMI1, KMI2, KMI2, KMIX, X1, or X2.

[0168] In some embodiments, the circular polyribonucleotide comprises at least one IRES flanking at least one (e.g., 2, 3, 4, 5, or more) expression sequence. In some embodiments, the IRES flanks both sides of at least one (e.g., 2, 3, 4, 5, or more) expression sequence. In some embodiments, the circular polyribonucleotide comprises one or more IRES sequences on one or both sides of each expression sequence, resulting in the spacing of the resulting peptides and / or polypeptides. Termination element

[0169] In certain embodiments, cyclic polyribonucleotide comprises one or more expressed sequences, and each expressed sequence may or may not have terminator.In certain embodiments, cyclic polyribonucleotide comprises one or more expressed sequences, and expressed sequence lacks terminator so that cyclic polyribonucleotide is translated continuously.Owing to lacking ribosome stagnation or falling off, the exclusion of terminator may cause rolling circle translation or the continuous expression of expression product such as peptide or polypeptide.In such embodiments, rolling circle translation expresses continuous expression product by each expressed sequence.In some other embodiments, the terminator of expressed sequence can be a part for staggered element.In certain embodiments, one or more expressed sequences in cyclic polyribonucleotide comprise terminator.But, in cyclic polyribonucleotide, carry out the expression of rolling circle translation or subsequent (such as second, third, fourth, fifth etc.) expressed sequence.In this case, when ribosome runs into terminator (such as stop codon) and stops translation, expression product can fall off from ribosome.In certain embodiments, when ribosome such as at least one subunit of ribosome keeps contact with cyclic polyribonucleotide, translation stops.

[0170] In certain embodiments, the cyclic polyribonucleotide includes a terminator at the end of one or more expressed sequences. In certain embodiments, one or more expressed sequences continuously comprise two or more terminators. In such embodiments, translation terminates and rolling circle translation terminates. In certain embodiments, ribosomes are completely separated from the cyclic polyribonucleotides. In some such embodiments, the generation of subsequent (for example, the second, third, fourth, fifth, etc.) expressed sequences in the cyclic polyribonucleotides may require ribosomes to reengage with the cyclic polyribonucleotides before initiating translation. Typically, terminator elements include nucleotide triplet in the frame that sends a translation termination signal, for example, UAA, UGA, UAG. In certain embodiments, one or more terminators in the cyclic polyribonucleotides are terminators of frame shifts, such as, but not limited to, can terminate the off-frame (off-frame) or -1 and +1 shifted reading frames (for example, hidden termination) of translation. The terminator elements of frame shifts include nucleotide triplet, TAA, TAG and TGA that appear in the second reading frame and the third reading frame of the expressed sequence. The terminator elements of frame shifts may be important to preventing the mRNA misreading that is usually harmful to cells. Staggered elements

[0171] In some embodiments, the circular polyribonucleotide comprises at least one stagger element adjacent to an expression sequence. In some embodiments, the circular polyribonucleotide comprises a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, resulting in, for example, the partitioning of the expression product of one or more peptides and / or one or more polypeptides. In some embodiments, the stagger element is part of 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 partitioned from a succeeding expression sequence by a stagger element of the circular polyribonucleotide. In some embodiments, the stagger element prevents (a) two rounds of translation of a single expression sequence or (b) one or more rounds of translation of two or more expression sequences from generating a single polypeptide. In some embodiments, the stagger element is a sequence that is partitioned from one or more expression sequences. In some embodiments, the stagger element comprises a portion of an expression sequence of one or more expression sequences.

[0172] In some embodiments, the circular polyribonucleotide comprises a stagger element. To avoid the production of a continuous expression product, such as a peptide or polypeptide, while maintaining rolling circle translation, a stagger element can be included to induce ribosome stalling during translation. In some embodiments, the stagger element is at the 3’ end of at least one of the one or more expression sequences. The stagger element can be configured to stall the ribosome during rolling circle translation of the circular polyribonucleotide. The stagger element can include, but is not limited to, a 2A-like or CHYSEL (cis-acting hydrolyzing enzyme element) sequence. In some embodiments, the stagger element encodes a sequence having a C-terminal consensus sequence of X1X2X3EX5NPGP, where X1 is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid. In some embodiments, the sequence comprises a non-conserved sequence of amino acids with a rich alpha-helix content followed by the consensus sequence -D(V / I)ExNPGP, where x = any amino acid. Some non-limiting examples of stagger elements include GDVESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, GDVEQNPGP, IESNPGP, GDIELNPGP, HDIETNPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP.

[0173] In certain embodiments, staggered element cracking expression product as herein described, as between G and the P of consensus sequence as herein described.As a limiting examples, cyclic polyribonucleotide comprises at least one staggered element with cracking expression product.In certain embodiments, cyclic polyribonucleotide comprises the staggered element adjacent to at least one expressed sequence.In certain embodiments, cyclic polyribonucleotide is included in the staggered element after each expressed sequence.In certain embodiments, cyclic polyribonucleotide comprises the staggered element that is present in one side or both sides of each expressed sequence, causes translating one or more individual peptides and / or polypeptide by each expressed sequence.

[0174] In certain embodiments, the staggered elements include one or more modified nucleotides or non-natural nucleotides that induce ribosomes to pause during translation. Non-natural nucleotides can include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Examples such as these are different from naturally occurring DNA or RNA by changing the molecular backbone. Exemplary modifications can include any modification and any combination thereof that can induce ribosomes to pause during translation, such as to sugar, core bases, internucleoside bonds (e.g., to the phosphate ester connected / to the phosphodiester bond / to the phosphodiester backbone). Some exemplary modifications provided herein are described elsewhere herein.

[0175] In some embodiments, the interleave element is present in other forms in the circular polyribonucleotide. For example, in some exemplary circular polyribonucleotides, the interleave element comprises a termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a first translation initiation sequence of a subsequent expression of the first expression sequence. In some examples, the first interleave element of the first expression sequence is upstream (5') of the first translation initiation sequence of a subsequent expression of the first expression sequence in the circular polyribonucleotide. In some cases, the first expression sequence and the subsequent expression sequence are two separate expression sequences in the circular polyribonucleotide. The distance between the first interleave element and the first translation initiation sequence can be such that the first expression sequence and its subsequent expression sequence are capable of being translated continuously. In some embodiments, the first interleave element comprises a termination element and separates the expression product of the first expression sequence from the expression product of its subsequent expression sequence, thereby producing discrete expression products. In some cases, the circular polyribonucleotide comprising the first interleave element upstream of the first translation initiation sequence of a subsequent sequence in the circular polyribonucleotide is translated continuously, while the corresponding circular polyribonucleotide comprising the interleave element upstream of the second translation initiation sequence of the second expression sequence of a subsequent sequence in the corresponding circular polyribonucleotide is not translated continuously. In some cases, there is only one expression sequence in the circular polyribonucleotide, and the first expression sequence and its subsequent expression sequence are the same expression sequence. In some exemplary circular polyribonucleotides, the interleave element comprises a first termination element of a first expression sequence in the circular polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from a downstream translation initiation sequence. In some such examples, the first interleave element in the circular polyribonucleotide is upstream (5') of the first translation initiation sequence of the first expression sequence. In some cases, the distance between the first interleave element and the first translation initiation sequence enables continuous translation of the first expression sequence and any subsequent expression sequence. In some embodiments, the first interleave element separates a round of expression product of the first expression sequence from a next round of expression product of the first expression sequence, thereby producing discrete expression products. In some cases, the circular polyribonucleotide comprising the first interleave element upstream of the first translation initiation sequence of the first sequence in the circular polyribonucleotide is translated continuously, while the corresponding circular polyribonucleotide comprising the interleave element upstream of the second translation initiation sequence of the second expression sequence in the corresponding circular polyribonucleotide is not translated continuously. In some cases, the distance between the second interleave element and the second translation initiation sequence in the corresponding circular polyribonucleotide is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the distance between the first interleave element and the first translation initiation sequence in the circular polyribonucleotide.In some cases, the distance between the first staggered element and the first translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or more. In certain embodiments, the distance between the first interlaced element and the first translation initiation is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt or greater than the distance between the first interlaced element and the first translation initiation.In certain embodiments, the circular polyribonucleotide comprises more than one expressed sequence. Regulatory nucleic acids

[0176] In certain embodiments, cyclic polyribonucleotide comprises the expressed sequence of one or more coding regulatory nucleic acids (for example, modifying the expression of endogenous gene and / or exogenous gene).In certain embodiments, the expressed sequence of cyclic polyribonucleotide provided herein can comprise and the sequence of regulatory nucleic acid image non-coding RNA such as but not limited to tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, γ RNA and hnRNA antisense.

[0177] In one embodiment, the regulatory nucleic acid targets a host gene. Regulatory nucleic acids may include, but are not limited to, nucleic acids that hybridize with endogenous genes (e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA as described elsewhere herein), nucleic acids that hybridize with exogenous nucleic acids (e.g., viral DNA or RNA), nucleic acids that hybridize with RNA, nucleic acids that interfere with gene transcription, nucleic acids that interfere with RNA translation, nucleic acids that stabilize RNA or destabilize RNA (e.g., by targeted degradation), and nucleic acids that regulate DNA or RNA binding factors. In one embodiment, the sequence is miRNA. In some embodiments, the regulatory nucleic acid targets the sense strand of a host gene. In some embodiments, the regulatory nucleic acid targets the antisense strand of a host gene.

[0178] In some embodiments, the circular polyribonucleotide comprises a regulatory nucleic acid, such as a guide RNA (gRNA). In some embodiments, the circular polyribonucleotide comprises or encodes a guide RNA. gRNA short synthetic RNAs consist of a "scaffold" sequence necessary to bind to the incomplete effector moiety and a user-defined ~20 nucleotide targeting sequence for the genomic target. In practice, guide RNA sequences are typically designed to have a length of 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides), and are complementary to the target nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for designing effective guide RNAs. Gene editing has also been achieved using chimeric "single guide RNAs" ("sgRNAs"), an engineered (synthetic) single RNA molecule that mimics the naturally occurring crRNA-tracrRNA complex and comprises a tracrRNA (for binding the nuclease) and at least one crRNA (to direct the nuclease to the edit target sequence). Chemically modified sgRNAs have also been shown to be effective in genome editing; see, e.g., Hendel et al. (2015) Nature Biotechnol. 33:985-991.

[0179] The gRNA can recognize a specific DNA sequence (e.g., a sequence adjacent to or within a promoter, enhancer, silencer, or repressor of a gene).

[0180] In one embodiment, the gRNA is used as part of a CRISPR system for gene editing. For purposes of gene editing, the circular polyribonucleotide can be designed to include one or more guide RNA sequences corresponding to a desired target DNA sequence; see, e.g., Cong et al. (2013) Science 339:819-823; Ran et al. (2013) Nature Protocols 8:2281-2308. For DNA cleavage, Cas9 requires at least about 16 or 17 nucleotides of the gRNA sequence; for Cpfl, at least about 16 nucleotides of the gRNA sequence, to achieve detectable DNA cleavage.

[0181] Some regulatory nucleic acids can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules include RNA or RNA-like structures, which generally include 15-50 base pairs (e.g., about 18-25 base pairs) and have a core base sequence identical (complementary) or almost identical (substantially complementary) to the coding sequence in the target gene expressed in the cell. RNAi molecules include, but are not limited to: short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), partial duplex and dicer substrates (U.S. Patent Nos. 8,084,599, 8,349,809 and 8,513,207).

[0182] In some embodiments, the cyclic polyribonucleotide comprises a regulatory nucleic acid, which is an RNA or RNA-like structure, typically between about 5-500 base pairs (depending on the specific RNA structure, e.g., miRNA 5-30bp, lncRNA 200-500bp) and can have a core base sequence that is identical (complementary) or almost identical (substantially complementary) to the coding sequence in a target gene expressed in the cell.

[0183] Long noncoding RNA (lncRNA) is defined as a non-protein-coding transcript longer than 100 nucleotides. This somewhat arbitrary restriction distinguishes lncRNA from small regulatory RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs), and other short RNAs. Typically, the majority (about 78%) of lncRNAs are characterized by tissue specificity. Divergent lncRNAs (accounting for a large proportion of about 20% of the total lncRNAs in mammalian genomes) that are transcribed in the opposite direction to nearby protein-coding genes may regulate the transcription of nearby genes. In one embodiment, the cyclic polyribonucleotides provided herein comprise the sense strand of the lncRNA. In one embodiment, the cyclic polyribonucleotides provided herein comprise the antisense strand of the lncRNA.

[0184] Circular polyribonucleotide can encode the whole or fragment of endogenous gene or gene product (for example, mRNA) complementary or completely complementary regulatory nucleic acid.Regulatory nucleic acid can be with the boundary between intron and exon, inside between exon or the sequence complementarity of adjacent exon, thereby prevent the newly generated nuclear RNA transcript of specific gene from maturing into the mRNA for transcribing.Can hybridize with the mRNA of this gene and stop its translation with the regulatory nucleic acid of specific gene complementation.Antisense regulatory nucleic acid can be DNA, RNA or its derivatives or hybrid.In certain embodiments, regulatory nucleic acid comprises the protein binding site that can be combined with the protein that participates in endogenous gene or exogenous gene expression regulation.

[0185] The length of the circular polyribonucleotide that can encode a regulatory nucleic acid that hybridizes to a transcript of interest is between 5 and 30 nucleotides, between about 10 and 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 targeted transcript should be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0186] The circular polyribonucleotide can encode a microRNA (miRNA) molecule that is identical to about 5 to about 25 contiguous nucleotides of the target gene. In some embodiments, the miRNA sequence targets an mRNA and begins with the dinucleotide AA, has a GC content of about 30-70% (about 30-60%, about 40-60%, or about 45-55%), and does not have a high percent identity to any nucleotide sequence other than the target in the mammalian genome into which it is to be introduced, e.g., as determined by a standard BLAST search.

[0187] In some embodiments, the circular polyribonucleotide comprises at least one miRNA, e.g., 2, 3, 4, 5, 6, or more. In some embodiments, the circular polyribonucleotide comprises a sequence encoding a miRNA that has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to any one of these nucleotide sequences or to a sequence complementary to the target sequence.

[0188] siRNAs and shRNAs are intermediates in the processing pathway of endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004). In some embodiments, siRNAs can be used as miRNAs, and vice versa (Zeng et al., Mol Cell 9: 1327-1333, 2002; Doench et al., Genes Dev 17:438-442, 2003). Like siRNAs, microRNAs use RISC to down-regulate target genes, but unlike siRNAs, most animal miRNAs do not cleave mRNAs. Instead, miRNAs reduce protein output through translational repression or poly-A removal and mRNA degradation (Wu et al., Proc Natl Acad Sci USA 103:4034-4039, 2006). Known miRNA binding sites are located within the mRNA 3'UTR; miRNAs appear to target sites that are nearly perfectly complementary to 2-8 nucleotides of the miRNA 5' end (Rajewsky, Nat Genet 38 Suppl:S8-13, 2006; Lim et al., Nature 433:769-773, 2005). This region is called the seed region. Because siRNAs and miRNAs are interchangeable, exogenous siRNAs down-regulate mRNAs with seed complementarity to the siRNA (Birmingham et al., Nat Methods 3:199-204, 2006). Multiple target sites within the 3'UTR produce stronger down-regulation (Doench et al., Genes Dev 17:438-442, 2003).

[0189] Lists of known miRNA sequences can be found in databases maintained by research organizations such as the Wellcome Trust Sanger Institute, the Penn Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and the European Molecule Biology Laboratory, among others. Known effective siRNA sequences and cognate binding sites are also well represented in the relevant literature. RNAi molecules are readily designed and produced by techniques known in the art. In addition, computational tools exist that increase the chances of finding motifs that are effective and specific (Lagana et al., Methods Mol. Bio. 2015, 1269:393-412).

[0190] Circular polyribonucleotides can modulate the expression of RNA encoded by a gene. Because multiple genes can share a degree of sequence homology with one another, in some embodiments, a circular polyribonucleotide can be designed to target a class of genes having sufficient sequence homology. In some embodiments, a circular polyribonucleotide can contain sequences that have complementarity to sequences shared among different gene targets or sequences that are unique to a specific gene target. In some embodiments, a circular polyribonucleotide can be designed to target a conserved region of RNA sequences that have homology among several genes, thereby targeting several genes in a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In some embodiments, a circular polyribonucleotide can be designed to target sequences that are unique to a particular RNA sequence of a single gene.

[0191] In some embodiments, the expression sequence is less than 5000 bp in length (e.g., less than about 5000 bp, 4000 bp, 3000 bp, 2000 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, or less). In some embodiments, the expression sequence independently or additionally has a length greater than 10 bp (e.g., 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, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 4 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, 5 kb, or greater).

[0192] In some embodiments, the expression sequence comprises one or more features described herein, e.g., a sequence encoding one or more peptides or proteins, one or more regulatory elements, one or more regulatory nucleic acids (e.g., one or more non-coding sequence RNAs), other expression sequences, and any combination thereof. Translation efficiency

[0193] In certain embodiments, the translation efficiency of the cyclic polyribonucleotide provided herein is greater than reference, for example linear counterpart, linear expression sequence or linear cyclic polyribonucleotide.In certain embodiments, the translation efficiency that the cyclic polyribonucleotide provided herein has is higher than the translation efficiency of reference 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 higher. In certain embodiments, the translation efficiency of cyclic polyribonucleotide is higher than the translation efficiency 10% of linear counterpart.In certain embodiments, the translation efficiency of cyclic polyribonucleotide is higher than the translation efficiency 300% of linear counterpart.

[0194] In certain embodiments, cyclic polyribonucleotide produces the expression product of stoichiometric ratio.Rolling circle translation produces expression product with the ratio that equates basically continuously.In certain embodiments, cyclic polyribonucleotide has stoichiometric translation efficiency so that expression product produces with the ratio that equates basically.In certain embodiments, cyclic polyribonucleotide has the stoichiometric translation efficiency of multiple expression product (for example from the product of 2,3,4,5,6,7,8,9,10,11,12 or more expressed sequences). Rolling Ring Translation

[0195] In certain embodiments, in case the translation of initial cyclic polyribonucleotide, before completing at least one round translation of cyclic polyribonucleotide, the ribosome that is attached to cyclic polyribonucleotide can not break away from cyclic polyribonucleotide.In certain embodiments, cyclic polyribonucleotide as herein described can rolling circle translation. In some embodiments, during rolling circle translation, once translation of the cyclic polyribonucleotide is initiated, at least at least 2 rounds, at least 3 rounds, at least 4 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 10 5 round or at least 10 6The ribosome bound to the circular polyribonucleotide does not dissociate from the circular polyribonucleotide prior to rolling circle translation.

[0196] In some embodiments, rolling circle translation of the circular polyribonucleotide results in the production of a polypeptide product that is derived from more than one round of translation of the circular polyribonucleotide (“continuous” expression product). In some embodiments, the circular polyribonucleotide comprises a stagger element, and rolling circle translation of the circular polyribonucleotide results in the production of a polypeptide product that is generated from one round of translation or less than one round of translation of the circular polyribonucleotide (“discrete” 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 least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the total polypeptide (moles / mole) generated during rolling circle translation of the circular polyribonucleotide is a discrete polypeptide. In some embodiments, the ratio of discrete product to total polypeptide is tested in an in vitro translation system. In some embodiments, the in vitro translation system for testing the ratio comprises rabbit reticulocyte lysate. In some embodiments, the ratio is tested in an in vivo translation system, such as a eukaryotic or prokaryotic, cultured cell or organism. Untranslated region

[0197] In some embodiments, the circular polyribonucleotide comprises an untranslated region (UTR). A UTR of a genomic region comprising a gene can be transcribed but not translated. In some embodiments, a UTR can be included upstream of a translation initiation sequence of an expression sequence described herein. In some embodiments, a UTR can be included downstream of an expression sequence described herein. In some examples, one UTR of a first expression sequence is the same or contiguous or overlapping with another UTR of a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, such as ZKSCAN1.

[0198] In some embodiments, the circular polyribonucleotide comprises a UTR with one or more stretches of adenosine and uridine embedded therein. These AU-rich signatures can increase the translational efficiency of the expression product.

[0199] The introduction, removal, or modification of UTR AU-rich elements (AREs) can be used to modulate the stability or immunogenicity of a circular polyribonucleotide. When engineering a particular circular polyribonucleotide, one or more copies of an ARE can be introduced into the circular polyribonucleotide, and these copies of the ARE can modulate translation and / or production of the expression product. Likewise, an ARE can be identified and removed or engineered into a circular polyribonucleotide to modulate intracellular stability, thereby affecting translation and production of the resulting protein.

[0200] It will be appreciated that any UTR from any gene can be incorporated into the respective flanking region of a circular polyribonucleotide. As a non-limiting example, UTRs or fragments thereof that can be incorporated are those listed in U.S. Provisional Application Nos. US 61 / 775,509 and US 61 / 829,372 or International Patent Application No. PCT / US2014 / 021522; the contents of each of which are incorporated by reference herein in their entirety. In addition, multiple wild-type UTRs of any known gene can be utilized. Artificial UTRs that are not variants of wild-type genes are also within the scope of the present application. These UTRs or portions thereof can be placed in the same orientation as the transcript from which they are selected, or can be changed in orientation or position. Thus, a 5' or 3' UTR can be inverted, shortened, lengthened, made chimeric with one or more other 5' or 3' UTRs. As used herein, the term "altered" with respect to a UTR sequence means that the 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 position as taught above, or can be altered by the inclusion of additional nucleotides, the deletion of nucleotides, the exchange of nucleotides, or transposition. Any of these alterations that result in an "altered" UTR, whether 3' or 5', encompass a variant UTR.

