Compositions comprising cyclic polyribonucleotides and uses thereof

Abstract

To provide a composition containing a cyclic polyribonucleotide and its use.SOLUTION: The present invention relates generally to pharmaceutical compositions and preparations of cyclic polyribonucleotides and uses thereof. In one aspect, the present invention provides: At least one structural element selected from a) an encryptogen; b) a staggerelement; c) a regulatory element; d) a replication element; f) a pseudo-double-stranded secondary structure; and g) an expression sequence; and at least one functional property selected from a) higher translation efficiency than a linear equivalent; b) stoichiometric translation efficiency of multiple translation products; c) lower immunogenicity than an equivalent lacking an encryptogen; d) increased half-life over a linear equivalent; and e) persistence during cell division.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] cross reference

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

[0002]

[0002] Certain cyclic polyribonucleotides are ubiquitously present in human tissues and cells, including those of healthy individuals. Summary of the Invention [Means for solving the problem]

[0003] In one aspect, the present invention provides a compound comprising: a) an encryptogen; b) a s tagger element; c) regulatory element; d) replication element; f) pseudoduplex secondary structure. and g) at least one structural element selected from an expression sequence and at least one functional property selected from: a) higher translation efficiency than a linear equivalent; b) stoichiometric translation efficiency of multiple translation products; c) lower immunogenicity than an equivalent lacking the encryptogen; d) increased half-life than a linear equivalent; and e) persistence during cell division.

[0004] In some embodiments, the circular polyribonucleotide is translatable. In one such embodiment, the pseudohelical structure comprises at least one double-stranded RNA segment along with at least one non-double-stranded segment. In another such embodiment, the pseudohelical structure comprises a first sequence and a second sequence linked by a repeating sequence, e.g., an A-rich sequence.

[0005]

[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 avoid an immune response, such as a CTL response.

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

[0007] In one embodiment, the cyclic polyribonucleotide has at least two-fold less immunogenicity than a counterpart lacking the encryptogen, as assessed, for example, by expression, signaling, or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, or IFN-β.

[0007]

[0008] In certain embodiments, the cyclic polyribonucleotide further comprises a riboswitch.

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

[0008]

[0010] In certain embodiments, the cyclic polyribonucleotide comprises a translation initiation sequence, eg, a GUG, CUG initiation codon, for expression under, eg, stress conditions.

[0011] In some embodiments, the cyclic polyribonucleotide comprises at least one expressed sequence, e.g., encoding a polypeptide. In one such embodiment, the expressed sequence encodes a peptide or polynucleotide. In some embodiments, the cyclic polyribonucleotide comprises multiple expressed sequences, which may be the same or different.

[0009]

[0012] In certain embodiments, the cyclic polyribonucleotide comprises a stagger element, eg, 2A.

[0013] In some embodiments, the cyclic polyribonucleotide comprises a regulatory nucleic acid, e.g., a non-coding RNA. In some embodiments, the cyclic polyribonucleotide comprises a regulatory element that alters expression of, e.g., an expression sequence.

[0010]

[0014] In one embodiment, the cyclic polyribonucleotide has a size ranging from about 20 bases to about 20 kb.

[0015] In certain embodiments, the circular polyribonucleotide is synthesized by circularization of a linear polynucleotide.

[0011]

[0016] In certain embodiments, the cyclic polyribonucleotide is substantially resistant to degradation, eg, exonucleases.

[0017] In one embodiment, the circular polyribonucleotide lacks at least one of: a) a 5'-UTR; b) a 3'-UTR; c) a poly-A sequence; d) a 5'-cap; e) a termination element; f) an internal ribosome entry site; g) susceptibility to exonuclease degradation; and h) binding to a cap-binding protein.

[0012]

[0018] In one aspect, the invention includes a method of making a composition comprising a cyclic polyribonucleotide described herein.

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

[0013]

[0020] In some embodiments, each of the one or more expressed sequences is separated from the subsequent expressed sequence by a stagger element in the circular polyribonucleotide, and rolling circle translation of the one or more expressed sequences produces at least two polypeptide molecules, e.g., the stagger element stalls or stalls the ribosome as the elongated polypeptide falls off the ribosome. In some embodiments, the stagger element prevents (a) two translations of a single expressed sequence, or (b) one or more translations of two or more expressed sequences from producing a single polypeptide. For example, the stagger element can prevent two or more translations of two or more expressed sequences from producing a single polypeptide, e.g., the stagger element stalls the ribosome and / or allows the elongated polypeptide to fall off the ribosome after completing one full rotation around the circular polyribonucleotide.

[0014]

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

[0022] In certain embodiments, the stagger element comprises a portion of one or more expressed sequences.

[0015]

[0023] In one aspect, the invention comprises a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide comprising one or more expressed sequences and capable of rolling circle translation, wherein 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 polypeptides produced during rolling circle translation of the circular polyribonucleotide are distinct polypeptides, each of the distinct polypeptides being produced from a single translation or less than a single translation of the one or more expressed sequences.

[0016]

[0024] In one embodiment, 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 polypeptides (mol / mol) produced during rolling circle translation of the circular polyribonucleotide are distinct polypeptides, and the quantitative ratio of the distinct products across all polypeptides is tested in an in vitro translation system.

[0017]

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

[0026] In one embodiment, the stagger element is downstream or 3'-terminal to at least one of the one or more expressed sequences, and the stagger element is configured to stall ribosomes during rolling circle translation of the circular polyribonucleotide.

[0018]

[0207] In one aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and one or more and a cyclic polyribonucleotide comprising a stagger element downstream or 3'-terminal of at least one of the one or more expressed sequences. In one embodiment, the stagger element is configured to stall ribosomes during rolling circle translation of the cyclic polyribonucleotide.

[0019]

[0028] In one embodiment, the stagger element encodes a peptide sequence selected from the group consisting of a 2A sequence and a 2A-like sequence.

[0029] In one embodiment, the stagger element encodes a sequence having a C-terminal sequence that is a GP.

[0020]

[0030] In one embodiment, the stagger element encodes a sequence with a C-terminal consensus sequence of D(V / I)ExNPG P, where x=any amino acid.

[0021]

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

[0022]

[0032] In one embodiment, the stagger element is downstream or 3' of each of one or more expression sequences.

[0033] In one embodiment, the stagger element of the first expressed sequence in the circular polyribonucleotide is downstream (5' end) of the first translation initiation sequence of the expressed sequence following the first expressed sequence in the circular polyribonucleotide, and the distance between the stagger element and the first translation initiation sequence allows for continuous translation of the first expressed sequence and the subsequent expressed sequence. In one embodiment, the stagger element comprises a termination element of the first expressed sequence in the circular polyribonucleotide that is located upstream (5' end) from the translation initiation sequence of the expressed sequence following the first expressed sequence in the circular polyribonucleotide, and this distance allows for continuous translation of the first expressed sequence and the subsequent expressed sequence.

[0023]

[0034] In one embodiment, a first stagger element is located upstream (5' end) of a first translation start sequence of a first expressed sequence in a continuously translated circular polyribonucleotide, and the corresponding circular polyribonucleotide comprises a second stagger element located upstream of a second translation start sequence of a second expressed sequence in a corresponding non-continuously translated circular polyribonucleotide, the second stagger element in the corresponding circular polyribonucleotide being located at a distance from the second translation start sequence that is, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold greater than the distance between the first stagger element and the first translation start in the circular polyribonucleotide. In one embodiment, the stagger element comprises a first termination element upstream (5' end) of a first translation start sequence of a first expressed sequence in the continuously translated circular polyribonucleotide, and the corresponding circular polyribonucleotide comprises a stagger element comprising a second termination element upstream from a second translation start sequence of a second expressed sequence in a corresponding circular polyribonucleotide that is not continuously translated, and the second termination element in the corresponding circular polyribonucleotide is located at a distance from the second translation start sequence that is, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold greater than the distance between the first termination element and the first translation start in the circular polyribonucleotide.

[0024]

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

[0025]

[0036] In certain embodiments, the circular polyribonucleotide comprises two or more expression sequences.

[0037] In certain embodiments, cyclic polyribonucleotides have 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-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater than their linear counterparts.

[0026]

[0038] In certain embodiments, cyclic polyribonucleotides have at least 5-fold higher translation efficiency than their linear counterparts.

[0039] In certain embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site.

[0027]

[0040] In certain embodiments, one or more expression sequences include a Kozak initiation sequence.

[0041] In certain embodiments, one or more of the expressed sequences encode a peptide.

[0042] In some embodiments, the cyclic polyribonucleotide comprises a regulatory nucleic acid, e.g., a non-coding RNA. In some embodiments, the cyclic polyribonucleotide comprises a regulatory element that alters expression of, e.g., an expression sequence.

[0028]

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

[0044] In one aspect, the invention includes a method of making the pharmaceutical compositions provided herein, comprising combining a cyclic polyribonucleotide described herein and a pharmaceutically acceptable carrier or excipient described herein.

[0029]

[0045] In one aspect, the invention includes methods of administering compositions comprising the cyclic polyribonucleotides described herein.

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

[0030]

[0047] In some embodiments, translation of at least a region of the circular polyribonucleotide is performed in vitro. In some embodiments, translation of at least a region of the circular polyribonucleotide is performed in vivo.

[0031]

[0048] In one aspect, the invention includes polynucleotides, eg, DNA vectors, that encode the circular polyribonucleotides provided herein.

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

[0032]

[0050] In certain embodiments, the method comprises splint ligation-mediated circularization of a linear polyribonucleotide.

[0051] In some embodiments, circularization, e.g., splint ligation-mediated circularization, 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, splint ligation-mediated circularization has an efficiency of about 40% to about 50%, or greater than 50%. 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. Terms used hereinafter generally should be understood in their ordinary sense unless otherwise indicated.

[0033]

[0053] Terms such as "obtainable by," "producible by," and the like are used to indicate that a claim or embodiment refers to the compound, composition, product, etc. per se, i.e., the compound, composition, product, etc. is obtainable or producible by a method described for making the compound, composition, product, etc., but that the compound, composition, product, etc. may also be obtained or produced by methods other than the method described. Terms such as "obtained by," "produced by," and the like indicate that the compound, composition, product is obtainable or produced by the specific method described. It should be understood that terms such as "obtainable by," "producible by," and the like also disclose terms such as "obtained by," "produced by," and the like, as preferred embodiments of "obtainable by," "producible by," and the like.

[0034]

[0054] It should be understood that the phrase "compound, composition, product, etc. for treatment, regulation, etc." refers to a compound, composition, product, etc. per se that is suitable for the indicated purpose of treatment, regulation, etc. The phrase "compound, composition, product, etc. for treatment, regulation, etc." further discloses that, as a preferred embodiment, such compound, composition, product, etc. is for use in treatment, regulation, etc.

[0035]

[0055] The phrases "a compound, composition, product, etc. for use in" or "the use of a compound, composition, product, etc. in the manufacture of a medicament, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for" indicate that such a compound, composition, product, etc. is for use in a therapeutic method that can be practiced on the human or animal body. They are considered to be equivalent disclosures of embodiments and claims relating to methods of treatment, etc. When an embodiment or claim thus refers to a "compound for use in treating a human or animal suspected of suffering from a disease," this is also considered to be a disclosure of "the use of a compound in the manufacture of a medicament for treating a human or animal suspected of suffering from a disease" or "a method of treatment by administering a compound to a human or animal suspected of suffering from a disease." The phrase "a compound, composition, product, etc. for treatment, modulation, etc." should be understood to refer to a compound, composition, product, etc. per se that is suitable for the indicated purpose of treatment, modulation, etc.