[0201] In one embodiment, a bi-, tri- or tetra-UTR, such as a 5' or 3' UTR, can be used. As used herein, a "bi" UTR is a case in which two copies of the same UTR are encoded in tandem or substantially in tandem. For example, a bi-beta globin 3' UTR can be used as described in U.S. Patent Publication No. 20100129877, the contents of which are incorporated by reference herein in their entirety. Poly A sequence

[0202] In some embodiments, the circular polyribonucleotide can comprise a poly-A sequence. In some embodiments, the poly-A sequence is greater than 10 nucleotides in length. In one embodiment, the poly-A sequence is greater than 15 nucleotides in length (e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A sequence is about 10 to about 3,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000, 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1,500, 50 to 2,000, 50 to 2,500, 50 to 3,000, 100 to 500, 100 to 750, 100 to 1,000, 100 to 1,500, 100 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,500, 1,500 to 3,000, 2,000 to 3,000, 2,000 to 2,5000, and 2,500 to 3,000).

[0203] In one embodiment, the polyA sequence is designed relative to the length of the entire circular polyribonucleotide. The 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 region), or based on the length of the final product of the circular polyribonucleotide expression. In this context, the polyA sequence can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the circular polyribonucleotide or a feature thereof. The polyA sequence can also be designed to be part of the circular polyribonucleotide to which it belongs. In this context, the polyA sequence can be 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 polyA sequence. Further, the conjugation of the engineered binding site and the circular polyribonucleotide to the polyA binding protein can enhance expression.

[0204] In one embodiment, the circular polyribonucleotide is designed to include a polyA-G quartet. G-quartets are circular arrays of hydrogen bonds of four guanine nucleotides and can be formed from G-rich sequences in DNA and RNA. In one embodiment, the G-quartet is incorporated at the end of the polyA sequence. The resulting circular polyribonucleotide construct is assayed for stability, protein yield, and / or other parameters, including half-life at different time points. In some embodiments, the protein yield resulting from the polyA-G quartet is equal to at least 75% of the protein yield resulting from a polyA sequence of 120 nucleotides alone.

[0205] In some embodiments, the circular polyribonucleotide comprises a polyA, lacks a polyA, or has a modified polyA to modulate one or more features of the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide lacking a polyA or having a modified polyA improves one or more functional features, such as immunogenicity, half-life, expression efficiency, and the like. RNA binding

[0206] In some embodiments, the circular polyribonucleotide comprises one or more RNA binding sites. MicroRNAs (or miRNAs) are short non-coding RNAs that can bind to the 3' UTR of a nucleic acid molecule and downregulate gene expression by decreasing the stability of the nucleic acid molecule or by inhibiting translation. The circular polyribonucleotide can comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences can correspond to any known microRNA, such as those taught in U.S. Publication US 2005 / 0261218 and U.S. Publication US 2005 / 0059005, the contents of which are incorporated by reference herein in their entirety.

[0207] A microRNA sequence includes a "seed" region, i.e., a sequence in the 2-8 position of a mature microRNA, which has perfect Watson-Crick complementarity to a miRNA target sequence. A microRNA seed can include positions 2-8 or 2-7 of a mature microRNA. In some embodiments, a microRNA seed can include 7 nucleotides (e.g., nucleotides 2-8 of a mature microRNA), with the seed complementation site in the corresponding miRNA target flanked by an adenine (A) opposite the microRNA position 1. In some embodiments, a microRNA seed can include 6 nucleotides (e.g., nucleotides 2-7 of a mature microRNA), with the seed complementation site in the corresponding miRNA target flanked by an adenine (A) opposite the microRNA position 1. See, e.g., Grimson A, Farh K, Johnston WK, Garrett-Engele P, Lim LP, Barrel DP; Mol Cell. 2007 Jul 6;27(l):91-105; each of which is incorporated by reference herein in its entirety.

[0208] The bases of a microRNA seed are substantially complementary to a target sequence. By engineering a microRNA target sequence into a circular polyribonucleotide, the circular polyribonucleotide can evade or be detected by the host immune system, can be modulated for degradation or modulated for translation, so long as the relevant microRNA is available. This process will reduce the risk of off-target effects upon delivery of the circular polyribonucleotide. The recognition of microRNAs, microRNA target regions, and their expression patterns and roles 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 (Dec. 20, 2011. doi: 10.1038 / leu.2011.356); Barrel, Cell 2009 136:215-233; Landgraf et al., Cell, 2007 129:1401-1414; each of which is incorporated by reference herein in its entirety).

[0209] Conversely, a microRNA binding site can be engineered out of (i.e., removed from) a circular polyribonucleotide to modulate protein expression in a particular tissue. Modulation of expression in a variety of tissues can be achieved by introducing or removing or one or several microRNA binding sites.

[0210] Examples of tissues where microRNAs are known to regulate mRNAs and thus protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), bone marrow cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-Id, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). MicroRNAs can also regulate complex biological processes, such as angiogenesis (miR-132) (Anand and Cheresh, Curr Opin Hematol 2011 18: 171-176; incorporated by reference in its entirety). In the circular polyribonucleotides described herein, binding sites for microRNAs associated with such processes can be removed or introduced to adapt the expression of the circular polyribonucleotide to the situation of the biologically relevant cell type or relevant biological process. MicroRNAs, miR sequences, and miR binding sites are listed in Table 9 of U.S. Provisional Application 61 / 753,661 filed January 17, 2013, Table 9 of U.S. Provisional Application 61 / 754,159 filed January 18, 2013, and Table 7 of U.S. Provisional Application No. 61 / 758,921 filed January 31, 2013, each of which is incorporated by reference in its entirety. In some embodiments, the microRNA binding site includes, for example, miR-7.

[0211] The circular polyribonucleotides disclosed herein can comprise a miRNA binding site that hybridizes to any miRNA, such as any of those disclosed in a miRNA database 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 genes have been disclosed in microRNA target gene data such as StarBase, StarScan, Cupid, TargetScan, TarBase, Diana-microT, miRecords, PicTar, PITA, RepTarm RNA22, miRTarBase, miRwalk, or MBSTAR.

[0212] By understanding the expression pattern of MicroRNA in different cell types, cyclic polyribonucleotide engineering approaches as herein described can be used for the expression that has more targeting in specific cell types or only under specific biological conditions.By introducing tissue-specific MicroRNA binding site, cyclic polyribonucleotide can be designed for optimal protein expression in tissue or under biological conditions.Listed example (Getner and Naldini, Tissue Antigens [tissue antigens] .2012,80:393-403 that uses MicroRNA to drive tissue or disease-specific gene expression; Incorporated herein in full by reference with it).

[0213] In addition, the microRNA seed site can be incorporated into the cyclic polyribonucleotide to regulate the expression in some cells, and this causes biology to improve.An example in this respect is the incorporation in the miR-142 site.The miR-142 site is incorporated into the expression in the scalable hematopoietic cell in cyclic polyribonucleotide as herein described, and can also reduce or eliminate the immunoreaction to the protein of cyclic polyribonucleotide encoding.

[0214] In some embodiments, the cyclic polyribonucleotide includes one or more large intergenic non-coding RNA (lincRNA) binding sites. Large intergenic non-coding RNA (lincRNA) constitutes most long non-coding RNA. LincRNA is a non-coding transcript, and in some embodiments, is more than about 200 nucleotides in length. In some embodiments, they have an exon-intron-exon structure, similar to protein-coding genes, but do not contain an open reading frame and do not encode proteins. More than 8,000 lincRNAs have been described recently and are considered to be the largest subclass of RNA, originating from the human non-coding transcriptome. Thousands of lincRNAs are known, and some appear to be key regulatory factors for various cellular processes. Determining the function of individual lincRNAs remains a challenge. Compared with coding genes, lincRNA expression has significant tissue specificity, and although the expression levels are similar to those of neighboring protein-coding genes, they are often co-expressed with neighboring genes.

[0215] In some embodiments, the cyclic polyribonucleotide comprises one or more lincRNAs, such as FIRRE, LINC00969, PVT1, LINC01608, JPX, LINC01572, LINC00355, C1orf132, C3orf35, RP11-734, LINC01608, CC-499B15.5, CASC15, LINC00937, RP11-191, etc., or other lincRNAs or lncRNAs, such as those from a known lncRNA database. Protein binding

[0216] In some embodiments, the circular polyribonucleotide comprises one or more protein binding sites, such that a protein, e.g., a ribosome, is able to bind to an internal site in the RNA sequence. By engineering a protein binding site, e.g., a ribosome binding site, into the circular polyribonucleotide, the circular polyribonucleotide can evade or be less detected by the host's immune system, modulate degradation, or modulate translation by masking the circular polyribonucleotide from host immune system components.

[0217] In some embodiments, the circular polyribonucleotide comprises at least one immune protein binding site, e.g., for evading an immune response, e.g., a CTL (cytotoxic T lymphocyte) response. In some embodiments, the immune protein binding site is a nucleotide sequence that binds to an immune protein and helps mask the circular polyribonucleotide as foreign. In some embodiments, the immune protein binding site is a nucleotide sequence that binds to an immune protein and helps hide the circular polyribonucleotide as foreign or extraneous.

[0218] The traditional mechanism of ribosome engagement with linear RNA involves the binding of the ribosome to the capped 5' end of the RNA. From the 5' end, the ribosome migrates to the start codon, whereupon the first peptide bond is formed. According to the present application, internal initiation of translation of a circular polyribonucleotide (i.e., cap-independent) does not require a free or capped end. Rather, the ribosome binds to an uncapped internal site, from which the ribosome begins polypeptide elongation at the start codon. In some embodiments, the circular polyribonucleotide comprises one or more RNA sequences comprising a ribosome binding site, e.g., a start codon.

[0219] Natural 5' UTRs have features that play a role in translation initiation. They carry signatures similar to Kozak sequences, which are well known to be involved in the process of ribosomes initiating translation of a variety of genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". 5' UTRs are also known to form secondary structures that are involved in the binding of elongation factors.

[0220] In some embodiments, the circular polyribonucleotide encodes a protein binding sequence that binds to a protein. In some embodiments, the protein binding sequence targets or localizes the circular polyribonucleotide to a particular target. In some embodiments, the protein binding sequence specifically binds to an arginine-rich region of a protein.

[0221] In some embodiments, the protein binding site includes, but is not limited to, a binding site for a protein such as ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, C ELF2, CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, ELAVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, LIN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2, POLR2A, PRPF8, PTBP1, RBFOX2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1, TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF1, WDR33, XRN2, YBX1, YTHDC1, YTHDF1, YTHDF2, YWHAG, ZC3H7B, PDK1, AKT1, and any other protein that binds RNA. cryptogen

[0222] As described herein, the circular polyribonucleotides comprise a cryptogen to reduce, evade, or avoid a cellular innate immune response. In one aspect, provided herein is a circular polyribonucleotide that, when delivered to a cell, results in a reduced immune response by the host as compared to the response elicited by a reference compound (e.g., a linear polynucleotide corresponding to the circular polyribonucleotide or a circular polyribonucleotide lacking a cryptogen). In some embodiments, the circular polyribonucleotide is less immunogenic than a counterpart lacking a cryptogen.

[0223] In some embodiments, the cryptid enhances stability. There is growing evidence that UTRs play regulatory roles in the stability and translation of nucleic acid molecules. Regulatory features of UTRs can be included in the cryptid to enhance the stability of the circular polyribonucleotide.

[0224] In some embodiments, a 5' or 3' UTR can constitute a cryptid in the circular polyribonucleotide. For example, removal or modification of UTR AU-rich element (ARE) can be used to modulate the stability or immunogenicity of the circular polyribonucleotide.

[0225] In some embodiments, modification of the expression sequence to remove AU-rich element (ARE) (e.g., translatable region) can be used to modulate the stability or immunogenicity of the circular polyribonucleotide.

[0226] In some embodiments, the cryptid comprises a miRNA binding site or binding site for any other non-coding RNA. For example, incorporation of a miR-142 site into the circular polyribonucleotide described herein can not only modulate expression in hematopoietic cells, but also reduce or eliminate an immune response to the protein encoded by the circular polyribonucleotide.

[0227] In some embodiments, the cryptid comprises one or more protein binding sites, enabling a protein (e.g., an immune protein) to bind to the RNA sequence. By engineering a protein binding site into the circular polyribonucleotide, the circular polyribonucleotide can evade or be less detected by the host's immune system, modulating degradation or modulating translation by masking the circular polyribonucleotide in a host immune system component. In some embodiments, the circular polyribonucleotide comprises at least one immune protein binding site, e.g., for evading an immune response, e.g., a CTL response. In some embodiments, the immune protein binding site is a nucleotide sequence that binds to an immune protein and helps mask the circular polyribonucleotide as foreign.

[0228] In some embodiments, the cryptid comprises one or more modified nucleotides. Exemplary modifications can include any modification to the sugar, nucleobase, internucleoside linkage (e.g., to a linked phosphate / phosphodiester bond / phosphodiester backbone) that can prevent or reduce an immune response to the circular polyribonucleotide, and any combination thereof. Some exemplary modifications provided herein are described in detail below.

[0229] In some embodiments, the circular polyribonucleotide comprises one or more modifications as described elsewhere herein to reduce the immune response of the host as compared to the response elicited by a reference compound, e.g., a circular polyribonucleotide lacking the modification. In particular, it has been shown that the addition of one or more inosine distinguishes RNA as endogenous or viral. See, e.g., Yu, Z, et al. (2015) RNA editing by ADAR1 marks dsRNA as“self” [RNA editing by ADAR1 marks dsRNA as“self”]. Cell Res [Cell Research] 25, 1283-1284, which is incorporated by reference herein in its entirety.

[0230] In some embodiments, the circular polyribonucleotide comprises one or more expression sequences of shRNAs or RNA sequences that can be processed into siRNAs, and the shRNAs or siRNAs target RIG-1 and reduce the expression of RIG-1. RIG-1 can sense exogenous circular RNA and cause degradation of the exogenous circular RNA. Thus, a circular polynucleotide with shRNAs, siRNAs, or any other regulatory nucleic acid sequence that targets RIG-1 can reduce immunity, e.g., host cell immunity, against the circular polyribonucleotide.

[0231] In some embodiments, the circular polyribonucleotide lacks a sequence, element, or structure that contributes to the circular polyribonucleotide reducing, evading, or avoiding a cellular innate immune response. In some such embodiments, the circular polyribonucleotide can lack a polyA sequence, a 5’ end, a 3’ end, a phosphate group, a hydroxyl group, or any combination thereof. Riboswitches

[0232] In some embodiments, the circular polyribonucleotide comprises one or more riboswitches.

[0233] Riboswitches are typically considered part of a circular polyribonucleotide, can directly bind a small target molecule, and binding of the riboswitch to the target affects RNA translation, stability, and activity of the expression product (Tucker B J, Breaker R R (2005) Curr Opin Struct Biol 15(3):342-8). Thus, depending on the presence or absence of the riboswitch target molecule, a circular polyribonucleotide comprising a riboswitch is directly involved in regulating its own activity. In some embodiments, the riboswitch has an aptamer-like affinity region for a separate molecule. Thus, in the broader context of the present invention, any aptamer contained in a non-coding nucleic acid can be used to sequester a molecule from a bulk volume. Downstream reporting via the activity event of the “(riboswitch) switch” can be particularly advantageous.

[0234] In some embodiments, a riboswitch can have an effect on gene expression, including but not limited to transcriptional termination, translational initiation inhibition, mRNA self-cleavage, and alteration of splicing pathways in eukaryotes. Riboswitches can control gene expression by triggering the binding or removal of a molecule. Thus, subjecting a circular polyribonucleotide comprising a riboswitch to conditions that activate, inactivate, or block the riboswitch alters expression. Expression can be altered due to, for example, transcriptional termination or blocking of ribosome binding to the RNA. Depending on the nature of the riboswitch, binding of the trigger molecule or analog thereof can decrease or prevent expression of the RNA molecule or promote or increase expression of the RNA molecule. Some examples of riboswitches are described herein.

[0235] In some embodiments, the riboswitch is a cobalamin riboswitch (also known as a B 12 -Box), which binds adenosylcobalamin (a coenzyme form of vitamin B 12 to regulate the biosynthesis and transport of cobalamin and similar metabolites.

[0236] In some embodiments, the riboswitch is a cyclic di-GMP riboswitch, which binds cyclic di-GMP to regulate a variety of genes. They are divided into two classes that are not structurally related, cyclic di-GMP-I and cyclic di-GMP-II.

[0237] In some embodiments, the riboswitch is an FMN riboswitch (also known as an RFN Box), which binds flavin mononucleotide (FMN) to regulate the biosynthesis and transport of riboflavin.

[0238] In some embodiments, the riboswitch is a glmS riboswitch, which self-cleaves when sufficient concentrations of glucosamine-6-phosphate are present.

[0239] In some embodiments, the riboswitch is a glutamine riboswitch, which binds glutamine to regulate genes involved in glutamine and nitrogen metabolism. They also bind a short peptide of unknown function. These riboswitches are divided into two classes that are structurally related: the glnA RNA motif and the downstream peptide motif.

[0240] In some embodiments, the riboswitch is a glycine riboswitch, which binds glycine to regulate genes of glycine metabolism. The glycine riboswitch contains two adjacent aptamer domains in the same mRNA and is the only natural RNA known to exhibit cooperative binding.

[0241] In some embodiments, the riboswitch is a lysine riboswitch (also known as an L-box), which binds lysine to regulate the biosynthesis, catabolism, and transport of lysine.

[0242] In some embodiments, the riboswitch is a PreQl riboswitch that binds preQ- nucleoside to regulate genes involved in the synthesis or transport of Q-nucleoside from the precursor. Two distinct classes of PreGI riboswitches are known: PreQl-l riboswitches and PreQl-ll riboswitches. The binding domain of PreQl-l riboswitches is unusually small in naturally occurring riboswitches. PreGI-II riboswitches are found only in certain species of Streptococcus and Lactococcus, have a completely different structure, and are larger.

[0243] In some embodiments, the riboswitch is a purine riboswitch that binds purines to regulate metabolism and transport of purines. Different versions of purine riboswitches bind either guanine (a version originally called G-box) or adenine. The specificity for guanine or adenine is entirely dependent on the Watson-Crick interaction with a single pyrimidine at position Y74 in the riboswitch. In guanine riboswitches, this residue is cytosine (i.e., C74), and in adenine, it is always uracil (i.e., U74). A cognate version of purine riboswitches binds deoxyguanosine, but with a more significant difference than a single nucleotide mutation.

[0244] In some embodiments, the riboswitch is a SAH riboswitch that binds S-adenosylhomocysteine to regulate genes involved in recycling this metabolite that is produced when S- adenosylmethionine is used in methylation reactions.

[0245] In some embodiments, the riboswitch is a SAM riboswitch that binds S- adenosylmethionine (SAM) to regulate biosynthesis and transport of methionine and SAM. Three different SAM riboswitches are known: SAM-I (originally called S-box), SAM-II, and S M K box riboswitches. SAM-I is widespread in bacteria, but SAM-II is found only in the alpha-, beta-, and a few gamma-proteobacteria. S M K box riboswitches. These three riboswitch variants have no obvious similarity in sequence or structure. A fourth variant, SAM-IV, appears to have a similar ligand-binding core as SAM-I but in a different scaffold.

[0246] In some embodiments, the riboswitch is a SAM-SAH riboswitch that binds SAM and SAH with similar affinities. Since they are always found in positions that regulate genes encoding methionine adenosyltransferases, it is proposed that only their binding to SAM is physiologically relevant.

[0247] In some embodiments, the riboswitch is a tetrahydrofolate riboswitch that binds tetrahydrofolate to regulate synthesis and transport genes.

[0248] In some embodiments, the riboswitch is a theophylline-binding riboswitch or a riboswitch that binds pyrophosphate thymidine.

[0249] In some embodiments, the riboswitch is a T. tengcongensis glmS catalytic riboswitch that senses glucosamine-6-phosphate (Klein and Ferre-D'Amare 2006).

[0250] In some embodiments, the riboswitch is a TPP riboswitch (also known as THI-box) that binds thiamine pyrophosphate (TPP) to regulate thiamine biosynthesis and transport, as well as transport of similar metabolites. This is the only riboswitch found to date in eukaryotic cells.

[0251] In some embodiments, the riboswitch is a Moco riboswitch that binds molybdenum cofactor to regulate genes involved in the biosynthesis and transport of this cofactor, as well as enzymes that use this cofactor or its derivatives as a cofactor.

[0252] In some embodiments, the riboswitch is an adenine-sensing add-A riboswitch found in the 5' UTR of the adenine deaminase-encoding gene of Vibrio cholerae. aptazymes

[0253] In some embodiments, the circular polyribonucleotide comprises an aptazyme. Aptazymes are switches for conditional expression in which an aptamer region serves as an allosteric control element and is coupled to a catalytic RNA region ("ribozyme" as described below). In some embodiments, aptazymes are active in cell type-specific translation. In some embodiments, aptazymes are active in cell state-specific translation (e.g., virus-infected cells or presence of viral nucleic acid or viral protein).

[0254] Ribozymes (from ribonucleases, also known as RNases or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but natural ribozymes that catalyze ribosome amino-transferase activity have also been found. It has recently been shown that catalytic RNAs can be "evolved" by in vitro methods [1. Agresti J J, Kelly B T, Jaschke A, Griffiths AD: Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization. Proc Natl Acad Sci 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., [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.], similar to the riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression. In the context of the present invention, it can be particularly advantageous to place a catalytic RNA or ribozyme in a larger non-coding RNA, which allows the ribozyme to exist in many copies within the cell for the purpose of chemical transformation of large volume molecules. Furthermore, it can be particularly advantageous to encode both an aptamer and a ribozyme in the same non-coding RNA.

[0255] Some non-limiting examples of ribozymes include hammerhead ribozymes, VL ribozymes, leadzymes, hairpin ribozymes.

[0256] In some embodiments, the aptazyme is a ribozyme that can cleave an RNA sequence and can be regulated due to binding of a ligand / regulator. The ribozyme can also be a self-cleaving ribozyme. In this way, the aptazyme combines the properties of a ribozyme and an aptamer. Aptazymes offer more advantages than conventional aptamers due to their potential for translational activity in vivo, in fact aptazymes have catalytic action to inactivate expression and, due to cleavage of their own or a heterologous transcript, the inactivation is irreversible.

[0257] In some embodiments, the aptazyme is included in the untranslated region of the circular polyribonucleotide and is inactive in the absence of the ligand / regulator, allowing expression of the transgene. Expression can be turned off (or down-regulated) by the addition of the ligand. It should be noted that aptazymes down-regulated in response to the presence of a particular regulator can be used in control systems where up-regulation of gene expression in response to the regulator is desired.

[0258] Aptazymes can also allow for the formation of a system for self-regulation of expression of the circular polyribonucleotide. For example, the protein product of the circular polyribonucleotide is a rate determining enzyme in the synthesis of a particular small molecule, modifications can be made to the small molecule to include an aptazyme selected to be more catalytically active in the presence of the molecule, providing a self-regulatory feedback loop for the synthesis of the small molecule. Alternatively, the aptazyme activity can be selected to be sensitive to accumulation of the protein product of the circular polyribonucleotide or any other cellular macromolecule.

[0259] In some embodiments, the circular polyribonucleotide can include an aptamer sequence. Some non-limiting examples include an RNA aptamer that binds lysozyme, Toggle-25t (an RNA aptamer including 2'fluoropyrimidine nucleotides that binds thrombin with high specificity and affinity), RNA Tat that binds human immunodeficiency virus trans-acting response element (HIV TAR), an RNA aptamer that binds heme, an RNA aptamer that binds interferon gamma, an RNA aptamer that binds vascular endothelial growth factor (VEGF), an RNA aptamer that binds prostate specific antigen (PSA), an RNA aptamer that binds dopamine, and an RNA aptamer that binds the non-canonical oncogene, heat shock factor 1 (HSF1). Cyclization

[0260] In one embodiment, a linear circular polyribonucleotide can be circularized or concatamerized. In some embodiments, a linear circular polyribonucleotide can be circularized in vitro prior to formulation and / or delivery. In some embodiments, a linear circular polyribonucleotide can be circularized within a cell. Extracellular cyclization

[0261] In some embodiments, a linear circular polyribonucleotide is circularized or concatenated using a chemical method to form a circular polyribonucleotide. In some chemical methods, the 5'-end and 3'-end of a nucleic acid (e.g., a linear circular polyribonucleotide) includes a chemically reactive group that, when the groups are in close proximity to each other, can form a new covalent bond between the 3'-end and 5'-end of the molecule. The 5'-end can contain an NHS ester reactive group and the 3'-end can contain a 3'-amino terminal nucleotide such that, in an organic solvent, the 3'-amino terminal nucleotide on the 3'-end of the linear RNA molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety, forming a new 5'- / 3'-amide bond.

[0262] In one embodiment, a DNA or RNA ligase can be used to enzymatically ligate a 5'- phosphorylated nucleic acid molecule (e.g., a linear circular polyribonucleotide) to a 3'-hydroxyl of a nucleic acid (e.g., a linear nucleic acid) to form a new phosphodiester bond. In an exemplary reaction, a linear circular polyribonucleotide is incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) at 37 °C for 1 hour according to the manufacturer's protocol. The ligation reaction can occur in the presence of a linear nucleic acid that is capable of base pairing with the juxtaposed 5'- and 3'- regions to aid the enzymatic ligation reaction. In one embodiment, the ligation is a splint ligation. For example, a splint ligase, such as For splint ligation, a single-stranded polynucleotide (splint), 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 when hybridized to the single-stranded splint. Thus, a splint ligase can catalyze the ligation of the two ends of a linear polyribonucleotide that are juxtaposed, generating a circular polyribonucleotide.

[0263] In one embodiment, a DNA or RNA ligase can be used for the synthesis of a circular polynucleotide. As one non-limiting example, the ligase can be a circ ligase or a circular ligase.

[0264] In one embodiment, the 5'- or 3'- end of a linear circular polyribonucleotide can encode a ligase ribozyme sequence such that, during in vitro transcription, the resulting linear circular polyribonucleotide includes an active ribozyme sequence that is capable of ligating the 5'-end of the linear circular polyribonucleotide to the 3'-end of the linear circular polyribonucleotide. The ligase ribozyme can be derived from a Group I intron, a hepatitis D virus, a hairpin ribozyme, or can be selected by SELEX (Systematic Evolution of Ligands by Exponential Enrichment). The ribozyme ligase reaction can require 1 hour to 24 hours at a temperature between 0 °C and 37 °C.

[0265] In one embodiment, can be by using at least one non-nucleic acid part with linear cyclic polyribonucleotide cyclization or concatenation.On the one hand, at least one non-nucleic acid part can with 5 ' end near the linear cyclic polyribonucleotide and / or 3 ' end near the region or characteristic reaction, with cyclization or concatenation linear cyclic polyribonucleotide.On the other hand, at least one non-nucleic acid part can be positioned at or be connected to or contiguous linear cyclic polyribonucleotide 5 ' end and / or 3 ' end.The non-nucleic acid part of imagination can be homology or heterology.As a limiting examples, the non-nucleic acid part can be key, as hydrophobic bond, ionic bond, biodegradable key and / or cleavable key.As another limiting examples, the non-nucleic acid part is a connecting portion.As another limiting examples again, the non-nucleic acid part can be oligonucleotide or peptide moiety, as suitable as described herein or non-nucleic acid linker.

[0266] In one embodiment, linear cyclic polyribonucleotide can be owing to non-nucleic acid part by cyclization or concatenation, causes being positioned at, adjacent to or be connected to the 5 ' of cyclic polyribonucleotide and the atom of 3 ' end, the attraction between the molecular surface.As a limiting examples, can by intermolecular force or intramolecular force with one or more linear cyclic polyribonucleotide cyclization or concatenation.The limiting examples of intermolecular force comprises dipole-dipole force, dipole induced dipole force, induced dipole induced dipole force, van der Waals force and dispersion force.The limiting examples of intramolecular force comprises covalent bond, metallic bond, ionic bond, resonant bond, grasping hydrogen bond (agnostic bond), dipole bond, puts together, super puts together and reverse key.

[0267] In one embodiment, linear cyclic polyribonucleotide can comprise ribozyme RNA sequence near 5 ' end and 3 ' end.When sequence is exposed to the remainder of ribozyme, ribozyme RNA sequence can be covalently connected to peptide.On the one hand, peptide is covalently connected to ribozyme RNA sequence near 5 ' end and 3 ' end and can contact each other, causes linear cyclic polyribonucleotide cyclization or concatenation.On the other hand, peptide is covalently connected to ribozyme RNA sequence 5 ' end and 3 ' end and can cause linear primary construct or linear mRNA to be connected back cyclization or concatenation using methods known in the art (such as but not limited to protein connection).The limiting examples of ribozyme in linear primary construct of the present invention or linear RNA, or the non-exhaustive listing of the method for mixing and / or covalently connecting peptide is described in U.S. Patent Application No. US20030082768, and this patent content is incorporated herein by reference in its full text.

[0268] In some embodiments, the linear circular polyribonucleotides can include a 5' triphosphate of the nucleic acid converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with an RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (dephosphoesterase). Alternatively, conversion of the 5' triphosphate of the linear circular polyribonucleotides to a 5' monophosphate can be accomplished by a two-step reaction comprising: (a) contacting the 5' nucleotide of the linear circular polyribonucleotides with a phosphatase (e.g., a thermosensitive phosphatase, shrimp alkaline phosphatase, or calf intestinal phosphatase) to remove all three phosphates; and (b) after step (a), contacting the 5' nucleotide with a kinase that adds a single phosphate (e.g., a polynucleotide kinase).

[0269] 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 about 90%, at least about 95%, or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%. Splicing elements

[0270] In some embodiments, the circular polyribonucleotides include at least one splicing element. In the circular polyribonucleotides provided herein, the splicing element can be a complete splicing element that can mediate splicing of the circular polyribonucleotide. Alternatively, the splicing element can also be a remaining splicing element from a completed splicing event. For example, in some cases, a splicing element of a linear polyribonucleotide can mediate a splicing event that results in circularization of the linear polyribonucleotide, such that the resulting circular polyribonucleotide includes a remaining splicing element from such splicing-mediated circularization event. In some cases, the remaining splicing element is unable to mediate any splicing. In other cases, the remaining splicing element can still mediate splicing in some cases. In some embodiments, the splicing element is adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide includes a splicing element adjacent to each expression sequence. In some embodiments, the splicing element is on one or both sides of each expression sequence, resulting in, for example, the partitioning of the expression products of one or more peptides and / or one or more polypeptides.

[0271] In some embodiments, the circular polyribonucleotide comprises an internal splicing element that, when copied, the splice ends are ligated together. Some examples can include mini-introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt), such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in cis sequence elements proximal to inverted splicing events (suptable4 enrichment motifs), such as sequences in the 200 bp before (upstream) or after (downstream) the backsplice site with flanking exons. In some embodiments, the circular polyribonucleotide comprises at least one repeat nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repeat nucleotide sequence can comprise a repeat sequence from an Alu family intron. In some embodiments, splicing-associated ribosome binding proteins can regulate biogenesis of the circular polyribonucleotide (e.g., blindin and quaking (QKI) splicing factors).

[0272] In some embodiments, the circular polyribonucleotide can comprise canonical splice sites at the head-to-tail junctions flanking the circular polyribonucleotide.

[0273] In some embodiments, the circular polyribonucleotide can comprise a bulge-helix-bulge motif, comprising a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage occurs at one site in the bulge region, generating a characteristic fragment with a 5'-hydroxyl and a 2',3'-cyclic phosphate end. Cyclization occurs by nucleophilic attack of the 5'-OH group onto the 2',3'-cyclic phosphate of the same molecule, forming a 3',5'-phosphodiester bridge.

[0274] In some embodiments, the circular polyribonucleotide can comprise a multimeric repeat RNA sequence with 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 a linear circular polyribonucleotide, thereby ligating the ends together.

[0275] In some embodiments, the circular polyribonucleotide can comprise a sequence that mediates self-ligation. In one embodiment, the circular polyribonucleotide can comprise an HDV sequence (e.g., an HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUU UUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCGAGCC) to self-ligate. In one embodiment, the circular polyribonucleotide can include a loop E sequence (e.g., in PSTVd) to self-ligate. In another embodiment, the circular polyribonucleotide can include a self-circularizing intron, such as a 5’ and 3’ splice junction, or a self-circularizing catalytic intron, such as a Type I, Type II, or Type III intron. Non-limiting examples of Type I intron self-splicing sequences can include a self-splicing replacement intron-exon sequence derived from the td gene of T4 bacteriophage and the intervening sequence (IVS) rRNA of Tetrahymena. Other circularization methods

[0276] In some embodiments, the linear circular polyribonucleotide can include a complementary sequence, including a repeated or non-repeated nucleic acid sequence within an individual intron or flanking an intron. A repeated nucleic acid sequence is a sequence that occurs within a segment of the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide includes a repeated nucleic acid sequence. In some embodiments, the repeated nucleic acid sequence includes a poly CA sequence or a poly UG sequence. In some embodiments, the circular polyribonucleotide includes at least one repeated nucleic acid sequence that hybridizes to a complementary repeated nucleic acid sequence in another segment of the circular polyribonucleotide, the hybridized segments forming an internal duplex. In some embodiments, the repeated nucleic acid sequence and the complementary repeated nucleic acid sequence of two separate circular polyribonucleotides hybridize to generate a single circularized polyribonucleotide, the hybridized segments forming an internal duplex. In some embodiments, the complementary sequence is located at the 5’ end and the 3’ end of the linear circular polyribonucleotide. In some embodiments, the complementary sequence includes about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.

[0277] In some embodiments, a circularization chemical method can be used to generate a circular polyribonucleotide. Such methods can include, but are not limited to, click chemistry (e.g., alkyne and azide based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, semi-imine-imine crosslinking, base modifications, and any combination thereof.

[0278] In some embodiments, a circularization enzymatic method can be used to generate a circular polyribonucleotide. In some embodiments, a ligase, such as a DNA or RNA ligase, can be used to generate a template for a circular polyribonucleotide or complement, a complementary strand of a circular polyribonucleotide, or a circular polyribonucleotide.

[0279] Cyclization of the circular polyribonucleotide can be accomplished by methods known in the art, for example, those described in Petkovic and Muller, "RNA circularization strategies in vivo and in vitro" Nucleic Acids Res, 2015, 43(4):2454-2465, and Muller and Appel, "In vitro circularization of RNA" RNA Biol, 2017, 14(8): 1018-1027. Replication element

[0280] The circular polyribonucleotide can encode a sequence and / or motif that can be used for replication. Replication of the circular polyribonucleotide can be carried out by generating a complementary circular polyribonucleotide. In some embodiments, the circular polyribonucleotide includes a motif that initiates transcription, where transcription is driven by an endogenous cellular machinery (DNA-dependent RNA polymerase) or an RNA-dependent RNA polymerase encoded by the circular polyribonucleotide. The product of the rolling circle transcription event can be cleaved by a ribozyme to generate a unit length of complementary or proliferating circular polyribonucleotide. The ribozyme can be encoded by the circular polyribonucleotide, its complement, or by a trans RNA sequence. In some embodiments, the encoded ribozyme can include a sequence or motif that modulates (inhibits or promotes) the activity of the ribozyme to control circular RNA proliferation. In some embodiments, the unit length sequence can be ligated into a circular form by a cellular RNA ligase. In some embodiments, the circular polyribonucleotide includes a replication element that facilitates self-amplification. Examples of such replication elements include, but are not limited to, the HDV replication domain, the RNA promoter of potato spindle tuber viroid (see, e.g., Kolonko 2005 Virology), and the replication-competent circular RNA sense and / or antisense ribozymes such as the antigenome 5'-CGGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGACGCACGUCCACUCGGAUGGCUAAGGGAGAGCCA-3' or genome 5'-UGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGACCGUCCCCUCGGUAAUGGCGAAUGGGACCCA-3'.

[0281] In some embodiments, the circular polyribonucleotide comprises at least one stagger element as described herein to aid in replication. The stagger element in the circular polyribonucleotide can cleave the long transcript resulting from replication of the circular polyribonucleotide to a specific length, which can then be circularized to form the complement of the circular polyribonucleotide.

[0282] In another embodiment, the circular polyribonucleotide comprises at least one ribozyme sequence to cleave the long transcript resulting from replication of the circular polyribonucleotide to a specific length, wherein another encoded ribozyme cleaves the transcript at the ribozyme sequence. Circularization forms the complement of the circular polyribonucleotide.

[0283] In some embodiments, the circular polyribonucleotide is substantially resistant to degradation, e.g., by exonucleases.

[0284] In some embodiments, the circular polyribonucleotide replicates within a cell. In some embodiments, the replication rate of the circular polyribonucleotide within a cell is 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 any percentage in between. In some embodiments, the circular polyribonucleotide replicates within a cell and is passed to a daughter cell. In some embodiments, a cell passes at least one circular polyribonucleotide to a daughter cell with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, a cell undergoing meiosis passes a circular polyribonucleotide to a daughter cell with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, a cell undergoing mitosis passes a circular polyribonucleotide to a daughter cell with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%.

[0285] In some embodiments, the circular polyribonucleotide replicates within a host cell. In one embodiment, the circular polyribonucleotide is capable of replicating in a mammalian cell, e.g., a human cell.

[0286] Although in some embodiments the circular polyribonucleotide replicates in the host cell, the circular polyribonucleotide is not integrated into the host's genome, e.g., is not integrated with the host's chromosome. In some embodiments, the circular polyribonucleotide has, e.g., a negligible recombination frequency with the host's chromosome. In some embodiments, the circular polyribonucleotide has a recombination frequency with, e.g., the host's chromosome, of, e.g., 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. Other Sequences

[0287] In some embodiments, the circular polyribonucleotide further comprises another nucleic acid sequence. In some embodiments, the circular polyribonucleotide can comprise other sequences, including DNA, RNA, or artificial nucleic acids. The other sequences can include, but are not limited to, genomic DNA, cDNA, or sequences encoding tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecules. In one embodiment, the circular polyribonucleotide comprises an siRNA to target a different locus of the same gene expression product as the circular polyribonucleotide. In one embodiment, the circular polyribonucleotide comprises an siRNA to target a different gene expression product than the circular polyribonucleotide.

[0288] 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 polyA sequence. In some 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 a susceptibility to degradation by exonucleases. In some embodiments, the fact that the circular polyribonucleotide lacks susceptibility to degradation can mean that the circular polyribonucleotide is not degraded by exonucleases, or is degraded to a limited extent in the presence of exonucleases comparable to or similar to the absence of exonucleases. 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.

[0289] In some embodiments, the cyclic polyribonucleotide lacks a 5'-UTR and can express a protein from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a 3'-UTR and can express a protein from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a poly A sequence and can express a protein from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a termination element and can express a protein from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site and can express a protein from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a cap and can express a protein from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a 5'-UTR, a 3'-UTR and an IRES and can express a protein from one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide further comprises one or more of the following sequences: sequences encoding one or more miRNAs, sequences encoding one or more replication proteins, sequences encoding exogenous genes, sequences encoding therapeutic agents, regulatory elements (e.g., translation regulators, e.g., translation enhancers or repressors), translation initiation sequences, one or more regulatory nucleic acids targeting endogenous genes (siRNA, lncRNA, shRNA), and sequences encoding therapeutic mRNA or proteins.

[0290] The length of the other sequences may be about 2 nt to about 10,000 nt, about 2 nt to about 5,000 nt, about 10 nt to about 100 nt, about 50 nt to about 150 nt, about 100 nt to about 200 nt, about 150 nt to about 250 nt, about 200 to about 300 nt, about 250 nt to about 350 nt, about 300 nt to about 500 nt, about 10 nt to about 1,000 nt, about 50 nt to about 1,000 nt, about 1000 nt to about 1,000 nt, about 1,000 nt to about 2,000 nt, about 2,000 nt to about 3,000 nt, about 3,000 nt to about 4,000 nt, about 4,000 nt to about 5,000 nt, or any range therebetween.

[0291] As a result of its circularization, a circular polyribonucleotide can comprise certain characteristics that distinguish it from linear RNA. For example, a circular polyribonucleotide is less susceptible to degradation by exonucleases than linear RNA. As such, a circular polyribonucleotide is more stable than linear RNA, particularly when incubated in the presence of an exonuclease. The increased stability of a circular polyribonucleotide compared to linear RNA makes the circular polyribonucleotide more useful as a cell transformation reagent for producing a polypeptide, and is easier and longer to store compared to linear RNA. The stability of a circular polyribonucleotide treated with an exonuclease can be tested using methods standard in the art that determine whether RNA degradation has occurred (e.g., by gel electrophoresis).

[0292] Further, unlike linear RNA, a circular polyribonucleotide is less susceptible to dephosphorylation when incubated with a phosphatase, such as calf intestinal phosphatase. Intervening sequence of nucleotides

[0293] In some embodiments, a circular polyribonucleotide comprises an intervening sequence.

[0294] In some embodiments, a circular polyribonucleotide comprises at least one intervening sequence. In some embodiments, a circular polyribonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or more intervening sequences.

[0295] In some embodiments, a circular polyribonucleotide comprises a ratio of circular polyribonucleotide intervening sequences to non-intervening sequences, such as expression sequences, of about 0.05: 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 1.25: 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.

[0296] In some embodiments, the spacer sequence comprises a ratio of the spacer sequence to the downstream (e.g., 3' of the spacer sequence) non-spacer element of a 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 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 upstream (e.g., 5’ of the spacer sequence) non-spacer element 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.

[0297] 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 1000 ribonucleotides.

[0298] In some embodiments, the spacer sequence can be a nucleic acid sequence or molecule with a low GC content, for example, 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%, In some embodiments, the spacer sequence comprises at least 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the total length of the spacer, or at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the consecutive nucleic acid residues of the spacer. In some embodiments, the spacer 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 therebetween of adenine ribonucleotides. In some embodiments, the spacer sequence comprises at least 5 or more consecutive adenine ribonucleotides. In some embodiments, the spacer sequence comprises at least 6 consecutive adenine ribonucleotides, at least 7 consecutive adenine ribonucleotides, at least 8 consecutive ribonucleotides, at least about 10 consecutive adenine ribonucleotides, 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 consecutive adenine ribonucleotides, at least about 95 consecutive adenine ribonucleotides. In some embodiments, the present invention comprises 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 consecutive adenine ribonucleotides, at least about 600 consecutive adenine ribonucleotides, at least about 700 consecutive adenine ribonucleotides, at least about 800 consecutive adenine ribonucleotides, at least about 900 consecutive adenine ribonucleotides, or at least about 1000 consecutive adenine ribonucleotides.

[0299] In some embodiments, a spacer sequence is positioned between one or more elements. In some embodiments, a spacer 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 40 kcal / 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 region can comprise a nucleic acid, such as DNA or RNA.