[0036]

[0056] The term "pharmaceutical composition" is also intended to disclose that the cyclic polyribonucleotide contained within the pharmaceutical composition can be used for therapeutic treatment of the human or animal body, and is therefore intended to be equivalent to "cyclic polyribonucleotide for use in therapy."

[0037]

[0057] The cyclic polyribonucleotides, compositions comprising such cyclic polyribonucleotides, methods of using such cyclic polyribonucleotides, etc. described herein are based in part on examples that demonstrate how cyclic polyribonucleotide effectors comprising different elements, such as a replication element, an expression sequence, a stagger element, and an encryptogen (see, e.g., Example 1), or an expression sequence, a stagger element, and a regulatory element (see, e.g., Examples 30 and 38), can be used to achieve different technical effects (e.g., increased translation efficiency over linear equivalents in Examples 1 and 38 and increased half-life over linear equivalents in Example 38). It is particularly based on these examples that the following description contemplates various variations of the specific findings and combinations contemplated in the examples.

[0038]

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

[0039]

[0059] As used herein, the term "encryptogen" may refer to a nucleic acid sequence or structure of a cyclic polyribonucleotide that serves to reduce, avoid, and / or evade detection by immune cells and / or reduce the induction of an immune response against the cyclic polyribonucleotide.

[0040]

[0060] As used herein, the term "expressed sequence" can refer to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide, or a regulatory nucleic acid. An exemplary expressed sequence that encodes a peptide or polypeptide can include multiple nucleotide triplets, each of which can encode an amino acid, and are referred to as a "codon."

[0041]

[0061] As used herein, the term "immunity protein binding site" may refer to a nucleotide sequence that binds to an immunity protein. In certain embodiments, the immunity protein binding site serves to mask the cyclic polyribonucleotide as being exogenous; for example, the immunity protein binding site may be bound by a protein (e.g., a competitive inhibitor) that prevents the cyclic polyribonucleotide from being recognized and bound by the immunity protein, thereby reducing or avoiding an immune response to the cyclic polyribonucleotide. As used herein, the term "immunity protein" may refer to, for example, any protein or peptide involved in an immune response to an immunogen, such as a cyclic polyribonucleotide. Non-limiting examples of immunity proteins include T cell receptors (TCRs), antibodies (immunoglobulins), major histocompatibility complex (MHC) proteins, complement proteins, and RNA-binding proteins.

[0042]

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

[0043]

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

[0044]

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

[0045]

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

[0066] As used herein, the term "repeated nucleotide sequence" can refer to repeated nucleic acid sequences within a stretch of DNA or RNA or throughout the genome. In some embodiments, the repetitive nucleotide sequence comprises a poly-CA or poly-TG (UG) sequence. In some embodiments, the repetitive nucleotide sequence comprises a repeated sequence in the Alu family of introns.

[0046]

[0067] As used herein, the term "replication element" may refer to sequences and / or motifs that are useful for replication or that initiate transcription of a circular polyribonucleotide.

[0047]

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

[0048]

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

[0049]

[0070] As used herein, the term " stoichiometric translation " can refer to the substantially equal production of expression products translated from cyclic polyribonucleotide.For example, in a cyclic polyribonucleotide having two expression sequences, the stoichiometric translation of cyclic polyribonucleotide can mean that the expression products of the two expression sequences can have substantially equal amounts, for example, the difference in amount (e.g., molar difference) between the two expression sequences can be about 0 or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%.

[0050]

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

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

[0051]

[0073] As used herein, the term "translation efficiency" can refer to the rate or amount of protein or peptide production from a ribonucleotide transcript. In certain embodiments, translation efficiency can be expressed as the amount of protein or peptide produced per given amount of transcript encoding the protein or peptide, for example, in a given translation system, for example, in an in vitro translation system such as a rabbit reticulocyte lysate or in an in vivo translation system such as a eukaryotic or prokaryotic cell, for example, over a given period of time.

[0052]

[0074] As used herein, the term "circularization efficiency" may refer to a measurement of the resulting circular polyribonucleotide compared to its starting material.

[0075] As used herein, the term "immunogenic" may refer to the potential for inducing an immune response against a substance. In some embodiments, an immune response can be induced when the immune system of an organism or a type of immune cell is exposed to an immunogenic substance. The term "non-immunogenic" may refer to the lack or absence of an immune response above a detectable threshold against a substance. In some embodiments, no immune response is detected when the immune system of an organism or a type of immune cell is exposed to a non-immunogenic substance. In some embodiments, the non-immunogenic cyclic polyribonucleotides provided herein do not induce an immune response above a predetermined threshold as measured by an immunogenicity assay. For example, when measuring antibodies elicited against a cyclic polyribonucleotide or an inflammatory marker using an immunogenicity assay, the non-immunogenic polyribonucleotides provided herein may result in the production of antibodies or markers at levels below the predetermined threshold. The predetermined threshold may be, for example, 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold or less of the level of antibodies or markers elicited by a control reference.

[0053] Incorporation 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.

[0054]

[0077] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments illustrated herein. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0055] [Figure 1]

[0078] FIG. 1 shows various exemplary circularization methods. [Figure 2]

[0079] FIG. 2 shows a schematic diagram of an exemplary in vitro generation process of circular RNA containing a start codon, an ORF (open reading frame) encoding GFP, a stagger element (2A), an encryptogen, and an IRES (internal ribosome entry site). [Figure 3]

[0080] FIG. 3 shows a schematic diagram of an exemplary in vivo generation process of circular RNA. [Figure 4]

[0081] FIG. 4 shows an exemplary circular RNA design containing a start codon, an ORF encoding GFP, a stagger element (2A), and an encryptogen. [Figure 5]

[0082] 5A and 5B are schematic diagrams showing the in vivo stoichiometric protein expression of two different circular RNAs. [Figure 6]

[0083] FIG. 6 shows a schematic diagram of a control circular RNA that has an intron and expresses GFP. [Figure 7]

[0084] FIG. 7 shows a schematic of an exemplary circular RNA with a synthetic riboswitch (red) that regulates the expression of GFP from the circular RNA in the presence or absence of a ligand for the riboswitch. [Figure 8]

[0085] FIG. 8 is a schematic diagram showing in vivo protein expression in a mouse model from exemplary circular RNAs. [Figure 9]

[0086] FIG. 9 is a schematic diagram showing the in vivo biodistribution of exemplary circular RNAs in a mouse model. [Figure 10]

[0087] FIG. 10 is a schematic diagram showing in vivo protein expression in a mouse model from an exemplary circular RNA with an encryptogen (intron). [Figure 11]

[0088] FIG. 11 shows a schematic diagram of an exemplary circular RNA with one double-stranded RNA segment that can be subjected to dot blot analysis for its structural information. [Figure 12]

[0089] FIG. 12 shows a schematic diagram of an exemplary circular RNA with a pseudohelical structure (HDVmin) that can be subjected to SHAPE analysis for its structural information. [Figure 13]

[0090] FIG. 13 shows a schematic diagram of an exemplary circular RNA with a functional pseudohelical structure (HDVmin) that exhibits HDAg binding activity. [Figure 14]

[0091] FIG. 14 is a schematic diagram showing transcription, self-cleavage, and ligation of an exemplary self-replicating circular RNA. [Figure 15]

[0092] FIG. 15 shows a schematic diagram of an exemplary circular RNA expressed in vivo and having improved in vivo stability. [Figure 16]

[0093] Figure 16 shows a schematic diagram of an exemplary circular RNA that is stored during mitosis and persists in daughter cells. A BrdU pulse is shown used to label dividing cells. [Figure 17]

[0094] FIG. 17 is an image of a denaturing PAGE gel showing the in vitro generation of various exemplary circular RNAs. [Figure 18]

[0095] FIG. 18 is a graph summarizing the circularization efficiency of various exemplary circular RNAs. [Figure 19]

[0096] FIG. 19 is a denaturing PAGE gel image showing the reduced susceptibility of an exemplary circular RNA to degradation compared to its linear counterpart. [Figure 20]

[0097] FIG. 20 is a denaturing PAGE gel image showing an exemplary circular RNA after an exemplary purification process. [Figure 21]

[0098] FIG. 21 is a Western blot image showing expression of Flag protein (approximately 15 kDa) by an exemplary circular RNA lacking an IRES, cap, 5′ and 3′ UTRs. [Figure 22]

[0099] FIG. 22 is a Western blot image showing rolling circle translation of an exemplary circular RNA. [Figure 23]

[0100] FIG. 23 shows Western blot images demonstrating the production of distinct proteins or continuous long peptides from various exemplary circular RNAs with and without exemplary stagger elements. [Figure 24]

[0101] FIG. 24A is a Western blot image showing a comparison of protein expression between various exemplary circular RNAs with exemplary stagger or termination elements (stop codons).

[0102] FIG. 24B is a graph summarizing signal intensities from Western blot analysis of protein products translated from two exemplary circular RNAs. [Figure 25]

[0103] FIG. 25 is a graph summarizing the luciferase activity of the translation products of exemplary circular RNAs and their linear equivalents compared to vehicle control RNA. [Figure 26]

[0104] FIG. 26 is a graph summarizing the amount of RNA at different collection time points in a time course experiment examining the half-life of an exemplary circular RNA. [Figure 27]

[0105] FIG. 27A is a graph showing qRT-PCR analysis of linear and circular RNA levels 24 hours after delivery into cells using primers that captured both linear and circular RNA.

[0106] Figure 27B is a graph showing qRT-PCR analysis of linear and circular RNA levels using primers specific for circular RNA. [Figure 28]

[0107] FIG. 28 is an image showing blots of cell lysates from circular and linear RNA probed for EGF protein and β-tubulin loading control. [Figure 29]

[0108] FIG. 29 is a graph showing qRT-PCR analysis of immune-related genes from 293T cells transfected with circular or linear RNA. [Figure 30]

[0109] FIG. 30 is a graph showing the luciferase activity of proteins expressed from circular RNA by rolling circle translation. [Figure 31]

[0110] FIG. 31 is a graph showing the luciferase activity of proteins expressed from circular or linear RNA. [Figure 32]

[0111] FIG. 32 is a graph showing the luciferase activity of proteins expressed from linear or circular RNA by rolling circle translation. [Figure 33]

[0112] FIG. 33 is a graph showing luciferase activity of proteins expressed from circular RNAs via IRES translation initiation. [Figure 34]

[0113] FIG. 34 is a graph showing luciferase activity of proteins expressed from circular RNAs by IRES-initiated and rolling circle translation. [Figure 35]

[0114] FIG. 35 is an image showing a protein blot of expression products from circular or linear RNA. [Figure 36]

[0115] FIG. 36 is an image showing a protein blot of expression products from circular or linear RNA. [Figure 37]

[0116] FIG. 37 shows the predicted structure of an exemplary circular RNA with a pseudo-double-stranded structure. [Figure 38]

[0117] FIG. 38 shows the predicted structure of an exemplary circular RNA with pseudohelical structure. [Figure 39]

[0118] FIG. 39 shows the predicted structure of an exemplary circular RNA with a pseudohelical structure linked to repeat sequences. [Figure 40]

[0119] FIG. 40 shows experimental data showing that degradation of an exemplary circular RNA by RNAse H produced nucleic acid degradation products consistent with circular RNA but not concatemeric RNA. [Figure 41]

[0120] FIG. 41 shows electrophoretic images of DNA of various lengths generated for the formation of various RNA lengths. [Figure 42]