[0300] In some embodiments, a spacer sequence can encode an RNA sequence, and preferably a protein or peptide sequence, including a secretion signal peptide.

[0301] In some embodiments, a spacer sequence can be non-coding. If the spacer region is a non-coding sequence, a translation initiation sequence can be provided in the coding sequence of the adjacent sequence. In some embodiments, it is envisioned that the first nucleic acid residue of the coding sequence can be the A residue of a translation initiation sequence such as AUG. If the spacer region encodes an RNA or protein or peptide sequence, a translation initiation sequence can be provided in the spacer sequence.

[0302] In some embodiments, a spacer region is operably linked to another sequence described herein. Non-nucleic acid linkers

[0303] The circular polyribonucleotides described herein can also comprise a non-nucleic acid linker. In some embodiments, the circular polyribonucleotides described herein have a non-nucleic acid linker between one or more sequences or elements described herein. In one embodiment, one or more sequences or elements described herein are linked to a linker. The non-nucleic acid linker can be a chemical bond, such as one or more covalent or non-covalent bonds. In some embodiments, the non-nucleic acid linker is a peptide linker or a protein linker. Such linkers can be between 2-30 amino acids, or longer. Linkers include any flexible, rigid, or cleavable linkers described herein.

[0304] The most commonly used flexible linker has a sequence composed primarily of stretches of Gly and Ser residues ("GS" linker). Flexible linkers can be useful for connecting domains that need to move or interact to some degree, and can include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, and thus reduce unfavorable interactions between the linker and the protein portions.

[0305] Rigid linkers are useful for maintaining a fixed distance between domains and maintaining their independent functions. Rigid linkers can also be useful when the spatial separation of domains is critical to maintaining the stability or biological activity of one or more components in the fusion. Rigid linkers can have an alpha helical structure or a Pro-rich sequence, (XP) n where X represents any amino acid, preferably Ala, Lys or Glu.

[0306] Cleavable linkers can release free functional domains in vivo. In some embodiments, the linker can be cleaved under specific conditions, for example in the presence of a reducing agent or a protease. In vivo cleavable linkers can exploit the reversible nature of disulfide bonds. One example includes a thrombin-sensitive sequence between two Cys residues (e.g., PRS). In vitro thrombin treatment of CPRSC results in cleavage of the thrombin-sensitive sequence, while the reversible disulfide bond remains intact. Such linkers are known and described in, for example, Chen et al., 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369. In vivo cleavage of linkers in fusion proteins can also be performed by proteases that are expressed in vivo under pathological conditions (e.g., cancer or inflammation), in specific cells or tissues, or within restricted certain cellular compartments. The specificity of many proteases provides for slow cleavage of the linker in restricted compartments.

[0307] Examples of linking molecules include hydrophobic linkers, such as negatively charged sulfonate groups; lipids, such as poly(--CH2--) hydrocarbon chains, such as polyethylene glycol (PEG) groups, unsaturated variants thereof, hydroxylated variants thereof, amidated or other N-containing variants, non-carbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecules capable of covalently linking two or more polypeptides. Non-covalent linkers (such as hydrophobic lipid globules to which a polypeptide is attached) can also be included, for example by a hydrophobic region of a polypeptide or a hydrophobic extension of a polypeptide, such as a series of residues rich in leucine, isoleucine, valine, or possibly also alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues. The polypeptides can be linked using charge-based chemistry, such that positively charged portions of a polypeptide are linked to negatively charged portions of another polypeptide or nucleic acid. Stability / half-life

[0308] In some embodiments, the circular polyribonucleotides provided herein have an increased half-life compared to a reference, e.g., a linear polyribonucleotide having the same nucleotide sequence but not circularized (linear counterpart). In some embodiments, the circular polyribonucleotide is substantially resistant to degradation by, e.g., exonucleases. In some embodiments, the circular polyribonucleotide is resistant to self-degradation. In some embodiments, the circular polyribonucleotide lacks an enzymatic cleavage site, e.g., a dicer cleavage site. In some embodiments, the circular polyribonucleotide has a half-life that is 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 about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 140%, at least about 150%, at least about 160%, at least about 180%, at least about 200%, at least about 300%, at least about 400%, at least about 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 (e.g., a linear counterpart).

[0309] In some embodiments, the circular polyribonucleotide persists in the cell during cell division. In some embodiments, the circular polyribonucleotide persists in the daughter cell after mitosis. In some embodiments, the circular polyribonucleotide replicates within the cell and is passed to the daughter cell. 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 transcription of the circular polyribonucleotide into a linear polyribonucleotide that is complementary to the circular polyribonucleotide (linear complement). In some embodiments, the linear complementary polyribonucleotide can be circularized in vivo in the cell to a complementary circular polyribonucleotide. In some embodiments, the complementary polyribonucleotide can further self-replicate into another circular polyribonucleotide having the same or similar nucleotide sequence as the starting circular polyribonucleotide. An exemplary self-replication element includes the HDV replication domain (as described in Beeharry et al., Virol, 2014, 450-451: 165-173). In some embodiments, the cell passes at least one circular polyribonucleotide to a daughter cell with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, a cell undergoing meiosis passes a circular polyribonucleotide to a daughter cell with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, a cell undergoing mitosis passes a circular polyribonucleotide to a daughter cell with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. Modifications

[0310] A circular polyribonucleotide can include one or more substitutions, insertions and / or additions, deletions, and covalent modifications included within the scope of the present invention relative to a reference sequence (in particular, a parental polyribonucleotide).

[0311] In some embodiments, the circular polyribonucleotide comprises one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the over one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). In some embodiments, the first isolated nucleic acid comprises a messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl_1_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, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-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-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine, N6-glycinamidoadenosine, N6-threonylaminoadenosine, 2-methylthio-N6-threonylaminoadenosine, N6,N6-dimethyladenosine, 7- methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 1-methyl-inosine, wyosine, wittenyosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0312] A circular polyribonucleotide can include any useful modification, such as to the sugar, nucleobase, or internucleoside linkage (e.g., to a phosphate linkage / to a phosphodiester linkage / to a phosphodiester backbone). One or more atoms of a pyrimidine nucleobase can be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halo group (e.g., chloro or fluoro). In certain embodiments, there is a modification (e.g., one or more modifications) in each sugar and internucleoside linkage. The modification can be a ribonucleic acid (RNA) modification to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Other modifications are described herein.

[0313] In some embodiments, the circular polyribonucleotide comprises at least one N(6) methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the N(6) methyladenosine (m6A) modification can decrease immunogenicity of the circular polyribonucleotide.

[0314] In some embodiments, the modifications can include chemical or cell-induced modifications. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan, “RNA modifications and structures cooperate to guide RNA-protein interactions,” Nat Reviews Mol Cell Biol, 2017, 18:202-210.

[0315] In some embodiments, chemical modifications to ribonucleotides of the circular polyribonucleotides can enhance immune evasion. The circular polyribonucleotides can be synthesized and / or modified by methods well known 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, by reference in its entirety (incorporated herein). Modifications include, for example, end modifications, such as 5’ end modifications (phosphorylation (mono-, di-, and tri-phosphorylated), conjugation, inverted linkage, etc.), 3’ end modifications (conjugation, DNA nucleotides, inverted linkage, etc.), base modifications (e.g., substitution with stable bases, unstable bases, or bases that pair with extended parent library bases), base removal (abasic nucleotides), or base conjugation. Modified ribonucleotide bases can also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications can modulate expression, immune response, stability, subcellular localization of the circular polyribonucleotides, to name a few functional effects. In some embodiments, the modifications include dual orthogonal nucleotides, e.g., unnatural bases. See, e.g., Kimoto et al., Chem Commun (Camb), 2017, 53: 12309, DOI: 10.1039 / c7cc06661a, which is hereby incorporated by reference in its entirety.

[0316] In some embodiments, sugar modifications (e.g., at the 2' position or 4' position) or sugar replacements and backbone modifications of one or more ribonucleotides of the circular polyribonucleotide can include modifications or replacements of the phosphodiester linkage. Particular examples of circular polyribonucleotides include, but are not limited to, circular polyribonucleotides that include modified backbones or non-natural internucleoside linkages (such as internucleoside modifications, including modifications or replacements of the phosphodiester linkage). Circular polyribonucleotides having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes mentioned in the art, modified RNAs that do not have a phosphorus atom in their internucleosidic backbone can also be considered oligonucleosides. In particular embodiments, the circular polyribonucleotide will include ribonucleotides having a phosphorus atom in their internucleosidic backbone.

[0317] Modified circular polyribonucleotide backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, amino-phosphoramidates such as 3'-amino-amino phosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. In some embodiments, the circular polyribonucleotide can be negatively charged or positively charged.

[0318] Modified nucleotides that can be incorporated into the circular polyribonucleotide can be modified on the internucleoside linkage (e.g., the phosphate backbone). Herein, the phrases "phosphate" and "phosphodiester" are used interchangeably in the context of a polynucleotide backbone. The backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides can include the wholesale replacement of the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenoates, phosphoroboranoates, boranophosphates, hydrogen phosphonates, phosphoramidates, diaminophosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The two non-linking oxygens of a phosphorodithioate are replaced with sulfur. The phosphate linker can also be modified by replacing the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates).

[0319] a-thio-substituted phosphate moieties are provided to confer stability to RNA and DNA polymers through unnatural thio-phosphate backbone linkages. Thio-phosphate DNA and RNA have enhanced nuclease resistance and thus have longer half-lives in cellular environments. Thio-phosphate linked to a circular polyribonucleotide is expected to reduce innate immune response by attenuating binding / activation of cellular innate immune molecules.

[0320] In particular embodiments, the modified nucleosides include a-thio-nucleosides (e.g., 5'-0-(l-thio-phosphate)-adenosine, 5'-0-(l-thio-phosphate)-cytidine (a-thiocytidine), 5'-0-(l-thio-phosphate)-guanosine, 5'-0-(l-thio-phosphate)-uridine, or 5'-0-(l-thio-phosphate)-pseudouridine).

[0321] Other internucleoside linkages that can be used in accordance with the present application are described herein, including internucleoside linkages that do not include a phosphorus atom.

[0322] In some embodiments, the circular polyribonucleotide can include one or more cytotoxic nucleosides. For example, a cytotoxic nucleoside can be incorporated into the circular polyribonucleotide, such as a bifunctional modification. Cytotoxic nucleosides can include, but are not limited to, combinations of adenosine arabinoside, 5-azacytidine, 4'-thio- adenosine arabinoside, cyclopentenylcytosine, cladrabine, clofarabine, cytarabine, cytosine arabinoside, l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, tegafur, and uracil, tegafur ((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), troxacitabine, terazoxime, 2'-deoxy-2'-methylene cytidine (DMDC), and 6-mercaptopurine. Other examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinopentofuranosylcytosine, N4- octadecyl-l-beta-D-arabinopentofuranosylcytosine, N4-palmitoyl-l-(2-C-cyano-2-deoxy- beta-D-arabinopentofuranosyl)cytosine, and P-4055 (cytarabine 5'-arachidonate).

[0323] A circular polyribonucleotide can or can not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purines or pyrimidines, or any one or more or all of A, G, U, C, I, pU) in the circular polyribonucleotide, or in a given predetermined sequence region thereof, can or can not be uniformly modified. In some embodiments, the circular polyribonucleotide includes pseudouridine. In some embodiments, the circular polyribonucleotide includes inosine, which can help the immune system characterize the circular polyribonucleotide as endogenous relative to viral RNA. Incorporation of inosine can also mediate improved RNA stability / decreased degradation. See, e.g., Yu, Z, et al. (2015) RNA editing by ADAR1 marks dsRNA as “self.” Cell Res 25, 1283-1284, which is incorporated by reference herein in its entirety.

[0324] In some embodiments, all nucleotides in a circular polyribonucleotide (or a given sequence region thereof) are modified. In some embodiments, modifications can include m6A, which can enhance expression; inosine, which can attenuate immune response; pseudouridine, which can increase RNA stability, translational readthrough (stutter elements); m5C, which can increase stability; and 2,2,7-trimethylguanosine, which can facilitate subcellular translocation (e.g., nuclear localization).

[0325] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at each position of the circular polyribonucleotide. One of ordinary skill in the art will understand that a nucleotide analog or other modification(s) can be located at any one or more positions of the circular polyribonucleotide such that the function of the circular polyribonucleotide is not substantially reduced. The modification can also be a non-coding region modification. The circular polyribonucleotide can comprise from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any intermediate percentage (e.g., 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%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). Structure

[0326] In certain embodiments, cyclic polyribonucleotide comprises higher order structure, such as secondary or tertiary structure.In certain embodiments, the complementary section of cyclic polyribonucleotide is folded into double-stranded section by itself, with hydrogen bonding pairing (such as, AU and CG).In certain embodiments, spiral, also referred to as stem, is formed intramolecularly, has the double-stranded section being connected to end ring.In certain embodiments, cyclic polyribonucleotide has at least one section with quasi-double-stranded secondary structure.In certain embodiments, the section with 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 nucleotides of pairing. In certain embodiments, cyclic polyribonucleotide has one or more sections (such as 2,3,4,5,6 or more) with quasi-double-stranded secondary structure.In certain embodiments, section is 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 Nucleotide.

[0327] In certain embodiments, the one or more sequences of cyclic polyribonucleotide comprise substantially stranded and double-stranded zone.In certain embodiments, the ratio of strand and double-stranded can affect the function of cyclic polyribonucleotide.

[0328] In certain embodiments, one or more sequences of cyclic polyribonucleotide are basically single-stranded.In certain embodiments, one or more sequences of the cyclic polyribonucleotide of single-stranded can comprise protein or RNA binding site basically.In certain embodiments, the cyclic polyribonucleotide sequence of single-stranded can be conformational flexibility to allow to increase interaction basically.In certain embodiments, purposefully with the sequence engineering approaches of cyclic polyribonucleotide to comprise this type of secondary structure, thereby in conjunction with or increase protein or nucleic acid combination.

[0329] In some embodiments, the circular polyribonucleotide sequence is substantially double stranded. In some embodiments, one or more sequences of the substantially double stranded circular polyribonucleotide can include a conformational recognition site, such as a riboswitch or aptazyme. In some embodiments, the substantially double stranded circular polyribonucleotide sequence can be conformationally rigid. In some such instances, the conformationally rigid sequence can sterically hinder a circular polyribonucleotide binding protein or nucleic acid. In some embodiments, the sequence of the circular polyribonucleotide is engineered to include such secondary structure purposefully, thereby avoiding or reducing protein or nucleic acid binding.

[0330] There are 16 possible base pairs, but 6 of these (AU, GU, GC, UA, UG, CG) can form actual base pairs. The rest are called mismatches and occur in the helix at very low frequency. In some embodiments, the structure of the circular polyribonucleotide is not easily disrupted, does not affect its function and has no fatal consequences, which provides the option to maintain the secondary structure. In some embodiments, the primary structure of the stem (i.e. its nucleotide sequence) can still be varied while still maintaining the helical region. The nature of the bases is secondary to the higher order structure, and substitutions can be made as long as they preserve 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, the segment with 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 with a quasi-helical structure (e.g. 2, 3, 4, 5, 6 or more). 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 comprises at least one of a U-rich or A-rich sequence or a combination thereof. In some embodiments, the U-rich and / or A-rich sequence is arranged in a manner that will produce a triple quasi-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 with a double quasi-helical structure (e.g. 2, 3, 4, 5, 6 or more). In some embodiments, the circular polyribonucleotide comprises at least one of a C-rich and / or G-rich sequence. In some embodiments, the C-rich and / or G-rich sequence is arranged in a manner that will produce a triple quasi-helical structure. In some embodiments, the circular polyribonucleotide has an intramolecular triple quasi-helical structure that aids in stabilization.

[0331] In some embodiments, the circular polyribonucleotide has two quasi-helical structures (e.g. separated by a phosphodiester bond) such that their terminal base pairs stack and the quasi-helical structures become collinear, resulting in a substructure of “coaxial stacking”.

[0332] In some embodiments, the circular polyribonucleotide comprises a tertiary structure having one or more motifs, e.g., a pseudoknot structure, a g-quadruplex, a helix, and a coaxial stack.

[0333] In some embodiments, the circular polyribonucleotide has at least one binding site, e.g., at least one protein binding site, at least one miRNA binding site, at least one IncRNA binding site, at least one tRNA binding site, 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 snoRNA binding site, at least one snRNA binding site, at least one exRNA binding site, at least one scaRNA binding site, at least one Y RNA binding site, at least one hnRNA binding site, and / or at least one tRNA motif. Delivery

[0334] The circular polyribonucleotides described herein can also be included in a pharmaceutical composition with a delivery vehicle.

[0335] The pharmaceutical compositions described herein can be, for example, formulations comprising a carrier (such as a pharmaceutical carrier and / or a polymeric carrier, e.g., a liposome) and delivered to a subject in need thereof (e.g., a human or a non-human agricultural animal or livestock, e.g., a cow, dog, cat, horse, poultry) by known methods. Such methods include, but are not limited to, transfection (e.g., lipid-mediated cationic polymers, calcium phosphate, dendrimers); electroporation or other membrane-disrupting methods (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, microparticle bombardment (“gene gun”), fugene, direct sonication loading, cell extrusion, phototransfection, protoplast fusion, piercing infection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. Delivery methods are also described in, e.g., 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. doi: 10.1089 / hum.2015.074; and Zuris et al., Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct 30;33(1):73-80.

[0336] The present disclosure further relates to a host or host cell comprising a circular polyribonucleotide described herein. In some embodiments, the host or host cell is a plant, an insect, a bacterium, a fungus, a vertebrate, a mammal (e.g., a human), or other organism or cell.

[0337] In some embodiments, the circular polyribonucleotide is non-immunogenic in a host. In some embodiments, the circular polyribonucleotide reduces or fails to elicit a host immune system response as compared to a response elicited by a reference compound (e.g., a linear polynucleotide corresponding to the circular polyribonucleotide or a circular polyribonucleotide lacking the encryption origin). Some immune responses include, but are not limited to, humoral immune responses (e.g., production of antigen-specific antibodies) and cell-mediated immune responses (e.g., lymphocyte proliferation).

[0338] In some embodiments, a host or host cell is contacted (e.g., delivered to or administered to) with a circular polyribonucleotide. In some embodiments, the host is a mammal, e.g., 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 host growth in culture is determined. If growth is increased or decreased in the presence of the circular polyribonucleotide, the circular polyribonucleotide or expression product or both are considered effective at increasing or decreasing growth of the host. Methods of production

[0339] In some embodiments, the circular polyribonucleotide comprises a non-naturally occurring deoxyribonucleic acid sequence, and can be produced using recombinant techniques (methods are described in detail below; e.g., derived in vitro using DNA plasmids) or chemical synthesis.

[0340] Within the scope of the present application, DNA molecules used to produce RNA circles can include DNA sequences of naturally occurring original nucleic acid sequences, modified versions thereof, or DNA sequences encoding synthetic polypeptides not typically found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using a variety of techniques, including but not limited to classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and / or mutagenesis of selected regions of nucleic acid sequences, synthesis of oligonucleotide mixtures, and ligation of mixture moieties to “build” mixtures of nucleic acid molecules, and combinations thereof.

[0341] Circular polyribonucleotides 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 circularized or concatamerized to produce a circular polyribonucleotide as described herein. The mechanism of circularization or concatamerization can occur through methods such as but not limited to chemical, enzymatic, splint ligation, or ribozyme catalysis. The newly formed 5’- / 3’-bond can be an intramolecular bond or an intermolecular bond.

[0342] Methods of making the circular polyribonucleotides described herein are described, for example, in Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); Zhao, Synthetic Biology: Tools and Applications, (1st ed.), Academic Press (2013); and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (1st ed.), Wiley-VCH (2012).

[0343] Various methods of synthesizing circular polyribonucleotides are also described in the art (see, e.g., U.S. Patent No. US6210931, U.S. Patent No. US5773244, U.S. Patent No. US5766903, U.S. Patent No. US5712128, U.S. Patent No. US5426180, U.S. Publication No. US20100137407, International Publication No. WO 1992001813, and International Publication No. WO 2010084371; the contents of each of which are incorporated herein by reference in their entirety).

[0344] In some embodiments, the circular polyribonucleotides can be cleaned after production to remove production impurities, such as free ribonucleic acids, linear or mini- defective RNAs, DNA, proteins, and the like. In some embodiments, the circular polyribonucleotides can be purified by any commonly known method in the art. Non-limiting examples of purification methods include column chromatography, gel excision, size exclusion, and the like. Pharmaceutical compositions

[0345] The present application includes compositions in combination with one or more pharmaceutically acceptable excipients. The pharmaceutical compositions can optionally include one or more additional active substances, such as therapeutically and / or prophylactically active substances. The pharmaceutical compositions of the present application can be sterile and / or pyrogen-free. General considerations in the formulation and / or production of pharmaceutical formulations can be found in, for example, Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0346] While the description of pharmaceutical compositions provided herein is primarily concerned with pharmaceutical compositions that are suitable for administration to humans, it is to be understood that such compositions are also suitable for administration to other animals, such as non-human animals, for example, non-human mammals. Modifications can be made in the pharmaceutical compositions described above that are suitable for administration to various animals, including humans, and are well known in the art. Such modifications would not require undue experimentation or reasonable modification of the above described compositions, and the skills required to make them are within the level of those in the skilled in veterinary pharmacology. Subjects to which administration of the pharmaceutical compositions are contemplated include, but are not limited to, humans and / or other primates; mammals, including mammals of commercial interest, such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including birds of commercial interest, such as domesticated fowl, chickens, ducks, geese, and / or turkeys.

[0347] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing into association active ingredient with the excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, bringing the product into a suitable form and / or packaging. Methods of expression

[0348] The present application includes methods of protein expression comprising translating at least one region of a circular polyribonucleotide provided herein.

[0349] In some embodiments, the method for protein expression comprises 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 polynucleotide into a polypeptide. In some embodiments, the method for protein expression comprises translation of the circular polyribonucleotide into a polypeptide having 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, 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, the method for protein expression comprises translation of the circular polyribonucleotide into a polypeptide 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 method comprises translation of the circular polyribonucleotide into a contiguous polypeptide provided herein, a discrete polypeptide provided herein, or both.

[0350] In some embodiments, translation of at least one region of the circular polyribonucleotide occurs in vitro, such as in a rabbit reticulocyte lysate. In some embodiments, translation of at least one region of the circular polyribonucleotide occurs in vivo, for example, following transfection of a eukaryotic cell or transformation of a prokaryotic cell, such as a bacterium.

[0351] In some aspects, the disclosure provides a method of expressing one or more expression sequences in a cell of a subject, the method comprising: administering a circular polyribonucleotide to the cell of the subject, wherein the circular polyribonucleotide comprises the one or more expression sequences; and expressing the one or more expression sequences of the circular polyribonucleotide in the cell. In some embodiments, the circular polyribonucleotide is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher 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 is reduced by no more than about 40% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23, or more days. In some embodiments, the expression of the one or more expression sequences in the cell is maintained at a level that varies by no more than about 40% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23, or more days. In some embodiments, the administration of the circular polyribonucleotide is performed using any of the delivery methods described herein. In some embodiments, the subject is administered the circular polyribonucleotide by intravenous injection. In some embodiments, the administration of the circular polyribonucleotide includes, but is not limited to, prenatal administration, neonatal administration, postnatal administration, oral, by injection (e.g., intravenous, intra-arterial, intraperitoneal, intradermal, subcutaneous, and intramuscular), ophthalmic administration, and intranasal administration.

[0352] In some embodiments, the method for protein expression includes modification, folding, or other post-translational modification of the translation product. In some embodiments, the method for protein expression includes in vivo post-translational modification, e.g., by cellular machinery.

[0353] All references and publications cited herein are incorporated by reference herein.

[0354] The above-described embodiments can be combined to achieve the aforementioned functional features. This is also illustrated by the following examples, which set forth exemplary combinations and achieved functional features. Table 1 provides an exemplary overview showing how the different elements described above can be combined and the functional features observed. Table 1. Exemplary elements in examples Examples

[0355] The following examples are provided to further illustrate some embodiments of the application, but are not intended to limit the scope of the application; it being understood by those of ordinary skill in the art that other procedures, methods or techniques known to those of ordinary skill in the art can alternatively be used. Example 1: In vitro circular RNA production

[0356] This example demonstrates the in vitro production of circular RNA.

[0357] The circular RNA is designed to have a start codon (SEQ ID NO: 1), one or more ORFs (SEQ ID NO: 2), one or more interlaced elements (SEQ ID NO: 3), one or more cryptogenes (SEQ ID NO: 4) and an IRES (SEQ ID NO: 5), such as Figure 2 Circularization enables rolling circle translation, multiple open reading frames (ORFs) with alternating staggered elements for discrete ORF expression and controllable protein stoichiometry, one or more cryptogens to attenuate or mitigate RNA immunogenicity, and an optional IRES to target the RNA for ribosome entry (without the need for a poly A sequence).

[0358] In this example, circular RNA was generated as follows. Unmodified linear RNA was synthesized by in vitro transcription from a DNA segment containing 5'- and 3'-ZKSCAN1 introns and an ORF encoding GFP linked to a 2A sequence using T7 RNA polymerase. The transcribed RNA was purified using an RNA purification system (QIAGEN), treated with alkaline phosphatase (Thermo Fisher Scientific, EF0652) according to the manufacturer's instructions, and purified again using an RNA purification system.

[0359] Splinted circular RNA was generated by treating transcribed linear RNA with a DNA splint using T4 DNA ligase (New England Bio, Inc., M0202M), and circular RNA was isolated after enrichment with RNase R. RNA quality was assessed by agarose gel or automated electrophoresis (Agilent). Example 2: In vivo circular RNA production, cell culture

[0360] This example demonstrates the in vivo generation of circular RNA.

[0361] GFP (SEQ ID NO: 2) is cloned into an expression vector, such as pcDNA3.1(+) (Addgene) (SEQ ID NO: 6). This vector is mutagenized to induce circular RNA production in cells (SEQ ID NO: 6 and as described in Kramer et al. 2015), as Figure 3 shown.

[0362] HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) containing high glucose supplemented with penicillin-streptomycin and 10% fetal bovine serum at 37°C and 5% CO. One microgram of the above expression plasmid was transfected using a lipid transfection reagent (Life Technologies), and total RNA was isolated from the transfected cells using a phenol-based RNA isolation reagent (Life Technologies) according to the manufacturer's instructions between 1 hour and 20 days after transfection.

[0363] To measure GFP circular RNA and mRNA levels, qPCR reverse transcription was performed using random hexamers. In short, for RT-qPCR, total RNA and RNA digested with RNase R from HeLa cells of the same source were used as templates for RT-PCR. To prepare cDNA for GFP mRNA and circular GFP RNA, reverse transcription was performed using reverse transcriptase (Super-Script II: RNase H; Invitrogen) and random hexamers according to the manufacturer's instructions. The amplified PCR products were analyzed using 6% PAGE and visualized by ethidium bromide staining. To estimate the enrichment factor, PCR products were quantified by optical density (ImageQuant; Molecular Dynamics), and the concentration of the total RNA sample was measured by UV absorbance.

[0364] Additional RNA measurements were performed using Northern blot analysis. Briefly, whole cell extracts were obtained using a phenol-based reagent (TRIzol), or cells were fractionated using a commercial kit (CelLytic NuCLEAR Extraction Kit, Sigma) to obtain nuclear and cytoplasmic protein extracts. To inhibit RNA polymerase II transcription, cells were treated with flavopiridol (final concentration 1 mM; Sigma) for 0-6 hours at 37°C. For RNase R treatment, 10 mg of total RNA was treated with 20 U of RNase R (Epicentre) at 37°C for 1 hour.

[0365] Northern blotting with oligonucleotide probes was performed as follows. Oligonucleotide probes, PCR primers were designed using standard primer design tools. A T7 promoter sequence was added to the reverse primer to obtain antisense probes in an in vitro transcription reaction. In vitro transcription was performed using T7 RNA polymerase with DIG-RNA labeling mix according to the manufacturer's instructions. DNA template was removed by DNase I digestion and the RNA probes were purified by phenol chloroform extraction followed by precipitation. Probes were used at 50 ng / ml. Total RNA (2-10 pg) was denatured using Glyoxal load dye (Ambion) and resolved on a 1.2% agarose gel in MOPS buffer. The gel was soaked in 1 x TBE for 20 min and transferred to a Hybond-N+ membrane (GE Healthcare) using a semi-dry blotting system (Bio-Rad) for 1 h (15 V). The membrane was dried and UV cross-linked (at 265 nm) at 120,000 pJ cm-2for 1 time. Pre-hybridization was performed at 68°C for 1 h and DIG-labeled in vitro transcribed RNA probes were hybridized overnight. The membrane was washed in 2 x SSC, 0.1% SDS at 68°C for 3 times for 30 min followed by 0.2 x SSC, 0.1% SDS at 68°C for 3 times (30 min). Immunodetection was performed with anti-DIG directly conjugated to alkaline phosphatase antibody. Immunoreactive bands were visualized using chemiluminescent alkaline phosphatase substrate (CDP star reagent) and an imaging detection and quantification system (LAS-4000 detection system). Example 3: Preparation and in vitro translation of circular RNA

[0366] This example demonstrates the detection of gene expression and gene products of circular RNAs.

[0367] In this example, circular RNAs were 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 human-derived cryptic origins (SEQ ID NO: 4), and with or without an IRES (SEQ ID NO: 5), see Figure 4 In this example, circular RNAs were generated in vitro or in cells as described in Example 1 and Example 2.

[0368] Cyclo-RNA was incubated in rabbit reticulocyte lysate (Promega, Fitchburg, WI, USA) at 30°C for 5 hours or overnight. The final composition of the reaction mixture included 70% rabbit reticulocyte lysate, 10 mM methionine and leucine, 20 mM amino acids except methionine and leucine, and 0.8 U / µL RNase inhibitor (Toyobo, Osaka, Japan). An aliquot was removed from the mixture and separated on a 10%-20% gradient polyacrylamide / sodium dodecyl sulfate (SDS) gel (Atto, Tokyo, Japan). The supernatant was removed and the pellet was dissolved in 2x SDS sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 30% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) at 70°C for 15 minutes. During this time hemoglobin was removed and proteins other than hemoglobin were concentrated.

[0369] After centrifugation at 1,400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel. Commercial standards (BioRad) were used as size markers. After semi-dry electrotransfer to a polyvinylidene difluoride (PVDF) membrane (Millipore), the blot was visualized using a chemiluminescence kit (Rockland).

[0370] GFP protein was expected to be visualized in the cell lysate and due to rolling circle translation, a higher amount than in linear RNA was expected to be detected in the cyclo-RNA. Example 4: Stoichiometric protein expression of circular RNA

[0371] This example demonstrates the ability of cyclo-RNA to stoichiometrically express proteins.

[0372] In this example, a cyclo-RNA was designed to include an encryption origin (SEQ ID NO: 4) and an ORF encoding GFP (SEQ ID NO: 2) and an ORF encoding RFP (SEQ ID NO: 8) with stagger elements flanking the GFP and RFP ORFs (SEQ ID NO: 3), see Figure 5 Another cyclo-RNA was designed similarly, but instead of flanking 2A sequences, it had a stop and start codon between the GFP and RFP ORFs. Cyclo-RNA was generated in vitro or in cells as described in Examples 1 and 2.

[0373] Cyclo-RNAs were incubated in rabbit reticulocyte lysate (Promega, Fitchburg, WI, USA) at 30°C for 5 hours or overnight. The final composition of the reaction mixture included 70% rabbit reticulocyte lysate, 10 mM methionine and leucine, 20 mM amino acids except methionine and leucine, and 0.8 U / µL RNase inhibitor (Toyobo, Osaka, Japan). Aliquots were removed from the mixture and separated on 10%-20% gradient polyacrylamide / sodium dodecyl sulfate (SDS) gels (Atto, Tokyo, Japan). The supernatant was removed and the pellet was dissolved in 2x SDS sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 30% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) at 70°C for 15 minutes. During this time hemoglobin was removed and proteins other than hemoglobin were concentrated.

[0374] After centrifugation at 1,400 x g for 5 minutes, the supernatant was analyzed on 10%-20% gradient polyacrylamide / SDS gels. Commercial standards (BioRad) were used as size markers. After semi-dry electrotransfer to polyvinylidene difluoride (PVDF) membranes (Millipore), blots were visualized using a chemiluminescent kit (Rockland).

[0375] It is expected that cyclo-RNAs with GFP and RFP ORFs not separated by a stop and start codon will have equal amounts of each protein, while cells treated with cyclo-RNAs including a start and stop codon between the ORFs will have different amounts of each protein. Example 5: Non-immunogenicity in cell culture

[0376] This example demonstrates in vivo evaluation of the immunogenicity of cyclo-RNAs after infection of cells.

[0377] In this example, cyclo-RNAs were designed to include cryptic exons, e.g., ZKSCAN1 introns and GFP ORFs. Additionally, control cyclo-RNAs were designed to include GFP ORFs with and without introns, see Figure 6 Cyclo-RNAs were generated in vitro or in cells as described in Examples 1 and 2. HeLa cells were transfected with 500 ng of cyclo-RNA.

[0378] Transfection of circular RNAs 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, polyethylene, DEAE-dextran) (Turbofect); (4) virus-like particles (L1 of HPV, VP1 of polyomavirus) (Tonges et al., 2006); (5) exosomes (Exo-Fect by SBI); (6) nanostructured calcium phosphate (nanoCaP) (Olton et al., 2006); (6) peptide transduction domains (TAT, polyR, SP, pVEC, SynBl, etc.) (Zhang et al., 2009); (7) vesicles (VSV-G, TAMEL) (Liu et al., 2017); (8) cell extrusion; (SQZ Biotechnologies); (9) nanoparticles (Neuhaus et al., 2016); and / or (10) magnetofection (Mair et al., 2009). Transfection methods were performed in cell culture medium (DMEM 10% FBS) and cells were subsequently cultured for 24-48 hours.

[0379] Relative expression of indicator RNA and transfecting RNA was measured by qRT-PCR 2-48 hours after transfection.

[0380] For qRT-PCR analysis, total RNA was isolated from cells using a phenol-based RNA isolation solution (TRIzol) and RNA isolation kit (Qiagen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using PCR master mix (Brilliant II SYBR Green qRT-PCR Master Mix) and a PCR cycler (LightCycler 480) in triplicate. mRNA levels of well-known innate immune modulators such as RIG-I, MDA5, OAS, OASL, and PKR were quantified and normalized to actin, GAPDH, or HPRT values. Relative expression of indicator RNA genes for circular RNA transfection was normalized by the level of transfecting RNA and compared to the expression level of circular RNA transfection cells that did not contain one or more cryptids.

[0381] In addition to qRT-PCR analysis, Western blot analysis and immunohistochemistry were used to assess GFP expression efficiency as described in Example 4 above.

[0382] GFP positive cells containing one or more cryptic origins are expected to show an attenuated immunogenic response.

[0383] In addition, (1) murine primary dendritic cells; (2) human embryonic kidney 293 cells stably expressing TLR-7, 8, or 9 (InvivoGen); (3) monocyte-derived dendritic cells (AllCells) or (4) unmanipulated 264.7 cells were transfected with DNA plasmids containing ZKSCAN1 or td introns to produce circular RNAs encoding GFP as described above. Six hours to 48 hours after transfection, cell culture supernatants were collected and used to measure cytokine expression using ELISA. At the time the cell culture supernatants were collected, cells were collected for Northern blot, gene expression array, and FACS analysis.

[0384] For ELISA, ELISA kits for interferon-beta (IFN-beta), chemokine (C-C motif) ligand 5 (CCL5), IL-12 (BD Biosciences), IFN-alpha, TNF-alpha, and IL-8 (Biosource International) were used. ELISA was performed according to the manufacturer's recommendations. Expression of the indicated cytokines by circular RNA transfected cells was compared to levels in control RNA transfected cells. Cells transfected with circular RNA having cryptic origins are expected to have reduced cytokine expression compared to control transfected cells.

[0385] Regarding Northern blot analysis. Samples were processed and analyzed as previously described. Probes were derived from plasmids and specific for the coding region of human IFN-alpha 13, IFN-beta (Open Biosystems), TNF-alpha, or GAPDH (ATCC). Cells transfected with circular RNA having cryptic origins are expected to have reduced cytokine expression compared to control transfected cells.

[0386] For gene expression arrays, RNA was isolated using phenol-based solution (TRIzol) and / or RNA isolation kit (RNeasy Qiagen). RNA was amplified and analyzed (e.g., Illumina HumanHT12 v4 chips in an Illumina BeadStation 500GX). Levels in mock treated cells were used as a baseline to calculate fold increase. Cells transfected with circular RNA having cryptic origins are expected to have reduced cytokine expression compared to control transfected cells.

[0387] For FACS analysis, cells were stained with directly conjugated antibodies against CD83 (Research Diagnostics Inc), HLA-DR, CD80, or CD86 and analyzed on a flow cytometer. It was expected that cells transfected with circRNA containing cryptogens would show reduced expression of these markers compared to control-transfected cells. Example 6: Riboswitches for Selective Expression

[0388] This example demonstrates the ability to control protein expression in vivo using circular RNA.

[0389] For this example, a circular RNA was designed that includes one or more cryptogenins (SEQ ID NO:4) and a synthetic riboswitch (SEQ ID NO:9) that regulates the expression of an 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 in vitro or in cells as described in Examples 1 and 2.

[0390] Theophylline induces activation of a riboswitch, resulting in an off-switch of gene expression (as described in Auslander et al., 2010). The riboswitch is expected to control GFP expression from the circular RNA. In the presence of theophylline, no GFP expression is expected to be observed.

[0391] HeLa cells were transfected with 500 ng of the described circular RNA encoding GFP under the control of a theophylline-dependent synthetic riboswitch (SEQ ID NO: 9) to assess selective expression. The transfection method is described in Example 5.

[0392] After 24 hours of incubation at 37°C and 5% CO2, cells were treated with or without theophylline at concentrations ranging from 1 nM to 3 mM. After 24 hours of continuous incubation, cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature and blocked and permeabilized with 10% FBS in PBS with 0.2% detergent for 45 minutes. Samples were then incubated with a primary antibody against GFP (Invitrogen) and secondary antibodies conjugated to Alexa 488 and DAPI (Invitrogen) in PBS containing 10% FBS and 0.1% detergent for 2 hours at room temperature or overnight at 4°C. Cells were then washed with PBS and subsequently analyzed for GFP expression using a fluorescence microscope. Example 7: In vivo expression

[0393] This example demonstrates the ability of circular RNA to express proteins in vivo.

[0394] For this example, the circular RNA was designed to include one or more cryptic origins (SEQ ID NO: 4) and ORFs encoding GFP (SEQ ID NO: 2) or RFP (SEQ ID NO: 8) or luciferase (SEQ ID NO: 10) with staggered elements flanking the GFP, RFP or luciferase ORFs (SEQ ID NO: 3), see Figure 8 The circular RNA was generated in vitro or in cells as described in Examples 1 and 2.

[0395] 6-8 week old male BALB / c mice received 300 mg / kg (6 mg) of circular RNA with GFP, RFP or luciferase ORFs (50 uL volume) as described herein or linear RNA as a control by intradermal (ID), intramuscular (IM), oral (PO), intraperitoneal (IP) or intravenous (IV) administration. Animals received a single dose or three injections (day 1, day 3, day 5).

[0396] Blood, heart, lung, spleen, kidney, liver and skin injection sites were collected from un-dosed control mice as well as at 2, 4, 8, 24, 48, 72, 96, 120, 168 and 264 hours post-dose (number = 4 mice / time point). Blood samples were collected from a neck vein puncture at study termination.

[0397] Circular RNA quantification was performed on serum and tissues using branched DNA (bDNA) quantification (Panomics / / Affymetrix). Standard curves using known amounts of RNA on each plate (added to untreated tissue samples) were used to quantify RNA in treated tissues. Calculated amounts in picograms (pg) were normalized to the amount of tissue weighed into the lysate applied to the plate. Protein expression (RFP or GFP) in each tissue was assessed by FACS or western blot as described in previous examples.

[0398] Independent groups of mice dosed with luciferase circular RNA were injected with 3 mg of luciferin at 6, 24, 48, 72 and 96 hours post-dose and the animals were imaged on an in vivo imaging system (IVIS Spectrum, PerkinElmer). Three animals were sacrificed and dissected at 6 hours post-dose and the muscle, skin, draining lymph nodes, liver and spleen were imaged in vitro.

[0399] It was expected that the mice would express GFP, RFP or luciferase in the treated tissues. Example 8: In vivo biodistribution

[0400] This example demonstrates the ability to control and measure the in vivo biodistribution of circular RNA.

[0401] In this example, mice were treated with a circular RNA encoding luciferase as described in Example 9. Briefly, the circular RNA was designed to include one or more cryptic origins (SEQ ID NO: 4) and an ORF encoding luciferase (SEQ ID NO: 10) with staggered elements flanking the luciferase ORF (SEQ ID NO: 3), see Figure 9 The circular RNA was generated in vitro or in cells as described in Examples 1 and 2.

[0402] At 6, 24, 48, 72, and 96 hours post-dose, mice were dosed with luciferase circular RNA (3 mg luciferin injected) and the animals were imaged on an in vivo imaging system (IVIS Spectrum, PerkinElmer). At 6 hours post-dose, three animals were sacrificed and dissected and the muscle, skin, draining lymph nodes, liver, and spleen were imaged in vitro

[0403] Serum and tissues were quantified for circular RNA using branched DNA (bDNA) quantification (Panomics / / Affymetrix). A standard curve using known amounts of RNA on each plate (added to untreated tissue samples) was used to quantify RNA in treated tissues. The calculated amount in picograms (pg) was normalized to the amount of tissue weighed into the lysate applied to the plate.

[0404] Male BALB / c mice, 6-8 weeks of age, were dosed with luciferase circular RNA by IM or ID administration at four dose levels: 10 mg, 2 mg, 0.4 mg, and 0.08 mg (number of animals per group = 6) in separate groups. At 6, 24, 48, 72, and 96 hours post-dose, the animals were injected with 3 mg luciferin and imaged on an in vivo imaging system (IVIS Spectrum, PerkinElmer). At 6 hours post-dose, three animals were sacrificed and dissected and the muscle, skin, draining lymph nodes, liver, and spleen were imaged in vitro. Luciferase expression in mouse tissues was also assessed and the tissue distribution of this expression was analyzed as described in Example 9.

[0405] It was expected that mice would show luciferase expression in treated tissues. Example 9: Non-immunogenicity in vivo

[0406] This example demonstrates in vivo assessment of the immunogenicity of circular RNA following infection of cells.

[0407] This example describes the quantification and comparison of immune responses following administration of circular RNA with cryptic origins, see Figure 10In one embodiment, any of the circular RNAs with cryptids will have a reduced (e.g., reduced compared to administration of control RNA) immunogenic response after one or more administrations of the circular RNA compared to a control.