[0121] FIG. 42 shows experimental data confirming RNA circularization using RNAse R treatment and qPCR analysis for circular linkages of various lengths. [Figure 43]

[0122] Figure 43 shows the generation of an exemplary circular RNA with a miRNA binding site. [Figure 44]

[0123] FIG. 44 shows the generation of an exemplary circular RNA by self-splicing. [Figure 45]

[0124] FIG. 45 shows the generation of an exemplary circular RNA with a protein binding site. [Figure 46]

[0125] FIG. 46 shows experimental data demonstrating the higher stability of circular RNA in dividing cells compared to linear controls. [Figure 47]

[0126] FIG. 47 shows experimental data demonstrating protein expression from and rolling circle translation of exemplary circular RNAs with multiple expression sequences. [Figure 48]

[0127] FIG. 48 shows experimental data demonstrating the reduced toxicity of exemplary circular RNAs to transfected cells compared to linear controls. [Figure 49]

[0128] FIG. 49 shows that exemplary circular RNAs were translated to higher levels compared to linear RNAs under stress conditions. [Figure 50]

[0129] Figure 50 shows the production of a circular RNA with a riboswitch. [Figure 51A]

[0130] FIG. 51A shows that the modified circular RNA was translated intracellularly. [Figure 51B] FIG. 51B shows that the modified circular RNA was translated intracellularly. [Figure 51C]Figure 51C shows that the modified circular RNA was translated intracellularly. [Figure 52A]

[0131] Figure 52A shows that the modified circular RNAs have reduced immunogenicity compared to unmodified circular RNAs to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Figure 52B] Figure 52B shows that the modified circular RNAs have reduced immunogenicity compared to unmodified circular RNAs to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Figure 52C] Figure 52C shows that the modified circular RNAs have reduced immunogenicity compared to unmodified circular RNAs to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Figure 53]

[0132] FIG. 53 shows that after injection into mice, circular RNA was detected at higher levels than linear RNA in the liver of mice at 3, 4 and 7 days after injection. [Figure 54A]

[0133] Figure 54A shows that after injection of circular or linear RNA expressing Gaussia luciferase into mice, Gaussia luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23 days after administration of circular RNA, while the activity was detected in plasma only at 1 and 2 days after administration of modified linear RNA. [Figure 54B] Figure 54B shows that after injection of circular or linear RNA expressing Gaussia luciferase into mice, Gaussia luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23 days after administration of circular RNA, while the activity was detected in plasma only at 1 and 2 days after administration of modified linear RNA. [Figure 55]

[0134] FIG. 55 shows that after injection of RNA, circular RNA, but not linear RNA, was detected in the liver and spleen 16 days after administration of RNA. [Figure 56]

[0135] FIG. 56 shows that after injection of RNA, linear RNA, but not circular RNA, was immunogenic as assessed by RIG-I, MDA-5, IFN-B, and OAS. DETAILED DESCRIPTION OF THE INVENTION

[0056]

[0136] The present invention relates generally to cyclic polyribonucleotide pharmaceutical compositions and preparations and uses thereof.

[0057] cyclic polyribonucleotide

[0137] In some aspects, the invention described herein includes compositions and methods for using and making cyclic polyribonucleotides, as well as delivery of cyclic polyribonucleotides. In some embodiments, cyclic polyribonucleotides are non-immunogenic in mammals, such as humans. In some embodiments, cyclic polyribonucleotides can replicate or replicate in cells derived from aquaculture animals (such as fish, crabs, shrimp, oysters, etc.), mammalian cells, such as cells derived from pet or zoo animals (such as cats, dogs, lizards, birds, lions, tigers, and bears), cells derived from livestock or working animals (such as horses, cows, pigs, and chickens), 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 certain embodiments, the invention includes cells comprising the cyclic polyribonucleotides described herein, wherein the cells are cells derived from aquaculture animals (such as fish, crabs, shrimp, oysters, etc.), mammalian cells, such as cells derived from pet or zoo animals (such as cats, dogs, lizards, birds, lions, tigers, and bears), cells derived from livestock or working animals (such as horses, cows, pigs, and chickens), 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 certain embodiments, the cells are modified to comprise cyclic polyribonucleotides.

[0058]

[0138] In certain embodiments, the cyclic polyribonucleotide comprises a sequence or an expression product.

[0139] In some embodiments, the cyclic polyribonucleotide has a half-life at least that of its linear counterpart, e.g., a linear expression sequence or a linear cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide has an increased half-life over that of its linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more. In certain embodiments, the cyclic polyribonucleotide has a half-life or persistence within cells of 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 in between. In certain embodiments, the cyclic polyribonucleotide has a half-life or persistence within the cell of about 10 minutes or less to about 7 days, or 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 less, or any time in between. In some embodiments, the cyclic polyribonucleotide has a half-life or persistence within the cell while the cell is dividing. In some embodiments, the cyclic polyribonucleotide has a half-life or persistence within the cell after division.In certain embodiments, the cyclic polyribonucleotide has a half-life or persistence in dividing cells of greater than about 10 minutes to about 30 days, or 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 in between.

[0059]

[0140] In certain embodiments, the cyclic polyribonucleotide modulates a cellular function, e.g., transiently or long-term. In certain embodiments, the cellular function, e.g., modulation, is stably altered, lasting 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 in between. In certain embodiments, the cellular function, such as regulation, is temporarily altered, for example, lasting from about 30 minutes or less to about 7 days, or 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 less, or any time in between.

[0060]

[0141] In certain embodiments, the cyclic polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments, the cyclic polyribonucleotide may be of a size sufficient to accommodate a binding site for a ribosome. Those skilled in the art will understand that the maximum size of a cyclic polyribonucleotide may be somewhat large within the technical constraints of generating and / or using the cyclic polyribonucleotide. Without being bound by theory, it is possible that multiple segments of RNA may be generated from DNA and their 5' and 3' free ends annealed to generate a "string" of RNA (which may ultimately be circularized when only one 5' free end and one 3' free end remain). In some embodiments, the maximum size of a cyclic polyribonucleotide may be limited by the ability to package and deliver RNA to a target.In certain embodiments, the size of the circular polyribonucleotide is sufficient to encode a useful polypeptide, and therefore lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, 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, or at least 100 nucleotides may be useful.

[0061]

[0142] In some embodiments, the cyclic polyribonucleotide comprises one or more elements described elsewhere herein. In some embodiments, the elements may be separated from one another by a spacer sequence or linker. In some embodiments, the elements may be separated from one another by 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000 nucleotides, or any amount of nucleotides therebetween. In some embodiments, one or more elements are contiguous with one another, e.g., lacking a spacer element. In some embodiments, one or more elements in the cyclic polyribonucleotide are conformationally flexible. In some embodiments, the conformational flexibility is due to the sequence being substantially free of secondary structure. In some embodiments, the cyclic polyribonucleotide comprises a secondary or tertiary structure corresponding to one or more desired functions or properties described herein, e.g., a binding site for a ribosome, e.g., translation, e.g., rolling circle translation.

[0062]

[0143] In some embodiments, a cyclic polyribonucleotide comprises specific sequence characteristics. For example, a cyclic polyribonucleotide may comprise a specific nucleotide composition. In some such embodiments, a cyclic polyribonucleotide may comprise one or more purine-rich regions (adenine or guanosine). In some such embodiments, a cyclic polyribonucleotide may comprise one or more purine-rich regions (adenine or guanosine). In some embodiments, a cyclic polyribonucleotide may comprise one or more AU-rich regions or elements (AREs). In some embodiments, a cyclic polyribonucleotide may comprise one or more adenine-rich regions.

[0063]

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

[0145] In certain embodiments, the cyclic polyribonucleotide comprises one or more of the modifications described elsewhere herein.

[0064]

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

[0065] Expression sequence peptide or polypeptide

[0147] In some embodiments, the cyclic polyribonucleotide comprises at least one expressed sequence encoding a peptide or polypeptide. Such peptides include, but are not limited to, small peptides, peptidomimetics (e.g., peptoids), amino acids, and amino acid analogs. The peptides can be linear or branched. Such peptides can have a molecular weight of less than about 5,000 g per mole, less than about 2,000 g per mole, less than about 1,000 g per mole, or less than about 500 g per mole, as well as salts, esters, and other pharmaceutically acceptable forms of such compounds. Such peptides include, but are not limited to, neurotransmitters, hormones, drugs, toxins, viral or microbial particles, synthetic molecules, and their agonists or antagonists.

[0066]

[0148] The polypeptide may be linear or branched, and may have a length of about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, or about 1,000 to about 2,500 amino acids, or any range therebetween. In certain embodiments, polypeptides having a length of less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or less may be useful.

[0067]

[0149] Some examples of peptides or polypeptides include, but are not limited to, fluorescent tags or markers, antigens, peptide therapeutics, synthetic or analog peptides derived from naturally occurring bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degradative or self-immolative peptides, and multiple degradative or self-immolative peptides. Peptides useful in the invention described herein also include antigen-binding peptides, such as antigen-binding antibodies or antibody-like fragments, such as single-chain antibodies, nanobodies (e.g., Steeland, (See, e.g., J. 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, nuclear, or intraorganellar antigens.

[0068]

[0150] In some embodiments, the circular polyribonucleotide comprises one or more RNA expression sequences, each of which can encode a polypeptide. The polypeptide can be produced in significant quantities. Thus, the polypeptide can be any protein molecule that can be produced. The polypeptide can be a polypeptide that is secreted from the cell or that is localized in the cytoplasm, nucleus, or membrane compartment 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., that regulates lipid or steroid production), an antigen, a tolerogen, a cytokine, a toxin, an enzyme whose absence is associated with disease, and a polypeptide that is not active in an animal (e.g., in the animal's intestine) until cleaved, and a hormone.

[0069]

[0151] In some embodiments, the cyclic polyribonucleotide comprises an expression sequence encoding a protein, such as a therapeutic protein. In some embodiments, the therapeutic protein that can be expressed from the cyclic polyribonucleotides disclosed herein has antioxidant activity, binding activity, cargo receptor activity, catalytic activity, molecular carrier activity, molecular function regulator activity, molecular transducer activity, nutrient retention activity, protein tag activity, structural molecule activity, toxin activity, transcriptional regulation activity, translational regulation activity, or transport activity. Some examples of therapeutic proteins include, but are not limited to, enzyme replacement proteins, replacement proteins, protein vaccinations, antigens (e.g., tumor antigens, viruses, bacteria), hormones, cytokines, antibodies, immunotherapy (e.g., cancer), reprogramming / transdifferentiation factors, transcription factors, chimeric antigen receptors, transposases or nucleases, immune effectors (e.g., affecting sensitivity to immune responses / signals), regulated death effector proteins (e.g., inducers of apoptosis or necrosis), non-lytic inhibitors of tumors (e.g., inhibitors of oncoproteins), epigenetic modifiers, epigenetic enzymes, transcription factors, DNA or protein modifying enzymes, DNA intercalators, efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors, competitive inhibitors for enzymes, protein synthesis effectors or inhibitors, nucleases, protein fragments or domains, ligands or receptors, and CRISPR systems or components thereof.

[0070]

[0152] In certain embodiments, exemplary proteins that can be expressed from the cyclic polyribonucleotides disclosed herein include human proteins, such as receptor-binding proteins, hormones, growth factors, growth factor receptor modulators, and regenerative proteins (e.g., proteins involved in proliferation and differentiation, e.g., therapeutic proteins for wound healing). In certain embodiments, exemplary proteins that can be expressed from the cyclic polyribonucleotides disclosed herein include EGF (epidermal growth factor). In certain embodiments, exemplary proteins that can be expressed from the cyclic polyribonucleotides disclosed herein include enzymes, such as oxidoreductase enzymes, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, and desaturases. In certain embodiments, exemplary proteins that can be expressed from the cyclic polyribonucleotides disclosed herein include intracellular or cytoplasmic proteins. In certain embodiments, the cyclic polyribonucleotides express NanoLuc® luciferase (nLuc). In certain embodiments, exemplary proteins that can be expressed from the cyclic polyribonucleotides disclosed herein include secreted proteins, such as secreted enzymes. In some cases, the cyclic polyribonucleotide expresses a secreted protein that may have a therapeutically effective short half-life in the blood, or may be a protein with an intracellular localization signal or a protein with a secretory signal peptide. In some embodiments, the cyclic polyribonucleotide expresses Gaussia luciferase (gLuc). In some cases, the cyclic polyribonucleotide expresses a non-human protein, such as a fluorescent protein, an energy transfer receptor, or a protein tag such as Flag, Myc, or His. In some embodiments, an exemplary protein that can be expressed from the cyclic polyribonucleotide is GFP.In certain embodiments, the cyclic polyribonucleotide is a tagged protein, e.g., a fusion protein or engineered protein containing a protein tag, such as 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); Softag 1 (SLAELLNAGLGGS); Softag 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).

[0071]

[0153] In some embodiments, the cyclic polyribonucleotide expresses an antibody, such as an antibody fragment or portion thereof. In some embodiments, the antibody expressed by the cyclic polyribonucleotide can be of any isotype, such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, the cyclic polyribonucleotide expresses a portion of an antibody, such as a light chain, a heavy chain, an Fc fragment, a CDR (complementarity-determining region), an Fv fragment, or a Fab fragment, or further portion thereof. In some embodiments, the cyclic polyribonucleotide expresses one or more portions of an antibody. For example, the cyclic polyribonucleotide can contain two or more expression sequences, each expressing a portion of an antibody, and the collection of these can constitute an antibody. In some cases, the cyclic polyribonucleotide contains one expression sequence encoding the heavy chain of the antibody and another expression sequence encoding the light chain of the antibody. In some cases, when the cyclic polyribonucleotide is expressed in a cell or in a cell-free environment, the light and heavy chains can be subjected to appropriate modification, folding, or other post-translational modification to form a functional antibody.

[0072] Adjustment element

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

[0073]

[0155] A regulatory element can include a sequence located adjacent to an expression sequence that encodes an expression product. The regulatory element can be operably linked to the adjacent sequence. The regulatory element can increase the amount of the expressed product compared to the amount of the expressed product in the absence of the regulatory element. Furthermore, one regulatory element can increase the amount of the expressed product for multiple expression sequences that are linked together. Thus, one regulatory element can promote the expression of one or more expression sequences. Multiple regulatory elements are well known to those skilled in the art.

[0074]

[0156] The regulatory elements provided herein may include alternative translation sequences. As used herein, the term "alternative translation sequence" may refer to a nucleic acid sequence that selectively initiates or activates the translation of an expression sequence in a circular polyribonucleotide, such as a specific riboswitch aptazyme. The regulatory elements may also include alternative degradation sequences. As used herein, the term "alternative degradation sequence" may refer to a nucleic acid sequence that initiates the degradation of a circular polyribonucleotide or an expression product of a circular polyribonucleotide. Exemplary alternative degradation sequences may include riboswitch aptazymes and miRNA binding sites.

[0075]

[0157] In some embodiments, the regulatory element is a translation modulator. The translation modulator can regulate translation of an expression sequence in the circular polyribonucleotide. The translation modulator can be a translation enhancer or suppressor. In some embodiments, the circular polyribonucleotide comprises at least one translation modulator adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide comprises a translation modulator adjacent to each expression sequence. In some embodiments, a translation modulator is present on one or both sides of each expression sequence, resulting in the separation of expression products, such as peptides and / or polypeptides.

[0076]

[0158] In some embodiments, the translation initiation sequence may function 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 under selective conditions, e.g., stress-induced conditions, from an alternative translation initiation sequence, e.g., a translation initiation sequence other than an AUG codon. As a non-limiting example, circular polyribonucleotide translation may initiate from an alternative translation initiation sequence, such as ACG. As another non-limiting example, circular polyribonucleotide translation may initiate from an alternative translation initiation sequence, CTG / CUG. As yet another non-limiting example, circular polyribonucleotide translation may initiate from an alternative translation initiation sequence, GTG / GUG. As yet another non-limiting example, a circular polyribonucleotide may initiate translation from a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence containing short stretches of repetitive RNA, e.g., CGG, GGGGCC, CAG, CTG.

[0077]

[0159] Nucleotides adjacent to the codon that initiates translation, including but not limited to the initiation codon or another initiation codon, are known to affect the translation efficiency, length, and / or structure of the circular polyribonucleotide. (See, for example, Matsuda and Mauro PLoS ONE, 2010 5:11, the entire contents of which are incorporated herein by reference.) Masking any of the nucleotides adjacent to the codon that initiates translation can be used to change the translation initiation position, translation efficiency, length, and / or structure of the circular polyribonucleotide.

[0078]

[0160] In one embodiment, a masking agent can be used near a start codon or an alternative start codon to mask or conceal the codon to reduce the likelihood of translation initiation at the masked start codon or an alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acid (LNA) oligonucleotides and exon junction complexes (EJCs). (See, for example, Matsuda and Mauro describing masking agents LNA oligonucleotides and EJCs (PLoS ONE, 2010 5:11), the entire contents of which are incorporated herein by reference.) 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 from an alternative start codon.

[0079]

[0161] In one embodiment, translation is initiated under selective conditions, such as, but not limited to, virus-induced selection in the presence of GRSF-1, and the circular polyribonucleotide contains a GRSF-1 binding site, see, e.g., http: / / jvi.asm.org / content / 76 / 20 / 10417.full.

[0080] Translation initiation sequence

[0162] In some embodiments, the cyclic polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the cyclic polyribonucleotide includes 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 separation of the expression products. In some embodiments, the cyclic polyribonucleotide includes at least one translation initiation sequence adjacent to an expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the cyclic polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single-stranded region of the cyclic polyribonucleotide.

[0081]

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

[0082]

[0164] In some embodiments, the cyclic polyribonucleotide may initiate from a codon other than the first initiation codon, such as AUG. Translation of the cyclic polyribonucleotide may initiate from an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, or TTG / UUG (see Touriol et al., Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro, PLoS ONE, 2010 5:11, the entire contents of each of which are incorporated herein by reference). In some embodiments, translation is initiated from an alternative translation initiation sequence under selective conditions, such as stress-induced conditions. As a non-limiting example, translation of the cyclic polyribonucleotide may initiate from an alternative translation initiation sequence, such as ACG. As another non-limiting example, translation of the cyclic polyribonucleotide may initiate from an alternative translation initiation sequence, such as CTG / CUG. As yet another non-limiting example, circular polyribonucleotide translation can initiate from an alternative translation initiation sequence, GTG / GUG. As yet another non-limiting example, circular polyribonucleotides can initiate translation from repeat-associated non-AUG (RAN) sequences, such as alternative translation initiation sequences that contain short stretches of repetitive RNA, e.g., CGG, GGGGCC, CAG, CTG.

[0083]

[0165] In one embodiment, translation is initiated by eukaryotic initiation factor 4A (eIF4A) processing by Rocaglates (translation is inhibited by blocking 43S scanning, leading to premature upstream translation initiation and reduced protein expression from transcripts with RocA-eIF4A target sequences; see, e.g., www.nature.com / articles / nature17978).

[0084] IRES

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

[0085]

[0167] In certain embodiments, the IRES element is derived at least in part from a virus, for example, it is derived from 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, HA The IRES element may be derived from a viral IRES element such as V_HM175, HCV_type_1a, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HRV-2, IAPV_IGRpred, idefix, 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 one embodiment, the IRES element is selected from the group consisting of 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, hsp 70, 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, In some embodiments, the IRES element is derived at least in part from a cellular IRES, such as 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, such as (GAAA)16, (PPT19)4, KMI1, KMI1, KMI2, KMI2, KMIX, X1, or X2.

[0086]

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

[0087] Termination element

[0169] In some embodiments, a cyclic polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination element. In some embodiments, a cyclic polyribonucleotide comprises one or more expression sequences, each of which lacks a termination element, allowing the cyclic polyribonucleotide to be translated continuously. Elimination of the termination element can result in rolling circle translation or continuous expression of expression products, such as peptides or polypeptides, due to the lack of ribosome stalling or shed. In such embodiments, rolling circle translation expresses continuous expression products through each expression sequence. In certain other embodiments, the termination element of an expression sequence can be part of a staggered element. In some embodiments, one or more expression sequences in a cyclic polyribonucleotide contain a termination element. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the cyclic polyribonucleotide occurs. In such cases, when a ribosome encounters a termination element, such as a stop codon, and terminates translation, the expression product can shed from the ribosome. In certain embodiments, translation is terminated while the ribosome, eg, at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.

[0088]

[0170] In some embodiments, the cyclic polyribonucleotide contains a termination element at the end of one or more expressed sequences. In some embodiments, the one or more expressed sequences contain two or more termination elements in succession. In such embodiments, translation is terminated, terminating rolling circle translation. In some embodiments, the ribosome completely disengages from the cyclic polyribonucleotide. In some such embodiments, generation of a subsequent expressed sequence (e.g., the second, third, fourth, fifth, etc.) in the cyclic polyribonucleotide may require the ribosome to re-engage with the cyclic polyribonucleotide before translation can begin. Generally, termination elements include in-frame nucleotide triplets, such as UAA, UGA, and UAG, that indicate translation termination. In some embodiments, one or more termination elements in the cyclic polyribonucleotide are frameshift termination elements, such as, but not limited to, off-frame or -1 and +1 shifted reading frames (e.g., cryptic stops), which can terminate translation. Frameshift termination elements contain the nucleotide triplets TAA, TAG, and TGA that appear in the second and third reading frames of an expressed sequence. Frameshift termination elements can be important in preventing misreading of mRNA, which is often harmful to the cell.

[0089] Stagger element

[0171] In some embodiments, the cyclic polyribonucleotide comprises at least one stagger element flanking the expression sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element flanking each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, resulting in separation of the expression products, e.g., peptides and / or polypeptides. In some embodiments, the stagger element is part of one or more expression sequences. In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, each of which is separated from the subsequent expression sequence by a stagger element in the cyclic polyribonucleotide. In some embodiments, the stagger element prevents (a) double translation of a single expression sequence, or (b) the generation of a single polypeptide from one or more translations of two or more expression sequences. In some embodiments, the stagger element is a sequence separated from one or more expression sequences. In some embodiments, the stagger element comprises a portion of the expression sequence of one or more expression sequences.

[0090]

[0172] In some embodiments, the circular polyribonucleotide contains a stagger element. To avoid the production of consecutive expression products, such as peptides or polypeptides, while maintaining rolling circle translation, the stagger element may be included to induce ribosome pausing during translation. In some embodiments, the stagger element is at the 3' end of at least one of the one or more expression sequences. The stagger element may be configured to stall ribosomes during rolling circle translation of the circular polyribonucleotide. Stagger elements include, but are not limited to, 2A-like or CHYSEL (cis-acting hydrolase element) sequences. In some embodiments, the stagger element encodes a sequence having a C-terminal consensus sequence of X1X2X3EX5NPGP (wherein X1 is absent or is G or H, X2 is absent or is D or G, X3 is D, V, I, S, or M, and X5 is any amino acid). In one embodiment, the sequence comprises a non-conserved sequence of amino acids with strong alpha helix propensity followed by the consensus sequence -D(V / I)ExNPG P, 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, HDIETPNPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP.