[0408] A measure of the immunogenicity of the circular RNA is the level of cytokines in serum.

[0409] In this example, cytokine serum levels are examined after one or more administrations of the circular RNA. The circular RNA of any of the preceding examples is administered intradermally (ID), intramuscularly (IM), orally (PO), intraperitoneally (IP), or intravenously (IV) to 6-8 week old BALB / c mice. Serum is drawn from different cohorts of mice that are systemically and / or locally injected, one or more injections with circular RNA with and without cryptids.

[0410] Collected serum samples are diluted 1-10 fold in PBS and analyzed for mouse IFN-α by enzyme-linked immunosorbent assay (PBL Biomedical Labs, Piscataway, NJ) and TNF-α (R&D Systems, Minneapolis, MN).

[0411] In addition to cytokine levels in serum, expression 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 is harvested at 1, 4, and 24 hours post administration. Samples are analyzed using the following techniques: qRT-PCR analysis, Northern blotting, or FACS analysis.

[0412] For qRT-PCR analysis, mRNA levels of RIG-I, MDA5, OAS, OASL, TNF-α, and PKR are quantified as described previously.

[0413] For Northern blot analysis, samples are processed and analyzed for IFN-α 13, IFN-β (Open Biosystems), TNF-α, or GAPDH (ATCC) as described above.

[0414] For FACS analysis, cells are stained with directly conjugated antibodies against CD83 (Research Diagnostics Inc), HLA-DR, CD80, or CD86 and analyzed on a flow cytometer.

[0415] In one embodiment, the circular RNA with the encryption origin will have reduced cytokine levels (as measured by ELISA, Northern blot, FACS and / or qRT-PCR) after one or more administrations compared to a control RNA. Example 10: Circular RNA contains at least one double-stranded RNA segment

[0416] This example demonstrates that the circular RNA comprises at least one double stranded RNA segment.

[0417] In this example, the circular RNA was synthesized by one of the aforementioned methods to comprise a GFP ORF and an IRES, see Figure 11 Dot blot assay using J2 and K1 monoclonal antibodies will be used to measure double stranded RNA structures of at least 40 bp in length. Circular RNAs (200 ng) were blotted onto a nylon membrane (Super-Over Nytran), dried, blocked with 5% non-fat dry milk in TBS-T buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4) and incubated with the dsRNA-specific mAb J2 or K1 (English & Scientific Consulting) for 60 min. The membrane was washed six times with TBS-T, then treated with HRP-conjugated donkey anti-mouse Ig (Jackson Immunology), washed six times and the dots were visualized using enhanced chemiluminescent Western blot detection reagents (Amersham).

[0418] It is expected that the circular RNA generates an internal quasi-double stranded RNA segment.

[0419] Example 11: Circular RNA comprises quasi-double stranded structure

[0420] This example demonstrates that the circular RNA comprises a quasi-double stranded structure.

[0421] In this example, the circular RNA was synthesized by one of the aforementioned methods, with or without the addition of HDVmin expression (Griffin et al., 2014). The RNA sequence forms a quasi-helical structure, see Figure 12 and was used as a positive control (as shown by Griffin et al. 2014).

[0422] To test whether the circular RNA structures contain functional quasi-duplex structures, we will determine the secondary structure using selective 2'OH acylation by primer extension (SHAPE) analysis. SHAPE assesses local backbone flexibility in RNA with single-nucleotide resolution. Reactivity of a base position to SHAPE electrophiles is related to secondary structure: positions that are base-paired are less reactive, while unpaired positions are more reactive.

[0423] SHAPE was performed on the circular RNAs, HDVmin, and on a linear RNA containing. SHAPE was performed essentially as reported by Wilkinson et al. 2006 and Griffin 2014 et al., with N-methyl indigo acid anhydride (NMIA) or benzoyl cyanide (BzCN), respectively. Briefly, for SHAPE with BzCN, 1 ul of 800 mM BzCN in dimethyl sulfoxide (DMSO) was added to a 20 ul reaction mixture containing 3 to 6 pmol RNA in 160 mM Tris (pH 8.0), 1 U / l RNase inhibitor (e.g. SuperaseIn RNase inhibitor), and incubated at 37 °C for 1 minute. Control reaction mixtures included 1 ul DMSO without BzCN. Following incubation with BzCN, the RNA was extracted using phenol chloroform and purified (e.g. using the RNA Clean & Concentrator-5 kit) according to the manufacturer’s instructions, and then resuspended in 6 ul 10 mM Tris (pH 8.0). The BzCN adducts were detected using a single-dye system. The RNA was annealed using primers labeled with 6-carboxyfluorescein (6-FAM). Primer extension was performed using a reverse transcriptase (SuperScript III - Invitrogen) according to the manufacturer’s recommendations, with the following modifications to the incubation conditions: 5 minutes at 42 °C, 30 minutes at 55 °C, 25 minutes at 65 °C, and 15 minutes at 75 °C. Two sequencing ladders were generated using 0.5 mM ddATP or 0.5 mM ddCTP in the primer extension reaction. The primer extension products were ethanol precipitated, washed to remove excess salts, and resolved by capillary electrophoresis along with a commercially available size standard (e.g. Liz size standard Genewiz Fragment Analysis Service).

[0424] Raw electropherograms were analyzed using a primary fragment analysis tool (e.g. Peak Scanner Applied Biosystems). The peaks at each position in the electropherogram were then integrated. For each RNA analyzed, a y-axis scaling was performed to correct for loading errors, in order to normalize the background of each primer extension reaction to the background of the negative control reaction performed on the BzCN untreated RNA. A signal decay correction was applied to the data of each reaction. The peaks were aligned to the ladder produced by the two sequencing reactions. At each position, the peak area of the negative control was subtracted from the peak area of the BzCN treated sample; then, the subtracted peak area was divided by the average of the highest 2% to 10% of subtracted peak areas, in order to convert these values to normalized SHAPE reactivities.

[0425] In addition to SHAPE analysis, we will also perform NMR (Marchanka et al., 2015), hydroxyl radical probing (Ding et al., 2012) or a combination of DMS and CMTC and Kethoxal (Tijerina et al., 2007 and Ziehler et al., 2001).

[0426] Cyclic RNAs are expected to have quasi-duplex structures. Example 12: Circular RNA includes a functional quasi-helical structure

[0427] This example shows that the cyclic RNA contains a functional quasi-helical structure.

[0428] In this example, the cyclic RNA was synthesized by one of the aforementioned methods, with the addition of the expression of 395L (Defenbaugh et al., 2009). The RNA sequence forms a quasi-helical structure (as shown above, by the RNA secondary structure folding algorithm mfold and Defenbaugh et al., 2009), Figure 13 This structure is essential for the formation of a complex with the hepatitis D antigen (HDAg).

[0429] Thus, to test whether the circular RNA structure contains a functional quasi- structure, we will incubate the circular RNA and linear RNA with HDAg-160 or HDAg-195 and analyze the binding using the EMSA assay. The binding reaction is performed in 25 ul including 10 mM Tris-HCl (pH 7.0), 25 mM KCl, 10 mM NaCl, 0.1 g / 1 bovine serum albumin (New England Biolabs), 5% glycerol, 0.5 mM DTT, 0.2 U / 1 RNase inhibitor (Applied Biosystems), and 1 mM phenylmethylsulfonyl fluoride solution. The circular RNA is incubated with the HDAg protein (obtained as described in Defenbaugh et al. 2009) at a concentration range of 0-110 nM. The reaction mixture is combined on ice, incubated at 37°C for 1 hour, and electrophoresed on a 6% native polyacrylamide gel in 0.5 Tris-borate-EDTA at 240 V for 2.5 hours. The levels of free and bound RNA are determined using a nucleic acid stain (e.g. gelred). Binding will be calculated as the intensity of unbound RNA subtracted from the total lane intensity minus background.

[0430] It is expected that the circular RNA will have a functional quasi-helical structure. Example 13: Self-transcription / replication

[0431] In this example, the circular RNA is synthesized by one of the aforementioned methods with the addition of one or more of the expression of HDV replication domains (as described in Beeharry et al., 2014), a ribozyme with replication competence of the antigenome, and a nuclear localization signal. These RNA sequences position the circular RNA in the nucleus where the host RNA polymerase will bind and transcribe the RNA. The RNA is then self-cleaved using the ribozyme. The RNA is then ligated and again self-replicates, see Figure 14 .

[0432] The circular RNA (1 microgram - 5 micrograms) is transfected into HeLa cells using the techniques described above. The HeLa cells are grown in Dulbecco's Modified Eagle Medium (DMEM) with high glucose (Life Technologies) supplemented with penicillin-streptomycin and 10% fetal bovine serum at 37°C and 5% CO2. Following transfection, the HeLa cells are incubated for 4 hours - 72 hours post-transfection, total RNA is then isolated from the transfected cells using the phenol-based RNA isolation reagent (Life Technologies) according to the manufacturer's instructions between 1 hour and 20 days post-transfection, and the total amount of the circular RNA encoding the HDV domains will be determined using qPCR as described herein and compared to the control circular RNA. Example 14: Circular RNA Stability / Half-Life

[0433] In this example, the circular RNA is synthesized by one of the aforementioned methods. The circular RNA is designed to include a cryptic origin (SEQ ID NO: 4) and an ORF encoding GFP (SEQ ID NO: 2) with stagger elements flanking the GFP ORF (SEQ ID NO: 3), see Figure 15 .

[0434] Human fibroblasts (e.g., IMR-90) are grown to confluence in Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) in tissue culture treated plates at 37°C in 5% C02. When fibroblasts reach confluence, they stop dividing due to contact inhibition (Leontieva et al., 2014). Lipofectin reagent (2 μL; Thermo Fisher Scientific) is added to a mixture of 1 μg of circular or linear RNA (as described above) and 145 μL of reduced serum medium (Opti-MEM I solution) in one well of a 12-well tissue culture treated plate. After incubation for 15 minutes at room temperature, about 1 x 10^5 cells suspended in DMEM containing 10% FBS are added to the circular RNA solution (as described above).

[0435] The cells are cultured and then harvested at 1, 2, 3, 4, 5, 10, 20, and 30 days post-circular RNA transfection. Cells are separated for q-rt-PCR and another subpopulation is used for FACS analysis. To measure GFP circular RNA and mRNA levels, qPCR reverse transcription is performed using random hexamers as described in Example 2. Cells are analyzed by FACS using GFP antibodies as described herein.

[0436] It is expected that the circular RNAs will persist in the cells for at least several days and that they retain functional expression of the GFP protein. Example 15: Preservation of circular RNAs in daughter cells

[0437] In this example, the circular RNA is synthesized by one of the aforementioned methods. The circular RNA is designed to include a cryptic origin (SEQ ID NO: 4) and an ORF encoding GFP (SEQ ID NO: 2) with stagger elements flanking the GFP ORF (SEQ ID NO: 3), see Figure 16 .

[0438] Human fibroblasts (e.g., IMR-90) were grown on tissue culture treated plates in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at 37°C in 5% C02. Cells were passaged regularly to maintain exponential growth. Lipofectin reagent (2 pL; Thermo Fisher Scientific) was added to a mixture of 1 pg of circular or linear RNA (as described above) and 145 pL of reduced serum medium (Opti-MEM I solution) in one well of a 12-well tissue culture treated plate. After incubation at room temperature for 15 minutes, approximately 1 x 105HeLa cells suspended in DMEM with 10% FBS were added to the circular RNA solution (as described above). After incubation at 37°C in 5% C02for 24 hours, cells were pulsed with BrdU (e.g., Sigma-Aldrich). BrdU labeling times were optimized according to the specific population doubling time of each cell, for example, IMR-90 human fibroblasts have a doubling time of 27 hours and were pulsed for 8-9 hours, as described by Elabd et al. 2013.

[0439] Cells were collected at days 1, 2, 3, 4, 5, and 10 after the BrdU pulse. One subpopulation of these cells was isolated for q-rt-PCR and another subpopulation for FACS analysis. To measure GFP circular RNA and mRNA levels, qPCR reverse transcription was performed using random hexamers as described in Example 2. Cells were analyzed by FACS using BrdU and GFP antibodies as described herein.

[0440] It is expected that the circular RNA will persist in daughter cells and that the daughter cells will express the GFP protein. Example 16: Circular RNA Circularization

[0441] This example demonstrates the use of splinted to generate circular RNA in vitro.

[0442] Non-naturally occurring circular RNAs can be engineered to include one or more desired properties and can be produced using recombinant DNA technology. As shown in the following examples, linear RNA is circularized using a splint.

[0443] CircRNA1 was designed to encode a triply FLAG-tagged EGF without a stop codon (264 nt). It has a Kozak sequence at the start codon for translation initiation (SEQ ID NO: 11). CircRNA2 has the same sequence as circular RNA1 except that it has a termination element (triple stop codon) (273 nt, SEQ ID NO: 12). Circular RNA3 was designed to encode a triply FLAG-tagged EGF flanked by an interlocking element (2A sequence, SEQ ID NO: 13) without a termination element (stop codon) (330 nt). CircRNA4 has the same sequence as circular RNA3 except that it has a termination element (triple stop codon) (339 nt).

[0444] In this example, circular RNAs were generated as follows. DNA templates for in vitro transcription were amplified from gBlocks gene fragments with forward primers with T7 promoters and 2-O-methylated nucleotides with reverse primers with corresponding sequences (IDT). The amplified DNA templates were gel purified with a DNA gel purification kit (Qiagen). 250-500 ng of purified DNA templates were subjected to in vitro transcription. Linear 5'-monophosphorylated in vitro transcripts were generated using T7 RNA polymerase from each DNA template with corresponding sequences in the presence of 7.5 mM GMP, 1.5 mM GTP, 7.5 mM UTP, 7.5 mM CTP, and 7.5 mM ATP. About 40 μg of linear RNA was generated in each reaction. After incubation, each reaction was treated with DNAse to remove the DNA template. The in vitro transcribed RNAs were precipitated with ethanol in the presence of 2.5 M ammonium acetate to remove unincorporated monomers.

[0445] The transcribed linear RNAs were circularized using T4 RNA ligase 2 on a 20 nt splint DNA oligo (SEQ ID NO: 14) as a template. The splint DNA was designed to anneal 10 nt to each 5' or 3' end of the linear RNA. After annealing with the splint DNA (3 μΜ), 1 μΜ of linear RNA was incubated with 0.5 U / μl T4 RNA ligase 2 at 37 °C for 4 hours. The mixture without T4 RNA ligase 2 was used as a negative control.

[0446] The circularization of linear RNAs was monitored by separating the RNAs on 6% denaturing PAGE. On a denaturing polyacrylamide gel, the slower migrating RNA band corresponds to the circular RNA, but not the linear RNA, due to its circular structure. As Figure 17As shown, the addition of ligase to the RNA mixture (+ lane) generated a new band, appearing above the linear RNA band, which was present in the mixture lacking ligase (- lane). The appearance of a slower migrating band in all RNA mixtures indicates that multiple constructs successfully underwent splint ligation (e.g., circularization) compared to the negative control. Example 17: RNA Circularization Efficiency

[0447] This example demonstrates the efficiency of RNA splint ligation circularization.

[0448] Non-naturally occurring circular RNAs engineered to include one or more desired properties can be generated using splint-mediated circularization. As shown in the following example, the efficiency of splint ligation circularization of linear RNA is higher than control.

[0449] CircRNAl, CircRNA2, CircRNA3, and CircRNA4 were also used here as described in Example 1. CircRNA5 was designed to encode a FLAG-tagged EGF, flanked by 2A sequences and followed by a FLAG-tagged nanoluciferase (873 nt, SEQ ID NO: 17). CircRNA6 has the same sequence as circular RNA 5 except that it includes a termination element (triple stop codon) between the EGF and nanoluciferase genes and a termination element (triple stop codon) at the end of the nanoluciferase sequence (762 nt, SEQ ID NO: 18).

[0450] In this example, to measure the efficiency of RNA circularization, six different sized linear RNAs (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 of linear or circular RNA on the gel were excised for purification. The excised RNA gel bands were crushed and the RNA was eluted overnight with 800 μΐ of 300 mM NaCl. The gel debris was removed by a centrifugal filter and the RNA was precipitated with ethanol in the presence of 0.3 M sodium acetate.

[0451] The efficiency of circularization was calculated by dividing the amount of circular RNA eluted by the total amount of RNA eluted (circular + linear RNA) and the results are depicted as Figure 18 in the graph.

[0452] The efficiency of using T4 RNA ligase 2 to ligate linear RNA to produce circular RNA was higher than control. The trend data indicates that larger constructs circularize at a higher rate. Example 18: Circular RNAs Lack Susceptibility to Degradation

[0453] This example demonstrates the susceptibility of circular RNA to degradation by RNase R compared to linear RNA.

[0454] Circular RNA is more resistant to exonuclease degradation than linear RNA due to the lack of 5' and 3' ends. As shown in the following example, circular RNA is less susceptible to degradation than its linear RNA counterpart.

[0455] CircRNA5 was generated and circularized as described in Example 2 for use in the assays described herein.

[0456] To test the circularization of CircRNA5, 20 ng / μΐ of linear or CircRNA5 was incubated with 2 U / μΐ of RNase R, a 3' to 5' exonuclease that digests linear RNA but not lariat or circular RNA structures, at 37°C for 30 minutes. Following incubation, the reaction mixture was analyzed by 6% denaturing PAGE.

[0457] The absence of the linear RNA band present in the lane lacking exonuclease in the CircRNA5 lane (see Figure 19 ) indicates that CircRNA5 shows a higher resistance to exonuclease treatment compared to the linear RNA control. Example 19: Isolation and purification of circular RNA

[0458] This example demonstrates the purification of circular RNA.

[0459] In certain embodiments, circular RNA can be isolated and purified prior to expression of the encoded protein product, as described in the previous examples. This example describes isolation using UREA gel separation. As shown in the following example, circular RNA is isolated and purified.

[0460] CircRNA1, CircRNA2, CircRNA3, CircRNA4, CircRNA5, and CircRNA6 as described in Example 2 were isolated as described herein.

[0461] In this example, linear and circular RNA were generated as described. To purify the circular RNA, the ligation mixture was resolved on a 6% denaturing PAGE and the RNA band corresponding to each of the circular RNAs was excised. The excised RNA gel fragments were crushed and the RNA was eluted with 800 μΐ of 300 mM NaCl overnight. The gel debris was removed by a centrifugal filter and the RNA was precipitated with ethanol in the presence of 0.3 M sodium acetate. The eluted circular RNA was analyzed by 6% denaturing PAGE, see Figure 20 .

[0462] A single band of circular RNA with variable size was visualized by PAGE. Example 20: Detection of protein expression

[0463] This example demonstrates in vitro protein expression of circular RNA.

[0464] Protein expression is the process by which mRNA generates a specific protein. This process includes transcription of DNA into messenger RNA (mRNA), followed by translation of the mRNA into a polypeptide chain, which is eventually folded into a functional protein and can be targeted to specific subcellular or extracellular locations.

[0465] As shown in the following examples, circular RNA sequences express proteins in vitro.

[0466] Circular RNA was designed to encode a triply FLAG-tagged EGF flanked by 2A sequences without a termination element (stop codon) (330 nt, SEQ ID NO: 19).

[0467] Linear RNA or circular RNA was incubated in a 25 μΐ volume of rabbit reticulocyte lysate for 5 hours at 30°C. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.8 U / μΐ RNAse inhibitor, and 1 μg linear RNA or circular RNA. After incubation, hemoglobin was removed by adding acetic acid (0.32 μΐ) and water (300 μΐ) to the reaction mixture (16 μΐ) and centrifugation at 20,817 x g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μΐ of 2x SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0468] After electrotransformation onto nitrocellulose membranes using the dry transfer method, the blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blots were visualized with an ECL kit (see Figure 21 ) and the Western blot band intensity was measured by ImageJ.

[0469] The detection of fluorescence indicates the presence of the expression product. Thus, it is shown that circular RNA drives protein expression. Example 21: IRES-independent expression

[0470] This example demonstrates that circular RNA drives expression in the absence of an IRES.

[0471] An IRES or internal ribosome entry site is an RNA element that allows translation initiation in a cap-independent manner. It is shown that circular RNA drives expression of Flag protein in the absence of an IRES.

[0472] The circular RNA was designed to encode a tripartite FLAG-tagged EGF flanked by 2A sequences without a termination element (stop codon) (330 nt, SEQ ID NO: 19).

[0473] Linear RNA or circular RNA was incubated in a 25 μl volume of rabbit reticulocyte lysate at 30°C for 5 hours. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μM amino acids, 0.8 U / μl RNase inhibitor, and 1 μg linear RNA or circular RNA. After incubation, hemoglobin was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 x g for 10 minutes at 15°C. The supernatant was removed and the pellet was dissolved in 30 °l of 2x SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0474] After electrotransfer to a nitrocellulose membrane using dry transfer, the blot was incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blot was visualized using an enhanced chemiluminescence (ECL) kit (see Figure 21 ) and measured Western blot band intensities by ImageJ.

[0475] Even in the absence of IRES, expression products were detected in the circular RNA reaction mixture. Example 22: Cap-independent expression

[0476] This example demonstrates that circular RNA can drive expression in the absence of a cap.

[0477] The cap is a specially modified nucleotide at the 5' end of an mRNA. The 5' cap contributes to the stability of linear mRNA and helps initiate translation. In the absence of the cap, circular RNAs drive expression.

[0478] The circular RNA was designed to encode a tripartite FLAG-tagged EGF flanked by 2A sequences without a termination element (stop codon) (330 nt, SEQ ID NO: 19).