[0091]

[0173] In some embodiments, the stagger elements described herein cleave the expression product, such as between G and P of the consensus sequences described herein. As one non-limiting example, a cyclic polyribonucleotide comprises at least one stagger element for cleaving the expression product. In some embodiments, the cyclic polyribonucleotide comprises a stagger element adjacent to at least one expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element after each expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element, and the stagger element is present on one or both sides of each expressed sequence to provide for translation of individual peptides and / or polypeptides from each expressed sequence.

[0092]

[0174] In some embodiments, the stagger element comprises one or more modified or non-natural nucleotides that induce ribosome pausing during translation. Non-natural nucleotides can include peptide nucleic acids (PNAs), morpholinos, and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Examples such as these are distinguished from natural DNA or RNA by changes to the backbone of the molecule. Exemplary modifications can include any modification to the sugar, nucleobase, internucleoside linkage (e.g., to the connecting phosphate / phosphodiester bond / phosphodiester backbone), and any combination thereof that can induce ribosome pausing during translation. Some of the exemplary modifications provided herein are described elsewhere herein.

[0093]

[0175] In some embodiments, the stagger element is present in the cyclic polyribonucleotide in another form. For example, in an exemplary cyclic polyribonucleotide, the stagger element comprises a nucleotide spacer sequence that separates the termination element from the termination element of the first expression sequence in the cyclic polyribonucleotide and the first translation initiation sequence of the expression following the first expression sequence. In some instances, the first stagger element of the first expression sequence is located upstream (5' end) of the first translation initiation sequence of the expression following the first expression sequence in the cyclic polyribonucleotide. In some cases, the first expression sequence and the expression sequence following the first expression sequence are two separate expression sequences in the cyclic polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence may allow for continuous translation of the first expression sequence and the subsequent expression sequence. In some embodiments, the first stagger element comprises a termination element that separates the expression product of the first expression sequence from the expression product of the subsequent expression sequence, thereby forming separate expression products. In some cases, a circular polyribonucleotide containing a first stagger element upstream of a first translation initiation sequence of a subsequent sequence in the circular polyribonucleotide is translated continuously, while a corresponding circular polyribonucleotide containing a stagger element of a second expressed sequence upstream of a second translation initiation sequence of an expressed sequence following a second expressed sequence is not translated continuously. In some cases, there is only one expressed sequence in the circular polyribonucleotide, and the first expressed sequence and its subsequent expressed sequence are the same expressed sequence. In some exemplary circular polyribonucleotides, the stagger element includes a first termination element of a first expressed sequence in the circular polyribonucleotide and a nucleotide spacer sequence that separates the termination element from the downstream translation initiation sequence. In some such examples, the first stagger element is upstream (5' end) of the first translation initiation sequence of the first expressed sequence in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence allows for continuous translation of the first expressed sequence and any subsequent expressed sequences. In one embodiment, the first stagger element separates the expression product of one round of the first expression sequence from the expression product of a subsequent round of the first expression sequence, thereby forming separate expression products.In some cases, a circular polyribonucleotide comprising a first stagger element upstream of a first translation initiation sequence of a first expressed sequence in a circular polyribonucleotide is translated continuously, while a corresponding circular polyribonucleotide comprising a stagger element upstream of a second translation initiation sequence of a second expressed sequence in the corresponding circular polyribonucleotide is not translated continuously. In some cases, the distance between the second stagger element and the second translation initiation sequence is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater in the corresponding circular polyribonucleotide than the distance between the first stagger element and the first translation initiation sequence in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or more. In some embodiments, the distance between the second 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 more greater than the distance between the first staggered element and the first translation start. In some embodiments, the circular polyribonucleotide comprises two or more expressed sequences.

[0094] regulatory nucleic acids

[0176] In some embodiments, the cyclic polyribonucleotides encode regulatory nucleic acids and include one or more expression sequences that regulate, for example, the expression of endogenous and / or exogenous genes. In some embodiments, the expression sequences of the cyclic polyribonucleotides provided herein can include sequences that are antisense to regulatory nucleic acids, such as non-coding RNAs, including, but not limited to, tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA, and hnRNA.

[0095]

[0177] In one embodiment, the regulatory nucleic acid targets a host gene. Regulatory nucleic acids include, but are not limited to, nucleic acids that hybridize to endogenous genes (e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA, as described elsewhere herein), nucleic acids that hybridize to foreign nucleic acids such as viral DNA or RNA, nucleic acids that hybridize to RNA, nucleic acids that interfere with gene transcription, nucleic acids that interfere with RNA translation, nucleic acids that stabilize RNA or destabilize RNA, such as by targeting 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.

[0096]

[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. gRNAs are short synthetic RNAs composed of a "scaffold" sequence necessary for binding to an imperfect effector moiety and a user-defined, approximately 20-nucleotide targeting sequence for a genomic target. In practice, guide RNA sequences are generally 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) in length and are designed to be complementary to the target nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for use in designing effective guide RNAs. Gene editing has also been performed using chimeric "single guide RNAs" ("sgRNAs"), engineered (synthetic) single RNA molecules that mimic the natural crRNA-tracrRNA complex and contain both a tracrRNA (to bind to a nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective for genome editing, see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991.

[0097]

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

[0098]

[0180] In one embodiment, gRNA is used as part of the CRISPR system for gene editing.For gene editing, circular polyribonucleotide can be designed to include one or more guide RNA sequences corresponding to desired target DNA sequence, for example, see Cong et al.(2013)Science,339:819-823;Ran et al.(2013)Nature Protocols,8:2281-2308.At least about 16 or 17 nucleotides of gRNA sequence are required by Cas9 to cause DNA cleavage; for Cpf1, at least about 16 nucleotides of gRNA sequence are required to achieve detectable DNA cleavage.

[0099]

[0181] Certain regulatory nucleic acids can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules typically contain 15-50 base pairs (e.g., about 18-25 base pairs) and comprise RNA or RNA-like structures with nucleobase sequences identical (complementary) or nearly identical (substantially complementary) to coding sequences in target genes expressed in cells. RNAi molecules include, but are not limited to, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), meroduplex, and dicer substrate. (U.S. Patent Nos. 8,084,599, 8,349,809 and 8,513,207).

[0100]

[0182] In certain embodiments, the circular polyribonucleotide comprises a regulatory nucleic acid, which is typically an RNA or RNA-like structure of about 5-500 base pairs (depending on the particular RNA structure, e.g., 5-30 bp for miRNA, 200-500 bp for lncRNA), and which may have a nucleobase sequence identical (complementary) or nearly identical (substantially complementary) to a coding sequence in a target gene expressed in a cell.

[0101]

[0183] Long non-coding RNAs (lncRNAs) are defined as non-protein-coding transcripts longer than 100 nucleotides. This somewhat arbitrary limitation distinguishes lncRNAs from small regulatory RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and other short RNAs. In general, the majority (approximately 78%) of lncRNAs are characterized as tissue-specific. Branched lncRNAs transcribed in the opposite direction to nearby protein-coding genes (accounting for a significant proportion, approximately 20% of all lncRNAs in mammalian genomes) can occasionally regulate the transcription of nearby genes. In one embodiment, the cyclic polyribonucleotides provided herein comprise the sense strand of a lncRNA. In one embodiment, the cyclic polyribonucleotides provided herein comprise the antisense strand of a lncRNA.

[0102]

[0184] A circular polyribonucleotide can encode a regulatory nucleic acid that is substantially complementary or completely complementary to all or a fragment of an endogenous gene or gene product (e.g., mRNA). The regulatory nucleic acid can complement sequences at boundaries between introns and exons, between exons, or adjacent to exons, preventing the maturation of a newly generated nuclear RNA transcript of a particular gene into mRNA for transcription. A regulatory nucleic acid complementary to a particular gene can hybridize with the mRNA for that gene and prevent its translation. An antisense regulatory nucleic acid can be DNA, RNA, or a derivative or hybrid thereof. In certain embodiments, the regulatory nucleic acid contains a protein-binding site capable of binding to a protein involved in regulating the expression of an endogenous or exogenous gene.

[0103]

[0185] The length of the circular polyribonucleotide can encode a regulatory nucleic acid that hybridizes to a transcript of interest that is about 5-30 nucleotides, about 10-30 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%.

[0104]

[0186] The cyclic polyribonucleotide can encode a microRNA (miRNA) molecule identical to about 5 to about 25 consecutive nucleotides of the target gene. In certain embodiments, the miRNA sequence targets an mRNA, starts with the dinucleotide AA, contains about 30-70% (about 30-60%, about 40-60%, or about 45-55%) GC-content, and does not share a high percentage of identity with any nucleotide sequence other than the target in the genome of the mammal into which it is to be introduced, as determined, for example, by a standard BLAST search.

[0105]

[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 having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to any one of the nucleotide sequences or a sequence complementary to a target sequence.

[0106]

[0188] siRNA and shRNA resemble intermediates in the processing pathway of endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004). In some embodiments, siRNA can function as miRNA, and vice versa (Zeng et al., Mol Cell 9:1327-1333, 2002; Doench et al., Genes Dev 17:438-442, 2003). Like siRNA, microRNAs downregulate target genes using RISC, but unlike siRNA, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein output by 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 with near-perfect complementarity to nucleotides 2-8 from the 5' end of the miRNA (Rajewsky, Nat Genet 38 Suppl:S8-13, 2006; Lim et al., Nature 433:769-773, 2005). This region is known as the seed region. Because siRNA and miRNA are interchangeable, exogenous siRNA downregulates mRNAs with seed complementarity to the siRNA (Birmingham et al., Nat Methods 3:199-204, 2006). Multiple target sites within a 3' UTR give stronger downregulation (Doench et al., Genes Dev 17:438-442, 2003).

[0107]

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

[0108]

[0190] Cyclic polyribonucleotides can regulate the expression of RNA encoded by genes. Because multiple genes may share a certain degree of sequence homology with each other, in some embodiments, cyclic polyribonucleotides can be designed to target a class of genes with sufficient sequence homology. In some embodiments, cyclic polyribonucleotides can contain sequences that are shared between different gene targets or that are complementary to sequences unique to a specific gene target. In some embodiments, cyclic polyribonucleotides can be designed to target conserved regions of RNA sequences that share 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, cyclic polyribonucleotides can be designed to target sequences unique to a specific RNA sequence of a single gene.

[0109]

[0191] In certain embodiments, the expressed sequence has a length of less than 5000 bp (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 certain embodiments, the expressed sequences may independently or additionally be longer 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, 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 more in length.

[0110]

[0192] In some embodiments, the expressed sequence comprises one or more of the features described herein, such as a sequence encoding one or more peptides or proteins, one or more regulatory elements, one or more regulatory nucleic acids, such as one or more non-coding RNAs, other expressed sequences, and any combination thereof.

[0111] Translation Efficiency

[0193] In certain embodiments, the translation efficiency of a cyclic polyribonucleotide provided herein is higher than that of a reference, eg, a linear equivalent, a linear expression sequence, or a linear cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotides provided herein have a translation efficiency that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2000%, 5000%, 10000%, 100000% or more higher than the translation efficiency of a reference. In some embodiments, the cyclic polyribonucleotides have a translation efficiency that is 10% higher than that of their linear counterparts. In one embodiment, cyclic polyribonucleotides have a translation efficiency that is 300% greater than that of their linear counterparts.