[0479] Linear RNA or circular RNA was incubated in a 25 μΐ volume of rabbit reticulocyte lysate for 5 hours at 30°C. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.8 U / μΐ RNase inhibitor, and 1 μg linear RNA or circular RNA. After incubation, hemoglobin was removed by adding acetic acid (0.32 μΐ) and water (300 μΐ) to the reaction mixture (16 μΐ) and centrifugation at 20,817 x g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μΐ 2x SDS sample buffer at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0480] After electrotransfer to nitrocellulose membranes using dry transfer, blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. Blots were visualized with an ECL kit (see Figure 21 ) and Western blot band intensities were measured by ImageJ.

[0481] Expression products were detected in the circular RNA reaction mixture even in the absence of cap. Example 23: Expression without 5'-UTR

[0482] This example demonstrates in vitro protein expression of circular RNA lacking a 5' untranslated region.

[0483] The 5' untranslated region (5'UTR) is the region immediately upstream of the start codon that aids in downstream protein translation of the RNA transcript.

[0484] As shown in the following examples, the 5'-untranslated region in the circular RNA sequence is not necessary for in vitro protein expression.

[0485] Circular RNA was designed to encode a triply-FLAG-tagged EGF flanked by 2A sequences without a termination element (stop codon) (330 nt, SEQ ID NO: 19).

[0486] Linear RNA or circular RNA was incubated in a 25 μΐ volume of rabbit reticulocyte lysate for 5 hours at 30°C. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.8 U / μΐ RNase inhibitor and 1 μg linear RNA or circular RNA. After incubation, hemoglobin was removed by adding acetic acid (0.32 μΐ) and water (300 μΐ) to the reaction mixture (16 μΐ) and centrifugation at 20,817 x g for 10 minutes at 15°C. The supernatant was removed and the pellet was dissolved in 30 μΐ of 2x SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0487] After electrotransformation onto nitrocellulose membranes using dry transfer method, the blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blots were visualized with an ECL kit (see Figure 21 ) and the Western blot band intensities were measured by ImageJ.

[0488] Expression products were detected in the circular RNA reaction mixture even in the absence of the 5'UTR. Example 24: Expression without 3'-UTR

[0489] This example demonstrates in vitro protein expression of circular RNA lacking the 3'-UTR.

[0490] The 3' untranslated region (3'-UTR) is the region immediately downstream of the translation termination codon and includes regulatory regions that can influence gene expression after transcription. The 3'-untranslated region can also play a role in gene expression by affecting the localization, stability, export and translation efficiency of the mRNA. In addition, the structural features of the 3'-UTR and the use of alternative polyadenylation can play a role in gene expression.

[0491] As shown in the following examples, the 3'-UTR in the circular RNA sequence is not essential for in vitro protein expression.

[0492] Circular RNA was designed to encode a triply FLAG-tagged EGF flanked by 2A sequences without a termination element (stop codon) (330 nt, SEQ ID NO: 19).

[0493] Linear RNA or circular RNA was incubated in a 25 μl volume of rabbit reticulocyte lysate at 30°C for 5 hours. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μM amino acids, 0.8 U / μl RNase inhibitor, and 1 μg linear RNA or circular RNA. After incubation, hemoglobin was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 x g for 10 minutes at 15°C. The supernatant was removed and the pellet was dissolved in 30 °l of 2x SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0494] After electrotransfer to a nitrocellulose membrane using dry transfer, the blot was incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blot was visualized using an ECL kit (see Figure 21 ) and measured Western blot band intensity by ImageJ.

[0495] Even in the absence of a 3'UTR, expression products were detected in the circular RNA reaction mixture. Example 25: Expression without stop codon

[0496] This example demonstrates the generation of a polypeptide product following rolling circle translation of a circular RNA lacking a stop codon.

[0497] Proteins are based on polypeptides, which consist of a unique sequence of amino acids. Each amino acid is encoded in mRNA by a nucleotide triplet called a codon. During protein translation, each codon in the mRNA corresponds to the addition of an amino acid to the growing polypeptide chain. A termination element, or stop codon, signals the end of this process by binding release factors, which causes the ribosomal subunits to dissociate, releasing the amino acid chain.

[0498] As shown in the examples below, circular RNA lacking a stop codon generates a large polypeptide product composed of repeated polypeptide sequences through rolling circle translation.

[0499] The circular RNA was designed to encode a triplet FLAG-tagged EGF without a termination element (stop codon) (264 nt, SEQ ID NO: 20) and including a Kozak sequence at the start codon to facilitate translation initiation.

[0500] Linear RNA or circular RNA was incubated in a 25 μΐ volume of rabbit reticulocyte lysate for 5 hours at 30°C. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.8 U / μΐ RNase inhibitor and 1 μg linear RNA or circular RNA. After incubation, hemoglobin was removed by adding acetic acid (0.32 μΐ) and water (300 μΐ) to the reaction mixture (16 μΐ) and centrifugation at 20,817 x g for 10 minutes at 15°C. The supernatant was removed and the pellet was dissolved in 30 μΐ of 2x SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0501] After electrotransfer to nitrocellulose membranes using dry transfer method, blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. Blots were visualized with ECL kit (see Figure 22 ) and Western blot band intensities were measured by ImageJ.

[0502] Expression products were detected in the circular RNA reaction mixture even in the absence of a stop codon. Example 26: Discrete protein expression without termination elements (stop codons)

[0503] This example demonstrates the generation of discrete protein products from circular RNA lacking a termination element (stop codon) for translation.

[0504] An interleave element (such as a 2A peptide) can comprise a short amino acid sequence of about 20 aa, allowing a single mRNA to produce equimolar levels of multiple genes. The interleave element can act by causing the ribosome to skip the peptide bond at the C-terminal end of the 2A element, resulting in a separation between the end of the 2A sequence and the next downstream peptide. The separation occurs between the glycine and proline residues found at the C-terminal end, and the upstream cistron is appended with some extra residues at the end, while the downstream cistron begins with a proline.

[0505] As shown in the following example, circular RNA lacking a terminal element (stop codon) generates large polypeptide polymers Figure 23 Left panel: no-interleave-circular RNA lane), and inclusion of a 2A sequence at the 3' end of the coding region results in discrete proteins of comparable size to those generated by the equivalent linear RNA construct Figure 23 Right panel: interleave-circular RNA lane).

[0506] The circular RNA was designed to encode a triply FLAG-tagged EGF without a termination element (stop codon) (264 nt, SEQ ID NO: 20) and without a shuffling element. The second circular RNA was designed to encode a triply FLAG-tagged EGF flanked by 2A sequences without a termination element (stop codon) (330 nt, SEQ ID NO: 19).

[0507] Linear or circular RNA was incubated in a 25 μΐ volume of rabbit reticulocyte lysate for 5 hours at 30°C. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.8 U / μΐ RNase inhibitor, and 1 μg linear or circular RNA. After incubation, hemoglobin was removed by adding acetic acid (0.32 μΐ) and water (300 μΐ) to the reaction mixture (16 μΐ) and centrifugation at 20,817 x g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μΐ of 2x SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0508] After electrotransformation onto nitrocellulose membranes using the dry transfer method, the blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blots were visualized with an ECL kit (see Figure 23 ) and the Western blot band intensities were measured by ImageJ.

[0509] The detection of discrete expression products indicates that circular RNA containing a shuffling element drives the expression of individual proteins even when a termination element (stop codon) is absent. Example 27: Rolling Ring Translation

[0510] This example demonstrates the use of a shuffling element to improve in vitro protein biosynthesis from circular RNA.

[0511] A non-naturally occurring circular RNA was engineered to contain a shuffling element to compare protein expression with a circular RNA lacking the shuffling element. As shown in the following example, the shuffling element overexpressed the protein compared to other identical circular RNA lacking this sequence.

[0512] The circular RNA was designed to encode a triply FLAG-tagged EGF with a termination element (e.g., three stop codons in tandem) (273 nt, SEQ ID NO: 21). The second circular RNA was designed to encode a triply FLAG-tagged EGF flanked by 2A sequences without a termination element (stop codon) (330 nt, SEQ ID NO: 19).

[0513] Linear RNA or circular RNA was incubated in a 25 μΐ volume of rabbit reticulocyte lysate for 5 hours at 30°C. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.8 U / μΐ RNAse inhibitor and 1 μg linear RNA or circular RNA. After incubation, hemoglobin was removed by adding acetic acid (0.32 μΐ) and water (300 μΐ) to the reaction mixture (16 μΐ) and centrifugation at 20,817 x g for 10 minutes at 15°C. The supernatant was removed and the pellet was dissolved in 30 μΐ of 2x SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0514] After electrotransformation onto nitrocellulose membranes using dry transfer method, the blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blots were visualized with an ECL kit (see Figure 24) and the Western blot band intensities were measured by ImageJ.

[0515] The detection of discrete expression products indicates that circular RNA comprising the shuffling element drives the expression of individual proteins even in the absence of a termination element (stop codon). Example 28: In vitro expression of biologically active proteins

[0516] This example demonstrates the in vitro biosynthesis of biologically active proteins from circular RNA.

[0517] Non-naturally occurring circular RNA was engineered to express a therapeutic protein with biological activity. As shown in the following example, circular RNA in reticulocyte lysate expressed a biologically active protein.

[0518] Circular RNA was designed to encode a FLAG-tagged EGF, flanked by 2A sequences and followed by a FLAG-tagged nanoluciferase (873 nt, SEQ ID NO: 17).

[0519] Linear RNA or circular RNA was incubated in a 25 μΐ volume of rabbit reticulocyte lysate for 5 hours at 30°C. The final composition of the reaction mixture contained 70% rabbit reticulocyte lysate, 20 μΜ amino acids, 0.8 U / μΐ RNAse inhibitor. Luciferase activity in the translation mixture was monitored using the Luciferase Assay System according to the manufacturer's protocol (Promega).

[0520] As shown in Figure 24, the detected fluorescence of both circular RNA and linear RNA was far higher than the control vehicle RNA, indicating the presence of expression products. Thus, it was shown that circular RNA expresses biologically active proteins. Figure 25 ​ Example 29: Circular RNAs with Longer Half-lives than Linear RNA Counterparts

[0521] This example demonstrates that engineered circular RNA has a longer half-life compared to linear RNA.

[0522] Circular RNA encoding therapeutic proteins provides recipient cells with the ability to produce higher levels of the encoded protein, resulting from, for example, an extended biological half-life compared to linear RNA. As shown in the following examples, circular RNA has a longer half-life in reticulocyte lysate than its linear RNA counterpart.

[0523] The circular RNA was designed to encode FLAG-tagged EGF flanked by 2A sequences and followed by FLAG-tagged nanoluciferase (873 nt, SEQ ID NO: 17).

[0524] In this example, a time course experiment was performed to monitor the stability of RNA. 100 ng of linear RNA or circular RNA was incubated with rabbit reticulocyte lysate and samples were collected at 1 hour, 5 hours, 18 hours, and 30 hours. Total RNA was isolated from the lysate using a phenol-based reagent (Invitrogen) and cDNA was generated by reverse transcription. qRT-PCR analysis was performed using a dye-based quantitative PCR reaction mixture (Bio-Rad).

[0525] like Figure 26 As shown, circular RNAs were detected at higher concentrations than linear RNAs at later time points. Therefore, circular RNAs are more stable or have an increased half-life compared to their linear counterparts. Example 30: Circular RNA exhibits a longer half-life in cells than linear RNA

[0526] This example demonstrates that circular RNA is delivered into cells and has an increased half-life in cells compared to linear RNA.

[0527] Non-naturally occurring circular RNAs are engineered to express biologically active therapeutic proteins. As shown in the examples below, circular RNAs are present at higher levels compared to their linear RNA counterparts, demonstrating that circular RNAs have longer half-lives.

[0528] In this example, circular RNA and linear RNA were designed to encode Kozak, EGF, which were flanked by 2A, stop or no stop (SEQ ID NO: 11, 19, 20, 21). To monitor the half-life of RNA in cells, 0.1 x 10 6One cell was seeded into each well of a 12-well plate. One day later, 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 the cells using a phenol-based extraction reagent (Invitrogen). Total RNA (500 ng) was subjected to reverse transcription to generate cDNA. qRT-PCR analysis was performed using a dye-based quantitative PCR mix (Bio-Rad). Primer sequences were as follows: primers for linear or circular RNA, F: ACGACGGTGTGTGCATGTAT, R: TTCCCACCACTTCAGGTCTC; primers for circular RNA, F: TACGCCTGCAACTGTGTTGT, R: TCGATGATCTTGTCGTCGTC.

[0529] Circular RNA and its linear counterpart were successfully transfected into 293T cells. Twenty-four hours later, the remaining circular and linear RNA were measured using qPCR. As shown in FIGS. 1A and IB, the circular RNA had a longer half-life in the cells compared to the linear RNA. Figure 27A and Figure 27B Example 31: Synthetic circular RNA was translated in cells and translated by rolling circle translation

[0530] This example demonstrates translation of synthetic circular RNA in cells.

[0531] As shown in the following example, circular and linear RNAs were designed to encode a Kozak 3xFLAG-EGF sequence without a termination element (SEQ ID NO: 11). The circular RNA was translated into a polymeric EGF, while the linear RNA was not, indicating that the cells performed rolling circle translation on the synthetic circular RNA.

[0532] In this example, 0.1 x 10 6 One cell was seeded into each well of a 12-well plate to monitor translation efficiency of linear or circular RNA in cells. One day later, 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 200 mΐ of RIPA buffer into each well. Next, 10 pg of cell lysate protein was analyzed on a 10%-20% gradient polyacrylamide / SDS gel. After electrotransfer to nitrocellulose membranes using dry transfer, the blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. As a loading control, an anti-beta tubulin antibody was used. The blots were visualized with an enhanced chemiluminescence (ECL) kit. Western blot band intensities were measured by ImageJ.

[0533] ​Circular RNA and its linear counterpart have been successfully transfected into 293T cells. Figure 28 The results showed that 24 hours after transfection, EGF protein was detected in cells transfected with circular RNA, but not in cells transfected with linear RNA. Therefore, circular RNA is translated in cells by rolling circle translation, as compared to linear RNA. Example 32: Synthetic circular RNA demonstrates reduced immunogenic gene expression in cells

[0534] This example demonstrates that circular RNA can be engineered to be less immunogenic than linear RNA.

[0535] Circular RNA encoding therapeutic proteins provides immunogenicity-related genes (RIG-I, MDA5, PKA and IFN-β) induction compared to linear RNA in recipient cells. RIG-I can recognize short 5' triphosphate uncapped double-stranded or single-stranded RNA, while MDA5 can recognize longer dsRNA. Both RIG-I and MDA5 can participate in activating MAVS and triggering antiviral responses. PKR can be activated by dsRNA and induced by interferons (such as IFN-β). As shown in the examples below, as evaluated by q-PCR expression of RIG-I, MDA5, PKR and IFN-β, circular RNA was shown to have immune-related gene activation reduced in 293T cells compared to similar linear RNA.

[0536] Circular RNA and linear RNA were designed to encode (1) Kozak, 3xFLAG-EGF sequence without termination element (SEQ ID NO: 11); (2) Kozak, 3xFLAG-EGF flanked by termination element (stop codon) (SEQ ID NO: 21); (3) Kozak, 3xFLAG-EGF flanked by 2A sequence (SEQ ID NO: 19); (4) Kozak, 3xFLAG-EGF sequence flanked by 2A sequence followed by termination element (stop codon) (SEQ ID NO: 20).

[0537] In this example, by adding 0.1x 10 6 Cells were seeded into each well of a 12-well plate to monitor the levels of innate immune response genes in the cells. One day later, 1 μg of linear RNA 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 the cells using a phenol-based extraction reagent (Invitrogen). Total RNA (500 ng) was reverse transcribed to generate cDNA. qRT-PCR analysis was performed using a dye-based quantitative PCR mixture (Bio-Rad).

[0538] Primer sequences used: Primer for GAPDH, F: AGGGCTGCTTTTAACTCTGGT, R: CCCCACTTGATTTTGGAGGGA; RIG-I, F: TGTGGGCAATGTCATCAAAA, R: GAAGCACTTGCTACCTCTTGC; MDA5, F: GGCACCATGGGAAGTGATT, R: ATTTGGTAAGGCCTGAGCTG; PKR, F: TCGCTGGTATCACTCGTCTG, R: GATTCTGAAGACCGCCAGAG; IFN-beta, F: CTCTCCTGTTGTGCTTCTCC, R: GTCAAAGTTCATCCTGTCCTTG.

[0539] As Figure 29 shown, qRT-PCR levels of immune-related genes in 293T cells transfected with circular RNA showed a decrease in RIG-I, MDA5, PKR, and IFN-beta compared to cells transfected with linear RNA. Thus, induction of immunogenicity-related genes in the recipient cell was reduced in cells transfected with circular RNA compared to cells transfected with linear RNA. Example 33: Increased expression of synthetic circular RNA in cells via rolling circle translation

[0540] This example demonstrates increased expression of synthetic circular RNA by rolling circle translation in cells.

[0541] Circular RNA was 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, stagger sequence (2A sequence), 3x FLAG-tagged nLUC sequence, stagger sequence (2A sequence), and stop codon; or nLUC stagger (SEQ ID NO: 24): EMCV IRES, stagger sequence (2A sequence), 3x FLAG-tagged nLUC sequence, and stagger sequence (2A sequence). As Figure 30 shown, both circular RNAs produced translation products with functional luciferase activity.

[0542] In this example, translation of circular RNA was monitored in cells. Specifically, 0.1 x 10 6Cells were plated into each well of a 12-well plate. One day later, 300 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). 24 hours later, cells were harvested by adding 100 μl of RIPA buffer. Nanoluciferase activity in the lysate was measured using a luciferase assay system according to the manufacturer's protocol (Promega).

[0543] like Figure 30 As shown, both circular RNAs expressed protein in cells. However, the circular RNA with the staggered element (e.g., 2A sequence) lacking a termination element (stop codon) produced higher levels of protein product with functional luciferase activity than the circular RNA with the termination element (stop codon). Example 34: Synthetic circular RNA translated in cells

[0544] This example demonstrates synthetic circular RNA translation in cells. Additionally, this example illustrates that circular RNA produces more expression products than its linear counterpart.

[0545] Circular RNA and its linear counterpart have been successfully transfected into 293T cells. Cells were transfected with the following: as a negative control, circular RNA encoding EGF (SEQ ID NO: 22): EMCV IRES, staggered sequence (2A sequence), 3x FLAG-tagged EGF sequence, staggered sequence (2A sequence); linear or circular nLUC (SEQ ID NO: 23): EMCV IRES, staggered sequence (2A sequence), 3x FLAG-tagged nLuc sequence, staggered sequence (2A sequence) and a stop codon. Figure 31 As shown, circular RNA is translated into nanoluciferase in cells.

[0546] Monitor linear RNA or circular RNA translation in cells. Specifically, 0.1x 10 6 Cells were plated into each well of a 12-well plate. After 1 day, 300 ng of linear RNA or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). After 24 hours, cells were harvested by adding 100 μl of RIPA buffer. According to the manufacturer's protocol (Promega), the nanoluciferase activity in the lysate was measured using a luciferase assay system.

[0547] like Figure 31 As shown, circular RNA translation products were detected in cells. Specifically, circular RNAs had higher luciferase activity levels or produced increased protein compared to their linear RNA counterparts. Example 35: Rolling circle translation of synthetic circular RNA in cells produces functional protein products

[0548] This example demonstrates the rolling circle translation of functional protein product from synthetic circular RNA in cells that lack a termination element (stop codon), e.g., with staggered elements that lack a termination element (stop codon). In addition, this example shows that circular RNA with staggered elements expresses more functional protein product than its linear counterpart.

[0549] Circular RNA and its linear counterpart were successfully transfected into 293T cells. Cells were transfected with the following: circular RNA EGF negative control (SEQ ID NO: 22); linear and circular nLUC (SEQ ID NO: 24): EMCV IRES, staggered sequence (2A sequence), 3x FLAG tagged nLuc sequence, staggered sequence (2A sequence). As shown in Figure 32 the circular RNA was translated in cells to nanoluciferase.

[0550] Linear RNA or circular RNA translation was monitored in cells. Specifically, 0.1 x 10 6 cells were plated into each well of a 12-well plate. After 1 day, 300 ng of linear RNA or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). After 24 hours, cells were harvested by adding 100 μΐ of RIPA buffer. Luciferase activity in the lysate was measured using a luciferase assay system according to the manufacturer’s protocol (Promega).

[0551] As shown in Figure 32 circular RNA translation products were detected in cells. In particular, circular RNA without a termination element (stop codon) produced higher levels of protein product with functional luciferase activity compared to its linear RNA counterpart. Example 36: Synthetic circular RNA in cells that initiates translation via IRES

[0552] This example demonstrates the translation initiation of synthetic circular RNA with an IRES in cells.

[0553] Circular RNA was designed to include either a kozak sequence or an IRES with a nanoluciferase gene or an EGF negative control gene. Cells were transfected with the following: EGF negative control (SEQ ID NO: 22), nLUC kozak (SEQ ID NO: 25): kozak sequence, lx FLAG tagged EGF sequence, staggered sequence (T2A sequence), lx FLAG tagged nLUC, staggered sequence (P2A sequence), and stop codon; or nLUC IRES (SEQ ID NO: 23): EMCV IRES, staggered sequence (2A sequence), 3x FLAG tagged nLUC sequence, staggered sequence (2A sequence), and stop codon. As shown in Figure 33As shown, circRNAs with IRES exhibited higher levels of luciferase activity, corresponding to higher protein levels, compared to circRNAs with Kozak sequences.