[0112]

[0194] In some embodiments, the cyclic polyribonucleotide produces expression products in a stoichiometric ratio. Rolling circle translation produces expression products sequentially in substantially equal ratios. In some embodiments, the cyclic polyribonucleotide has a stoichiometric translation efficiency such that expression products are produced in substantially equal ratios. In some embodiments, the cyclic polyribonucleotide has a stoichiometric translation efficiency for multiple expression products, for example, products from two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more expressed sequences.

[0113] Rolling circle translation

[0195] In some embodiments, once the translation of the cyclic polyribonucleotide is initiated, the ribosome bound to the cyclic polyribonucleotide does not leave the cyclic polyribonucleotide before completing at least one translation of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide described herein is capable of rolling circle translation. In one embodiment, during rolling circle translation, once translation of the circular polyribonucleotide is initiated, the ribosome bound to the circular polyribonucleotide is cleaved 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 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times, at least 5000 times, at least 10 ... 5 or at least 10 times 6 It does not leave the circular polyribonucleotide before completing its translation cycle.

[0114]

[0196] In some embodiments, rolling circle translation of a cyclic polyribonucleotide results in the production of polypeptide products ("contiguous" expression products) translated from two or more translations of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide comprises a staggered element, and rolling circle translation of the cyclic polyribonucleotide results in the production of polypeptide products ("distinct" expression products) generated from a single translation or less than one translation of the cyclic polyribonucleotide. In some embodiments, the cyclic 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 polypeptides (mol / mol) produced during rolling circle translation of the cyclic polyribonucleotide are distinct polypeptides. In some embodiments, the quantitative ratio of distinct products across all polypeptides is tested in an in vitro translation system. In some embodiments, the in vitro translation system used to test the quantity ratio comprises rabbit reticulocyte lysate. In some embodiments, the quantity ratio is tested in an in vivo translation system, such as a eukaryotic or prokaryotic cell, a cultured cell, or a cell within an organism.

[0115] Untranslated region

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

[0116]

[0198] In certain embodiments, the cyclic polyribonucleotide comprises a UTR with one or more stretches of adenosines and uridines embedded therein. These AU-rich signatures may increase the turnover rate of the expression product.

[0117]

[0199] The introduction, removal, or modification of UTR AU-rich elements (AREs) can be useful for adjusting the stability or immunogenicity of cyclic polyribonucleotides. When modifying a particular cyclic polyribonucleotide, one or more copies of AREs can be introduced into the cyclic polyribonucleotide, and the copies of AREs can regulate the translation and / or production of the expression product. Similarly, AREs can be identified and removed or modified in cyclic polyribonucleotides to adjust intracellular stability, thereby affecting the resulting protein translation and production.

[0118]

[0200] It should be understood that any UTR from any gene can be incorporated into each flanking region of the circular polyribonucleotide. Non-limiting examples of UTRs or fragments thereof that can be incorporated include those listed in U.S. Provisional Patent Application Nos. 61 / 775,509 and 61 / 829,372, or International Patent Application No. PCT / US2014 / 021522, the entire contents of each of which are incorporated herein by reference. Furthermore, multiple wild-type UTRs from any known gene can be used. It is also within the scope of the present invention to provide artificial UTRs that are not mutant versions of wild-type genes. These UTRs or portions thereof can be positioned in the same orientation as the transcript from which they were selected, or their orientation or position can be modified. Thus, 5' or 3' UTRs can be inverted, shortened, extended, or chimerized with one or more other 5' or 3' UTRs. As used herein, the term "modified," when referring to a UTR sequence, means that the UTR has been altered in some way relative to the reference sequence. For example, the 3' or 5' UTR may be modified relative to the wild-type or native UTR by a change in orientation or position as taught above, or may be modified by the inclusion of additional nucleotides, deletion of nucleotides, nucleotide exchanges, or rearrangements. Any of these changes, which produce an "modified" UTR (whether 3' or 5'), include mutant UTRs.

[0119]

[0201] In one embodiment, a double, triple, or quadruple UTR, such as a 5' or 3' UTR, can be used. As used herein, a "double" UTR is one in which two copies of the same UTR are encoded contiguously or substantially contiguously. For example, a double β-globin 3' UTR can be used, as described in U.S. Patent Application Publication No. 20100129877, the entire contents of which are incorporated herein by reference.

[0120] Poly(A) sequence

[0202] In some embodiments, the cyclic polyribonucleotide may comprise a poly-A sequence. In some embodiments, the length of the poly-A sequence is greater than 10 nucleotides. In one embodiment, the length of the poly-A sequence is greater than 15 nucleotides (e.g., at least 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 or more). In one embodiment, the poly-A sequence is from 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, 00, 100-2,000, 100-2,500, 100-3,000, 500-750, 500-1,000, 500-1,500, 500-2,000, 500-2,500, 500-3,000, 1,000-1,500, 1,000-2,000, 1,000-2,500, 1,000-3,000, 1,500-2,000, 1,500-2,500, 1,500-3,000, 2,000-3,000, 2,000-2,500, and 2,500-3,000).

[0121]

[0203] In one embodiment, the poly-A sequence is designed for the length of the entire cyclic polyribonucleotide. This design can be based on the length of the coding region, the length of a particular feature or region (such as the first or adjacent region), or the final product expressed from the cyclic polyribonucleotide. In this regard, the poly-A sequence can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the cyclic polyribonucleotide or its feature. The poly-A sequence can also be designed as part of the cyclic polyribonucleotide to which it belongs. In this regard, the poly-A sequence can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the entire length of the construct or the entire length of the construct minus the poly-A sequence. Furthermore, modified binding sites and conjugation of the cyclic polyribonucleotide for poly-A binding proteins can enhance expression.

[0122]

[0204] In one embodiment, the cyclic polyribonucleotide is designed to contain a poly-AG quartet. A G-quartet is a cyclic hydrogen-bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In one embodiment, the G-quartet is incorporated at the end of a poly-A sequence. The resulting cyclic polyribonucleotide construct is assayed for stability, protein production, and / or other parameters, including half-life at various time points. In one embodiment, the poly-AG quartet results in protein production that is at least 75% of that observed using a 120-nucleotide poly-A sequence alone.

[0123]

[0205] In some embodiments, the cyclic polyribonucleotide comprises a poly A, lacks a poly A, or has a modified poly A to adjust one or more properties of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide lacking a poly A or having a modified poly A improves one or more functional properties, such as immunogenicity, half-life, expression efficiency, etc.

[0124] RNA binding

[0206] In some embodiments, the cyclic polyribonucleotide comprises one or more RNA binding sites. MicroRNA (or miRNA) is a short non-coding RNA that binds to the 3'UTR of a nucleic acid molecule and downregulates gene expression by reducing nucleic acid molecule stability or inhibiting translation. The cyclic polyribonucleotide may comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNA, such as those taught in U.S. Patent Application Publication No. 2005 / 0261218 and U.S. Patent Application Publication No. 2005 / 0059005, the entire contents of which are incorporated herein by reference.

[0125]

[0207] The microRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which has perfect Watson-Crick complementarity to the miRNA target sequence. The microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA. In some embodiments, the microRNA seed may comprise seven nucleotides (e.g., nucleotides 2-8 of the mature microRNA), where the seed-complementary site in the corresponding miRNA target is adjacent to the adenine (A) opposite microRNA position 1. In some embodiments, the microRNA seed may comprise six nucleotides (e.g., nucleotides 2-7 of the mature microRNA), where the seed-complementary site in the corresponding miRNA target is adjacent to the adenine (A) opposite 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(1):91-105, each of which is incorporated herein by reference in its entirety.

[0126]

[0208] The bases of the microRNA seed are substantially complementary to the target sequence. By engineering a microRNA target sequence into a cyclic polyribonucleotide, the cyclic polyribonucleotide can evade or be detected by the host's immune system, and can have regulated degradation or regulated translation, provided that the corresponding microRNA is available. This process reduces the risk of off-target effects during cyclic polyribonucleotide delivery. The identification of microRNAs, microRNA target regions, and their expression patterns and roles in biology have been reported (Bonauer et al., Curr Drug Targets, 2009, pp. 111-114, each of which is incorporated herein by reference in its entirety). 2010 11:943-949;Anand and Cheresh Curr Opin Hematol 2011 18:171-176;Contreras and Rao Leukemia 2012 26:404-413(2011 Dec 20.doi:10.1038 / leu.2011.356);Barrel Cell 2009 136:215-233;Landgraf et al, Cell, 2007 129:1401-1414).

[0127]

[0209] Conversely, microRNA binding sites can be engineered out of (i.e., removed from) the cyclic polyribonucleotide to regulate protein expression in specific tissues. Regulation of expression in multiple tissues can be achieved through the introduction or removal of one or several microRNA binding sites.

[0128]

[0210] Examples of tissues in which microRNAs are known to regulate mRNA and thereby 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-ld, 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, which is incorporated herein by reference in its entirety). In the cyclic polyribonucleotides described herein, binding sites for microRNAs involved in such processes can be removed or introduced to regulate expression from the cyclic polyribonucleotide for biologically relevant cell types or in association with relevant biological processes. Lists of microRNAs, miR sequences, and miR binding sites are listed in Table 9 of U.S. Provisional Patent Application No. 61 / 753,661, filed January 17, 2013; Table 9 of U.S. Provisional Patent Application No. 61 / 754,159, filed January 18, 2013; and Table 7 of U.S. Provisional Patent Application No. 61 / 758,921, filed January 31, 2013, each of which is incorporated herein by reference in its entirety. In certain embodiments, the microRNA binding site comprises, for example, miR-7.

[0129]

[0211] The cyclic polyribonucleotides disclosed herein can include an miRNA-binding site that hybridizes to any miRNA, such as any of those disclosed in an 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 complementary to an miRNA whose target gene is disclosed in a microRNA-target gene database such as StarBase, StarScan, Cupid, TargetScan, TarBase, Diana-microT, miRecords, PicTar, PITA, RepTarm RNA22, miRTarBase, miRwalk, or MBSTAR.

[0130]

[0212] By understanding the expression patterns of microRNAs in various cell types, the cyclic polyribonucleotides described herein can be engineered for more targeted expression in specific cell types or only under specific biological conditions. By introducing tissue-specific microRNA binding sites, cyclic polyribonucleotides can be designed for optimal protein expression in tissues or in relation to biological conditions. Examples of the use of microRNAs to drive tissue- or disease-specific gene expression have been listed (Getner and Naldini, Tissue Antigens. 2012, 80:393-403, which is incorporated herein by reference in its entirety).

[0131]

[0213] Furthermore, microRNA seed sites can be incorporated into cyclic polyribonucleotides to regulate expression in specific cells, resulting in biological improvements. An example of this is the incorporation of miR-142 sites. Incorporation of miR-142 sites into the cyclic polyribonucleotides described herein can not only regulate expression in hematopoietic cells, but also reduce or eliminate immune responses to proteins encoded in the cyclic polyribonucleotides.

[0132]

[0214] In some embodiments, the circular polyribonucleotide contains one or more large intergenic non-coding RNA (lincRNA) binding sites. Large intergenic non-coding RNAs (lincRNAs) constitute the majority of long non-coding RNAs. LincRNAs are non-coding transcripts, and in some embodiments, are greater than about 200 nucleotides in length. In some embodiments, they have an exon-intron-exon structure similar to that of protein-coding genes, but do not contain open reading frames and do not encode proteins. More than 8,000 lincRNAs have been described recently and are believed to be the largest subclass of RNA derived from the non-coding transcriptome in humans. Thousands of lincRNAs are known, and several appear to be master regulators of diverse cellular processes. Determining the function of individual lincRNAs remains a challenge. Compared to coding genes, lincRNA expression is highly tissue-specific, and they are typically co-expressed with their neighboring genes, although not to the same extent as pairs of adjacent protein-coding genes.

[0133]

[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, or other lincRNAs or lncRNAs, such as those from known lncRNA databases.

[0134] Protein binding

[0216] In some embodiments, the cyclic polyribonucleotide comprises one or more protein binding sites that allow proteins, such as ribosomes, to bind to internal sites in the RNA sequence. By engineering a protein binding site, such as a ribosome binding site, into the cyclic polyribonucleotide, the cyclic polyribonucleotide can evade or have reduced detection by the host's immune system by masking the cyclic polyribonucleotide from components of the host's immune system, and can have regulated degradation or regulated translation.

[0135]

[0217] In certain embodiments, the cyclic polyribonucleotide comprises at least one immunity protein binding site, for example, to avoid an immune response, such as a CTL (cytotoxic T lymphocyte) response. In certain embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and serves to mask the cyclic polyribonucleotide as being exogenous. In certain embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and serves to mask the cyclic polyribonucleotide as being exogenous or foreign.

[0136]

[0218] The traditional mechanism of ribosome engagement with linear RNA involves the ribosome binding to the capped 5' end of the RNA. From the 5' end, the first peptide bond is formed as soon as the ribosome moves to the start codon. According to the present invention, internal initiation (i.e., cap-independent) of translation of a circular polyribonucleotide does not require a free or capped end. Rather, the ribosome binds to an uncapped internal site, thereby initiating polypeptide elongation at the start codon. In one embodiment, the circular polyribonucleotide comprises one or more RNA sequences that include a ribosome binding site, e.g., an start codon.

[0137]

[0219] Natural 5' UTRs have characteristics that play a role in translation initiation. They have signatures such as the Kozak sequence, which is commonly known to be involved in the process by which the ribosome initiates translation of many genes. The Kozak sequence has 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 participate in secondary structures involved in elongation factor binding.

[0138]

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

[0139]

[0221] In certain embodiments, the protein binding site may include, but is not limited to, ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, EL AVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC , HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, LIN28A, LIN28 B, m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2, POLR2A, PRPF8, PTBP1, RBF OX2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SR Binding sites for proteins such as SF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1, TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF1, WDR33, XRN2, YBX1, YTHDC1, YTHDF1, YTHDF2, YWHAG, ZC3H7B, PDK1, AKT1 and any other protein that binds to RNA.

[0140] Encryptogen

[0222] As described herein, cyclic polyribonucleotides contain an encryptogen to reduce, avoid, or circumvent the natural immune response of cells. In one aspect, provided herein are cyclic polyribonucleotides that, when delivered to cells, result in a reduced immune response from the host compared to the response elicited by a reference compound, such as a linear polynucleotide corresponding to the described cyclic polyribonucleotide or a cyclic polyribonucleotide lacking the encryptogen. In some embodiments, the cyclic polyribonucleotide has lower immunogenicity than its counterpart lacking the encryptogen.

[0141]

[0223] In some embodiments, the encryptogen improves stability. There is increasing evidence of the regulatory role that UTRs play in the stability of nucleic acid molecules and translation. The regulatory features of UTRs can be included in the encryptogen to improve the stability of the circular polyribonucleotide.

[0142]

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

[0143]

[0225] In certain embodiments, modification or removal of expressed sequences, such as AU-rich elements (AREs) in translatable regions, may be useful to modulate the stability or immunogenicity of circular polyribonucleotides.

[0144]

[0226] In some embodiments, the encryptogen contains a miRNA binding site or a binding site for any other non-coding RNA. For example, incorporation of a miR-142 site into the cyclic polyribonucleotides described herein can not only regulate expression in hematopoietic cells, but also reduce or eliminate immune responses against proteins encoded in the cyclic polyribonucleotides.

[0145]

[0227] In certain embodiments, the encryptogen contains one or more protein binding sites that allow a protein, such as an immune protein, to bind to the RNA sequence. By engineering a protein binding site into the cyclic polyribonucleotide, the cyclic polyribonucleotide can evade the host's immune system or have reduced detection by the host's immune system by masking the cyclic polyribonucleotide from components of the host's immune system, and can have regulated degradation or regulated translation. In certain embodiments, the cyclic polyribonucleotide contains at least one immune protein binding site, for example, to avoid an immune response, such as a CTL response. In certain embodiments, the immune protein binding site is a nucleotide sequence that binds to an immune protein and serves to mask the cyclic polyribonucleotide as exogenous.

[0146]

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

[0147]

[0229] In some embodiments, the cyclic polyribonucleotide comprises one or more modifications described elsewhere herein to reduce the immune response from the host compared to the response caused by a reference compound, for example, a cyclic polyribonucleotide lacking the modification. In particular, the addition of one or more inosines has been shown to distinguish RNA as endogenous to viruses. See, for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as "self". Cell Res. 25, 1283-1284, the entire contents of which are incorporated by reference.

[0148]

[0230] In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences for shRNA or an RNA sequence that can be processed into siRNA, and the shRNA or siRNA targets RIG-1 and reduces the expression of RIG-1. RIG-1 can sense the exogenous cyclic RNA and cause the exogenous cyclic RNA to be degraded. Therefore, a cyclic polynucleotide having a sequence for RIG-1-targeting shRNA, siRNA, or any other regulatory nucleic acid can reduce immunity, for example, host cell immunity, to the cyclic polyribonucleotide.

[0149]

[0231] In some embodiments, the cyclic polyribonucleotide lacks a sequence, element, or structure that assists the cyclic polyribonucleotide in reducing, avoiding, or evading a cellular innate immune response. In some such embodiments, the cyclic polyribonucleotide can lack a poly sequence, a 5' end, a 3' end, a phosphate group, a hydroxyl group, or any combination thereof.

[0150] Riboswitches

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

[0151]

[0233] Riboswitches are typically thought of as portions of a circular polyribonucleotide that can directly bind to a small target molecule, and target binding affects RNA translation, expression product stability, and activity (Tucker BJ, Breaker RR (2005), Curr Opin Struct Biol 15(3):342-8). Thus, a circular polyribonucleotide containing a riboswitch is directly involved in regulating its own activity in response to the presence or absence of its target molecule. In certain embodiments, a riboswitch has an aptamer-like region of affinity for a distinct molecule. Thus, in the broader context of the present invention, any aptamer contained within a non-coding nucleic acid can be used to sequester molecules from bulk volume. Downstream reporting of events by "(ribo)switch" activity can be particularly advantageous.

[0152]

[0234] In certain embodiments, riboswitches can affect gene expression, including, but not limited to, transcription termination, inhibition of translation initiation, mRNA self-cleavage, and alteration of splicing pathways in eukaryotes. Riboswitches can function to control gene expression through the binding or removal of a trigger molecule. Thus, a circular polyribonucleotide containing a riboswitch is subjected to conditions that activate, inactivate, or block the riboswitch, resulting in altered expression. Expression can be altered, for example, as a result of ribosomes binding to the RNA terminating or blocking transcription. Binding of a trigger molecule or its analog can reduce or prevent expression of the RNA molecule, or promote or increase expression of the RNA molecule, depending on the properties of the riboswitch. Some examples of riboswitches are described herein.

[0153]

[0235] In some embodiments, the riboswitch is a cobalamin riboswitch (B 12 element), which is adenosylcobalamin (vitamin B 12 It binds to cobalamin (the coenzyme form of cobalamin) and regulates the biosynthesis and transport of cobalamin and similar metabolites.

[0154]

[0236] In one embodiment, the riboswitch is a cyclic di-GMP riboswitch, which binds cyclic di-GMP to regulate various genes. There are two non-structurally related types: cyclic di-GMP-l and cyclic di-GMP-ll.

[0155]

[0237] In some embodiments, the riboswitch is an FMN riboswitch (also called an RFN element), which binds flavin mononucleotide (FMN) and regulates riboflavin biosynthesis and transport.

[0156]

[0238] In one embodiment, the riboswitch is a glmS riboswitch, which cleaves itself in the presence of sufficient concentrations of glucosamine-6-phosphate.

[0239] In one embodiment, the riboswitch is a glutamine riboswitch, which binds glutamine and regulates genes involved in glutamine and nitrogen metabolism. They also bind short peptides of unknown function. Such riboswitches fall into two structurally related types: the glnA RNA motif and the downstream-peptide motif.

[0157]

[0240] In one embodiment, the riboswitch is a glycine riboswitch, which binds glycine and regulates glycine metabolic genes. It is the only known natural RNA that contains two adjacent aptamer domains in the same mRNA and exhibits cooperative binding.

[0158]

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

[0159]

[0242] In one embodiment, the riboswitch is a PreQ1 riboswitch, which binds to pre-queosin and regulates genes involved in the synthesis or transport of this precursor to queosin. Two distinct types of PreGI riboswitches are known: PreQ1-l riboswitches and PreQ1-ll riboswitches. The binding domain of the PreQ1-l riboswitch is very small among naturally occurring riboswitches. The PreGI-II riboswitch, found only in certain species of Streptococcus and Lactococcus, has a completely different structure and is larger.

[0160]

[0243] In one embodiment, the riboswitch is a purine riboswitch, which binds to purines and regulates purine metabolism and transport. Different forms of purine riboswitches bind to guanine (a form originally known as a G-box) or adenine. Specificity for either guanine or adenine depends entirely on Watson-Crick interactions with a single pyrimidine in the riboswitch at position Y74. In guanine riboswitches, this residue is cytosine (i.e., C74); in adenine residues, it is always uracil (i.e., U74). Homologous types of purine riboswitches bind deoxyguanosine, but with significant differences beyond single nucleotide mutations.

[0161]

[0244] In one embodiment, the riboswitch is a SAH riboswitch, which binds S-adenosylhomocysteine ​​and regulates genes involved in recycling this metabolite, which is produced when S-adenosylmethionine is used in a methylation reaction.

[0162]

[0245] In some embodiments, the riboswitch is a SAM riboswitch, which binds S-adenosylmethionine (SAM) and regulates methionine and SAM biosynthesis and transport. Three different SAM riboswitches are known: SAM-I (originally called S-box), SAM-II, and S M K-box riboswitch. SAM-I is widespread in bacteria, while SAM-II is found only in α-, β-, and a few γ-proteobacteria. M K-box riboswitches are essential for the regulation of Lactobacillales ) are found only in SAM-IV riboswitches. These three species of riboswitches have no obvious similarities in sequence or structure. A fourth species, SAM-IV, appears to have a ligand-binding core similar to that of SAM-I, but in a different scaffold.

[0163]

[0246] In one embodiment, the riboswitch is a SAM-SAH riboswitch, which binds to both SAM and SAH with similar affinity, and it has been proposed that only their binding to SAM is physiologically relevant because they are always found in locations that regulate genes encoding methionine adenosyltransferase.

[0164]

[0247] In some embodiments, the riboswitch is a tetrahydrofolate riboswitch, which binds tetrahydrofolate and regulates synthesis and transport genes.

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

[0165]

[0249] In one embodiment, the riboswitch is a riboswitch derived from T. tengucongensis (T. tengucongensis). gcongensis) glmS catalytic riboswitch, which senses glucosamine-6-phosphate (Klein and Ferre-D'Amare 2006).