[0554] In this example, the translation of circular RNA was monitored in cells. 6 Cells were plated into each well of a 12-well plate. One day later, 300 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). 24 hours later, cells were harvested by adding 100 μl of RIPA buffer. Nanoluciferase activity in the lysate was measured using a luciferase assay system according to the manufacturer's protocol (Promega).

[0555] like Figure 33 As shown, circular RNA initiating protein expression with IRES produced higher levels of protein product with functional luciferase activity compared to circular RNA initiating protein expression with Kozak. Example 37: Rolling Circle Translation of Synthetic Circular RNA in Cells

[0556] This example demonstrates higher protein production from synthetic circular RNA in cells via rolling circle translation, using an IRES to initiate protein production.

[0557] Circular RNA was designed to include a Kozak sequence or IRES with the nanoluciferase gene or an EGF negative control with or without a termination element (stop codon). Cells were transfected with the following: EGF negative control (SEQ ID NO: 22); nLUC IRES stop (SEQ ID NO: 23): EMCV IRES, staggered sequence (2A sequence), 3x FLAG-tagged nLUC sequence, staggered sequence (2A sequence), and stop codon; or nLUC IRES staggered (SEQ ID NO: 24): EMCV IRES, staggered sequence (2A sequence), 3x FLAG-tagged nLUC sequence and staggered sequence (2A sequence). Figure 34 As shown, the expression products produced by both circular RNAs exhibited rolling circle translation, and the circular RNA initiated without the termination element IRES (e.g., without a Kozak sequence) produced higher levels of protein products with functional luciferase activity (exhibiting rolling circle translation) than the circular RNA with the termination element IRES (e.g., with a Kozak sequence).

[0558] In this example, the translation of circular RNA was monitored in cells. 6One cell was plated into each well of a 12-well plate. One day later, 300 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). After 24 hours, cells were harvested by adding 100 μΐ of RIPA buffer. Nanoluc activity in the lysate was measured using the luciferase assay system according to the manufacturer's protocol (Promega).

[0559] As shown in Figure 34 , circular RNA was translated into protein in cells by a rolling circle method derived from the two circular RNAs. However, the circular RNA lacks a termination element (stop codon). Nevertheless, rolling circle translation of the circular RNA initiated with an IRES resulted in higher protein production and produced more protein product with functional luciferase activity compared to the circular RNA with a termination element Kozak translation initiation. Example 38: Increased protein expression of circular RNA

[0560] This example demonstrates synthetic circular RNA translation in cells. In addition, this example shows that circular RNA produces more expression product with the correct molecular weight than its linear counterpart.

[0561] Linear and circular RNA were designed to include a nanoluc gene with a termination element (stop codon). Cells were transfected with: vehicle: transfection reagent only; linear nLUC (SEQ ID NO: 23): EMCV IRES, shuffle element (2A sequence), 3x FLAG-tagged nLuc sequence, shuffle element (2A sequence), and termination element (stop codon); or circular nLUC (SEQ ID NO: 23): EMCV IRES, shuffle element (2A sequence), 3x FLAG-tagged nLuc sequence, shuffle element (2A sequence), and termination element (stop codon). As shown in Figure 35 , the circular RNA produced higher levels of protein with the correct molecular weight compared to the linear RNA.

[0562] After 24 hours, cells were harvested by adding 100 μΐ of RIPA buffer. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel.

[0563] After electroporation onto nitrocellulose membranes using dry transfer method, blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. Blots were visualized with an ECL kit and Western blot band intensities were measured by ImageJ.

[0564] As shown in Figure 35As shown, circular RNA is translated into protein in cells. Specifically, circular RNA produces higher levels of protein with the correct molecular weight than its linear RNA counterpart. Example 39: Rolling circle translation of synthetic circular RNA in cells produces discrete protein products

[0565] This example demonstrates that discrete protein products are translated in cells by rolling translation of a synthetic circular RNA that lacks a termination element (stop codon), e.g., has a staggered element in place of the termination element (stop codon). In addition, this example shows that the circular RNA with the staggered element expresses more protein product with the correct molecular weight than its linear counterpart.

[0566] Circular RNA was designed to include the nanoluciferase gene with a staggered element instead of the termination element (stop codon). Cells were transfected with the following: vehicle: transfection reagent only; linear nLUC (SEQ ID NO: 24): EMCV IRES, staggered element (2A sequence), 3x FLAG-tagged nLuc sequence and staggered element (2A sequence); or circular nLUC (SEQ ID NO: 24): EMCV IRES, staggered element (2A sequence), 3x FLAG-tagged nLuc sequence and staggered element (2A sequence). Figure 36 As shown, circular RNAs produce higher levels of proteins with the correct molecular weight compared to linear RNAs.

[0567] After 24 hours, cells were harvested by adding 100 μl of RIPA buffer. After centrifugation at 1400 xg for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0568] After electrotransfer to a nitrocellulose membrane using dry transfer, the blot was incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blot was visualized with an ECL kit and Western blot band intensity was measured using ImageJ.

[0569] like Figure 36 As shown, circular RNA translation products were detected in cells. In particular, circular RNAs without a termination element (stop codon) produced higher levels of discrete protein products with the correct molecular weight than their linear RNA counterparts. Example 40: Preparation of circular RNA with a quasi-double-stranded helical structure

[0570] This example demonstrates a circular RNA with both quasi-duplex and helical structures.

[0571] Non-naturally occurring circular RNAs were engineered to adopt a quasi-double-stranded helical structure. Similar structures were shown to be involved in the condensation of naturally occurring circular RNAs, which have a uniquely long in vivo half-life (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).

[0572] In this example, circular RNA was designed to encode EMCV IRES, Nluc tagged as ORF with 3XFLAG and staggered sequence (EMCV 2A3XFLAG Nluc 2A no stop). To evaluate RNA secondary structure, a thermodynamic RNA structure prediction tool (RNAfold) (Vienna RNA) was used. In addition, RNA modeling algorithms were used to analyze RNA tertiary structure.

[0573] like Figure 37 and Figure 38 As shown, circular RNA is modeled as adopting a quasi-double-stranded helical structure. Example 41: Preparation of circular RNA having a quasi-helical structure linked to a repetitive sequence

[0574] This example demonstrates that circular RNA can be designed to have a quasi-helical structure connected to a repeat sequence.

[0575] Non-naturally occurring circular RNAs were engineered to adopt a quasi-helical structure connected to a repetitive sequence. Similar structures were shown to be involved in the condensation of naturally occurring circular RNAs, which have a uniquely long in vivo half-life (Griffin et al., 2014, Guedj et al., 2014).

[0576] In this example, a circular RNA was designed to encode EMCV IRES, Nluc, and a spacer region including a repeat sequence (SEQ ID NO: 26).To assess the tertiary structure of the RNA, an RNA modeling algorithm was used.

[0577] like Figure 39 As shown, circular RNA is modeled as adopting a quasi-helical structure. Example 42: Circular RNA is circular rather than concatenated

[0578] This example demonstrates circular RNA degradation by RNase H, where the resulting nucleic acid degradation products are consistent with both circular RNA and non-stranded RNA.

[0579] When incubated with ligase, the RNA is unable to react or form intramolecular or intermolecular bonds, resulting in circular (without free ends) or concatenated RNA, respectively. Treatment of each type of RNA with complementary DNA primers and RNase H (a nonspecific endonuclease that recognizes DNA / RNA duplexes) is expected to produce a unique number of degradation products of specific sizes depending on the starting RNA material.

[0580] As shown in the examples below, based on the amount and size of RNA produced by RNase H degradation, it was shown that the ligated RNA was circular rather than concatemerized.

[0581] Circular RNA and linear RNA containing EMCV T2A3XFLAG-Nluc P2A were generated.

[0582] To test the circularization state of RNA (1299 nt), 0.05 pmol / μl linear RNA or circular RNA was incubated with 0.25 U / μl RNase H (an endoribonuclease that digests DNA / RNA duplexes) and 0.3 pmol / μl oligomer against 1037-1046 nt RNA (CACCGCTCAGGACAATCCTT, SEQ ID NO: 55) at 37° C. for 20 minutes. After incubation, the reaction mixture was analyzed by 6% denaturing PAGE.

[0583] For the linear RNA used above, two cleavage products of 1041 nt and 258 nt are expected to be obtained after DNA primer binding and subsequent cleavage by RNase H. Concatemers are expected to produce three cleavage products of 258 nt, 1041 nt, and 1299 nt. Circularity is expected to produce a single cleavage product of 1299 nt.

[0584] Incubation of the linear RNA lane with RNase endonuclease yielded the expected two bands of 1041 nt and 258 nt, whereas a single band of 1299 nt was produced in the circular RNA lane (see Figure 40 ), indicating that circular RNA is actually circular rather than concatenated. Example 43: Preparation of large circRNA

[0585] This example demonstrates the generation of circular polyribonucleotides ranging from about 20 bases to about 6.2 kb.

[0586] Depending on the desired function, non-naturally occurring circular RNAs engineered to contain one or more desired properties are generated in a range of sizes. As shown in the examples below, linear RNAs up to 6200 nt were circularized.

[0587] Plasmid pCDNA3.1 / CAT (6.2 kb) was used herein. Primers were designed to anneal to pCDNA3.1 / CAT at regular intervals to generate DNA oligonucleotides corresponding to 500nt, 1000nt, 2000nt, 4000nt, 5000nt, and 6200nt. In vitro transcription was performed on the indicator DNA oligonucleotides to generate linear RNAs of corresponding sizes. Splint DNA was used to generate circular RNAs from these RNA oligonucleotides.

[0588] To measure the circularization efficiency of RNA, six linear RNAs of different sizes (500nt, 1000nt, 2000nt, 4000nt, 5000nt, and 6200nt) were generated. They were circularized using DNA splints and T4 DNA ligase 2. As a control, a reaction was performed in the absence of T4 RNA ligase. Half of the circularized samples were treated with RNase R to remove the linear RNA.

[0589] To monitor circularization efficiency, each sample was analyzed using qPCR. Figure 41 As shown in Figure 2, circular RNA is generated from DNA of various lengths. Figure 42 As shown, RNA circularization was confirmed using RNase R treatment and qPCR analysis of circular junctions. This example demonstrates the generation of circular RNAs of various lengths. Example 44: Circular RNA Engineered with Protein Binding Sites

[0590] This example demonstrates the generation of circular RNA with a protein binding site.

[0591] In this example, a circular RNA was designed to include CVB3IRES (SEQ ID NO:56) and an ORF encoding Gaussia luciferase (Gluc) (SEQ ID NO:57), followed by at least one protein binding site. For a specific example, the HuR binding sequence of the Sindbis virus 3'UTR (SEQ ID NO:52) was used to test the effect of protein binding on the immunogenicity of the circular RNA. The HuR binding sequence contains two elements, URE (U-rich element; SEQ ID NO:50) and CSE (conserved sequence element; SEQ ID NO:51). Circular RNAs without HuR binding sequence or with URE were used as controls. A portion of the autocatalytic intron and exon sequence of Anabaena is located before CVB3IRES (SEQ ID NO:56). Circular RNAs were generated in vitro as described. Figure 45 As shown, circular RNA containing the HuR binding site was generated.

[0592] In order to monitor the effect of RNA binding proteins on the immunogenicity of circular RNA, cells were plated into each well of a 96-well plate. After 1 day, 500 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). Translation efficiency / RNA stability / immunogenicity were monitored every day for up to 72 hours. Culture medium was harvested to monitor Gluc activity. Cell lysates for measuring RNA levels were prepared using a kit that can measure relative gene expression by real-time RT-PCR (Invitrogen).

[0593] Translation efficiency was monitored by measuring Gluc activity using the Gaussia Luciferase Rapid Assay Kit according to the manufacturer's instructions (Pierce).

[0594] For qRT-PCR analysis, cell lysate was used to prepare a kit to generate cDNA according to the manufacturer's instructions (Invitrogen). qRT-PCR analysis was performed in triplicate using PCR premix (Brilliant II SYBR Green qRT-PCR Master Mix) and PCR cycler (LightCycler 480). The stability of circular RNA was measured by primers for Nluc. The mRNA levels of well-known innate immune regulators (such as RIG-I, MDA5, OAS, OASL and PKR) were quantified and normalized to actin values. Example 45: Preparation of circular RNA with regulatory nucleic acid sites

[0595] This example demonstrates the in vitro generation of circular RNAs with regulatory RNA binding sites.

[0596] Different cell types possess unique nucleic acid regulatory mechanisms that target specific RNA sequences. Encoding these specific sequences within circular RNAs can confer unique properties in different cell types. As shown in the following example, circular RNAs were engineered to encode microRNA binding sites.

[0597] In this example, the circular RNA includes a sequence encoding the WT EMCV IRES, the mir692 microRNA binding site (GAGGUGCUCAAAGAGAU), and two spacer elements flanking the IRES-ORF.

[0598] Circular RNA is generated in vitro. Except for the T7 RNA polymerase promoter for driving transcription, unmodified linear RNA is transcribed in vitro from a DNA template comprising all the motifs listed above. The transcribed RNA is purified with an RNA purification kit (New England Biolabs, T2050), treated with RNA5'-phosphohydrolase (RppH) (New England Biolabs, M0356) according to the manufacturer's instructions, and purified again with an RNA purification column. The RNA processed by RppH is cyclized using splint DNA (GGCTATTCCCAATAGCCGTT) and T4 RNA ligase 2 (New England Biolabs, M0239). Circular RNA is 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.

[0599] like Figure 43 As shown, circular RNAs with miRNA binding sites were generated. Example 46: Self-splicing of circular RNA

[0600] This example demonstrates the ability to generate circular RNA by self-splicing.

[0601] For this example, the circular RNA includes a CVB3 IRES, an ORF encoding Gaussia luciferase (GLuc), and two spacer elements flanking the IRES-ORFs.

[0602] Circular RNA is generated in vitro. Unmodified linear RNA is transcribed in vitro from a DNA template containing all the motifs listed above. The in vitro transcription reaction includes 1 μg template DNA, T7 RNA polymerase promoter, 10X T7 reaction buffer, 7.5 mM ATP, 7.5 mM CTP, 7.5 mM GTP, 7.5 mM UTP, 10 mM DTT, 40 U RNase inhibitor, and T7 enzyme. Transcription is carried out at 37°C for 4 hours. The transcribed RNA is treated with 1 U of DNase I at 37°C for 15 minutes. To facilitate circularization by self-splicing, additional GTP is added to a final concentration of 2 mM and incubated at 55°C for 15 minutes. The RNA is then column purified and visualized by UREA-PAGE.

[0603] Figure 44 Circular RNA generated by self-splicing is shown. Example 47: Circular RNA with Splicing Elements Containing Encryptogens

[0604] This example demonstrates circular RNA engineered to have reduced immunogenicity.

[0605] For this example, the circular RNA includes a CVB3 IRES, an ORF encoding Gaussia luciferase (GLuc), and two spacer elements flanking the IRES-ORF, which contain a splice element that is a portion of the Anabaena autocatalytic intron and exon sequence (SEQ ID NO: 59).

[0606] Circular RNAs were generated in vitro.

[0607] In this example, the levels of innate immune response genes in cells were monitored by seeding cells into each well of a 12-well plate. One day later, 1 μg of linear RNA 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 the cells using a phenol-based extraction reagent (Invitrogen). The total RNA (500 ng) was reverse transcribed to generate cDNA. qRT-PCR analysis was performed using a dye-based quantitative PCR mix (Bio-Rad).

[0608] The qRT-PCR levels of immune-related genes in BJ cells transfected with circular RNAs containing a splice element were expected to show a decrease in RIG-I, MDA5, PKR, and IFN-β compared to linear RNA-transfected cells. Thus, the induction of immunogenicity-related genes in recipient cells was expected to be reduced in circular RNA-transfected cells compared to linear RNA-transfected cells. Example 48: Persistence of circular RNAs during cell division

[0609] This example demonstrates the persistence of circular polyribonucleotides during cell division. Non-naturally occurring circular RNAs engineered to contain one or more desired properties can persist in cells through cell division without being degraded. As shown in the following example, a circular RNA expressing Gaussia luciferase (GLuc) was monitored in HeLa cells over 72 hours.

[0610] In this example, the 1307 nt circular RNA includes a CVB3 IRES, an ORF encoding Gaussia luciferase (GLuc), and two spacer elements flanking the IRES-ORF.

[0611] Cyclic RNA persistence in cell division was monitored in HeLa cells. 96-well plates of 5000 cells / well were transfected with cell suspensions transfected with cyclic RNA. Bright cell imaging was performed in an Avos imager (Thermo Fisher Scientific) and cell counts were performed using luminescent cell viability assay (Promega) at 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours. Gaussia luciferase activity was monitored daily as a measure of protein expression by using Gaussia luciferase activity assay (Thermo Scientific Pierce) and gLuc expression was monitored daily in supernatant removed from the wells every 24 hours. 50 μΐ of IX Gluc substrate was added to 5 μΐ of plasma for Gluc luciferase activity assay. Plates were read immediately after mixing on a luminometer (Promega).

[0612] The protein expression level of the detected cyclic RNA was higher than linear RNA in cells undergoing division. Figure 46 At all measured time points, cells with cyclic RNA had a higher rate of cell division compared to linear RNA. This example demonstrates the increase in detection of cyclic RNA compared to its linear RNA counterpart during cell division. Example 49: Rolling circle translation generates multiple expressed sequences

[0613] This example demonstrates the ability of cyclic RNA to express multiple proteins from a single construct. Additionally, this example demonstrates rolling circle translation of a cyclic RNA encoding multiple ORFs. This example further demonstrates the expression of two proteins from a single construct.

[0614] A cyclic RNA (mtEMCV T2A3XFLAG-GFP F2A3XFLAG-Nluc P2A IS spacer) was designed for rolling circle translation to include an EMCV IRES (SEQ ID NO: 58) and an ORF encoding GFP with a 3XFLAG tag and an ORF encoding nanoluciferase (Nluc) with a 3XFLAG tag. Stagger elements (2A) flank the GFP and Nluc ORFs. Another cyclic RNA was similarly designed but included a triple stop codon between the Nluc ORF and the spacer. A portion of the Anabaena autocatalytic intron and exon sequence was included prior to the EMCV IRES. The cyclic RNA was generated in vitro as described.

[0615] Protein expression of circular RNAs was monitored in vitro or in cells. For in vitro analysis, circular RNAs were incubated in rabbit reticulocyte lysate (Promega, Fitchburg, WI, USA) at 30°C for 3 hours. The final composition of the reaction mixture included 70% rabbit reticulocyte lysate, 20 μM complete amino acids, and 0.8 U / μL RNase inhibitor (Toyobo, Osaka, Japan).

[0616] After incubation, hemoglobin was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 x g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 2x SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 x g for 5 minutes, the supernatant was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0617] For analysis in cells, cells were plated into each well of a 12-well plate to monitor the translation efficiency of circular RNA in the cells. One day later, 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 200 μl of RIPA buffer to each well. Next, 10 μg of cell lysate protein was analyzed on a 10%-20% gradient polyacrylamide / SDS gel.

[0618] After electroporation of samples from reticulocyte lysates and cells onto nitrocellulose membranes using dry transfer, the blots were incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. As a loading control, anti-β-tubulin antibody was used. Blots were visualized using an enhanced chemiluminescence (ECL) kit. Western blot band intensities were measured using ImageJ.

[0619] like Figure 47 As shown, circular RNAs encoding GFP and nLuc produce two protein products. Translation of the circular RNA without a triplet stop codon produces more of both protein products than does the circular RNA with a triplet stop codon. Finally, the circular RNAs with and without triplet stop codons expressed proteins at a ratio of 1:3.24 and 1:3.37, respectively. Example 50: Circular RNA Shows Lower Toxicity Than Linear RNA

[0620] This example demonstrates that circula...

Claims

1. A method for performing rolling circle translation on one or more expressed sequences, the method comprising Express one or more expressed sequences of cyclic polyribonucleotide by rolling circle translation, wherein this cyclic polyribonucleotide comprises these one or more expressed sequences, is positioned at the staggered element of at least one 3 ' end in these expressed sequences, and lacks termination element.

2. method as claimed in claim 1, wherein this staggered element is configured to make ribosome stagnation during the rolling circle translation of this cyclic polyribonucleotide.

3. The method of claim 1, wherein the staggered element encodes a sequence having a C-terminal consensus sequence of D(V / I)ExNPGP, where x = any amino acid.

4. method as claimed in claim 1, wherein expressing at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% discrete polypeptides in the total polypeptide (mole / mole) that generates during the rolling circle translation of these cyclic polyribonucleotide, and wherein each in these discrete polypeptides is all to be generated by single expressed sequence.

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

6. the method for claim 1, wherein this cyclic polyribonucleotide lacks internal ribosome entry site.

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

8. the method for claim 1, wherein this cyclic polyribonucleotide also comprises at least one structural element that is selected from following: (a) Encryption source; (b) regulatory elements; (c) replicating elements; and (d) Quasi-double-stranded secondary structure.

9. The method of claim 1, wherein the cyclic polyribonucleotide comprises at least one functional characteristic selected from the group consisting of: (i) higher translation efficiency than its linear counterpart; (ii) stoichiometric translation efficiency of various translation products; (iii) less immunogenic than its counterpart lacking the cryptogen; (iv) increased half-life compared to its linear counterpart; and (v) Persistence during cell division.

10. The method of claim 1, wherein expressing the one or more expression sequences produces an expression product that is at least 5-fold higher than its linear counterpart.

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