[0166]

[0250] In one embodiment, the riboswitch is a TPP riboswitch (also called a THI-box), which binds thiamine pyrophosphate (TPP) and regulates thiamine biosynthesis and transport and transport of similar metabolites, a riboswitch previously found only in eukaryotes.

[0167]

[0251] In some embodiments, the riboswitch is a Moco riboswitch, which binds a molybdenum cofactor and regulates genes involved in the biosynthesis and transport of this coenzyme and enzymes that use it or a derivative thereof as a cofactor.

[0168]

[0252] In one embodiment, the riboswitch is a riboswitch encoding a gene encoding Vibrio vulnificus. Adenine residues in the 5'UTR of the adenine deaminase-encoding gene of B. vulnificus It is known as the add-A riboswitch.

[0169] Aptazymes

[0253] In some embodiments, the cyclic polyribonucleotide comprises an aptazyme. Aptazymes are switches for conditional expression in which an aptamer region is used as an allosteric control element and bound to a region of a catalytic RNA ("ribozyme," described below). In some embodiments, the aptazyme is active in cell-type-specific translation. In some embodiments, the aptazyme is active in cell-state-specific translation, for example, in virus-infected cells or in the presence of viral nucleic acids or viral proteins.

[0170]

[0254] Ribozymes (derived from ribonucleic acid enzymes, also called RNA enzymes or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of ribosomes. More recently, it has been shown that catalytic RNAs can be "generated" by in vitro methods [1. Agresti JJ, Kelly BT, Jaschke A,Griffiths AD:Selection of ribozymes that catalyze multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization.Proc Natl Acad Sci USA 2005, 102:16170-16175; 2. Sooter LJ, Riedel T, Davidson EA, Levy M, Cox JC, Ellington AD: Toward automated nucleic acid enzyme selection. Biological Chemistry 2001, 382(9):1327-1334]. Winkler et al. have shown that ribozymes and their reaction products can regulate gene expression, similar to the riboswitch activity described above [Winkler WC, Nahvi A, Roth A, Collins JA, Breaker RR: Control of gene expression by a natural metabolite-responsive ribozyme. Nature 2004, 428:281-286]. In the context of the present invention, it may be particularly advantageous to place the catalytic RNA or ribozyme within a larger non-coding RNA so that the ribozyme is present in many copies within the cell for chemical conversion of molecules from bulk volume. Furthermore, it may be particularly advantageous to encode both the aptamer and the ribozyme within the same non-coding RNA.

[0171]

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

[0256] In some embodiments, aptazymes are ribozymes that can cleave RNA sequences and can be regulated as a result of binding ligands / modulators. Ribozymes can also be self-cleaving ribozymes. Thus, they combine the properties of ribozymes and aptamers. Aptazymes offer advantages over conventional aptamers due to their potential for transactivation, and they act catalytically to inactivate expression, and this inactivation is irreversible due to their own cleavage or heterologous transcription products.

[0172]

[0257] In some embodiments, the aptazyme is contained in the untranslated region of the circular polyribonucleotide and is inactive in the absence of a ligand / modulator, allowing transgene expression. Expression can be stopped (or downregulated) by adding a ligand. It should be noted that aptazymes that are downregulated in response to the presence of a specific modulator can be used in regulatory systems where upregulation of gene expression in response to a modulator is desired.

[0173]

[0258] Aptazymes may also enable the development of systems for the automatic regulation of cyclic polyribonucleotide expression. For example, the protein product of cyclic polyribonucleotide may be modified to contain an aptazyme that is a rate-limiting enzyme in the synthesis of a particular small molecule and is selected to have increased catalytic activity in the presence of that molecule, thereby providing a self-regulatory feedback loop for that synthesis. Alternatively, aptazyme activity may be selected to be sensitive to the accumulation of the protein product from cyclic polyribonucleotide or any other cellular macromolecule.

[0174]

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

[0175] cyclization

[0260] In one embodiment, the linear cyclic polyribonucleotide can be circularized or concatemerized. In some embodiments, the linear cyclic polyribonucleotide can be circularized in vitro before formulation and / or delivery. In some embodiments, the linear cyclic polyribonucleotide can be circularized intracellularly.

[0176] Extracellular cyclization

[0261] In some embodiments, linear circular polyribonucleotides are circularized or concatemerized using chemical methods to form circular polyribonucleotides. In some chemical methods, the 5'-end and 3'-end of a nucleic acid (e.g., linear circular polyribonucleotide) contain chemically reactive groups that, when brought close to each other, can form a new covalent bond between the 5'-end and 3'-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 at the 3'-end of the linear RNA molecule undergoes nucleophilic attack on the 5'-NHS-ester moiety to form a new 5'- / 3'-amide bond.

[0177]

[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 the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) to form a new phosphorodiester bond. In an example reaction, a linear circular polyribonucleotide is incubated with 1-10 units of T4 RNA ligase for 1 hour at 37°C according to the manufacturer's protocol (New England BioLabs, Ipswich, MA). The ligation reaction can occur in the presence of a linear nucleic acid capable of base pairing with both the 5'- and 3'-regions aligned to support the enzymatic ligation reaction. In one embodiment, the ligation is splint ligation. For example, a splint ligase, such as Splint® Ligase, can be used for splint ligation. In splint ligation, a single-stranded polynucleotide (splint), such as a single-stranded RNA, can be designed to hybridize with both ends of a linear polyribonucleotide such that the two ends can be juxtaposed upon hybridization with the single-stranded splint. Thus, a splint ligase can catalyze the ligation of the two juxtaposed ends of the linear polyribonucleotide to generate a circular polyribonucleotide.

[0178]

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

[0179]

[0264] In one embodiment, either the 5' or 3' end of the linear circular polyribonucleotide can encode a ligase ribozyme sequence such that during in vitro transcription, the resulting linear circular polyribonucleotide contains an active ribozyme sequence 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, hepatitis delta virus, a hairpin ribozyme, or selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction can be carried out at a temperature of 0 to 37°C for 1 to 24 hours.

[0180]

[0265] In one embodiment, a linear circular polyribonucleotide can be circularized or concatemerized by using at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety can react with a region or feature near the 5' end and / or near the 3' end of the linear circular polyribonucleotide to circularize or concatemerize the linear circular polyribonucleotide. In another aspect, the at least one non-nucleic acid moiety can be located at, linked to, or near the 5' end and / or 3' end of the linear circular polyribonucleotide. Possible non-nucleic acid moieties can be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety can be a bond such as a hydrophobic bond, an ionic bond, a biodegradable bond, and / or a cleavable bond. As another non-limiting example, the non-nucleic acid moiety can be a ligation moiety. As yet another non-limiting example, the non-nucleic acid moiety can be an oligonucleotide or peptide moiety, such as an aptamer or a non-nucleic acid linker described herein.

[0181]

[0266] In one embodiment, linear circular polyribonucleotides can be circularized or concatemerized by non-nucleic acid moieties that induce attractive forces between atoms or molecular surfaces at, near, or attached to the 5' and 3' ends of the linear circular polyribonucleotide. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, agnostic bonds, dipole bonds, conjugation, hyperconjugation, and antibonding.

[0182]

[0267] In one embodiment, a linear circular polyribonucleotide may contain ribozyme RNA sequences near the 5' end and near the 3' end. The ribozyme RNA sequences may be covalently linked to a peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, peptides covalently linked to the ribozyme RNA sequences near the 5' end and near the 3' end may bind to each other to circularize or concatemerize the linear circular polyribonucleotide. In another aspect, peptides covalently linked to the ribozyme RNA near the 5' end and near the 3' end may circularize or concatemerize a linear primary construct or linear mRNA after subjecting them to ligation using various methods known in the art, including, but not limited to, protein ligation. A non-limiting list of non-limiting examples of ribozymes or methods for incorporating and / or covalently linking peptides for use in the linear primary constructs or linear RNAs of the present invention is described in U.S. Patent Application Publication No. 20030082768, the entire contents of which are incorporated herein by reference.

[0183]

[0268] In certain embodiments, a linear circular polyribonucleotide may contain a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate by, for example, contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase (apyrase). Alternatively, converting the 5' triphosphate of a linear circular polyribonucleotide to a 5' monophosphate may be carried out by a two-step reaction comprising: (a) contacting the 5' nucleotide of the linear circular polyribonucleotide with a phosphatase (e.g., Antarctic phosphatase, shrimp alkaline phosphatase, or calf intestinal phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., polynucleotide kinase) that adds one phosphate.

[0184]

[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%.

[0185] Splicing Elements

[0270] In some embodiments, the cyclic polyribonucleotide comprises at least one splicing element. In the cyclic polyribonucleotide provided herein, the splicing element can be a complete splicing element that can mediate the splicing of the cyclic polyribonucleotide. Alternatively, the splicing element can also be a residual splicing element from a completed splicing event. For example, in some cases, the splicing element of a linear polyribonucleotide can mediate a splicing event that results in the circularization of the linear polyribonucleotide, and the resulting cyclic polyribonucleotide therefore comprises a residual splicing element from such a circularization event via splicing. In some cases, the residual splicing element cannot mediate splicing. In other cases, the residual splicing element can still mediate splicing under certain circumstances. In some embodiments, the splicing element is adjacent to at least one expression sequence. In some embodiments, the cyclic polyribonucleotide comprises a splicing element adjacent to each expression sequence. In certain embodiments, splicing elements are located on one or both sides of each expressed sequence, resulting in the separation of expression products, eg, peptides and / or polypeptides.

[0186]

[0271] In some embodiments, the circular polyribonucleotide contains an internal splicing element that, upon replication, joins the spliced ​​ends. Some examples include splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences in adjacent introns, Alu elements in adjacent introns, and small introns (<100 nt) containing motifs found in cis-sequence elements proximal to backsplice events (suptable 4-enriched motifs), such as sequences 200 bp before (upstream) or after (downstream) a backsplice site with an adjacent exon. In some embodiments, the circular polyribonucleotide contains at least one repeated nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repeated nucleotide sequence may include a repeated sequence from the Alu family of introns. In certain embodiments, ribosome binding proteins involved in splicing can regulate circular polyribonucleotide biosynthesis (eg, Muscleblind and Quaking (QKI) splicing factors).

[0187]

[0272] In certain embodiments, the cyclic polyribonucleotide may include a canonical splice site adjacent to the head-to-tail junction of the cyclic polyribonucleotide. .

[0188]

[0273] In certain embodiments, a cyclic polyribonucleotide can contain a bulge-helix-bulge motif, which includes a four-base-pair stem flanked by two three-nucleotide bulges. Cleavage occurs in the bulge region, generating characteristic fragments with terminal 5'-hydroxyl groups and 2',3'-cyclic phosphates. Circularization proceeds by nucleophilic attack of the 5'-OH group on the 2',3'-cyclic phosphate of the same molecule, forming a 3',5'-phosphodiester bridge.

[0189]

[0274] In some embodiments, the circular polyribonucleotide can comprise a multimeric repeat RNA sequence having an HPR element, which comprises a 2',3'-cyclic phosphate and a 5'-OH terminus, and the HPR element self-processes the 5' and 3' termini of the linear circular polyribonucleotide, thereby ligating the termini together.

[0190]

[0275] In some embodiments, the cyclic polyribonucleotide may contain a sequence that mediates self-ligation. In one embodiment, the cyclic polyribonucleotide may contain an HDV sequence (e.g., an HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCGAGCC). In one embodiment, the circular polyribonucleotide can include a loop E sequence (e.g., in PSTVd) for self-ligation. 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 Group I, Group II, or Group III intron. Non-limiting examples of Group I intron self-splicing sequences can include the self-splicing replacement intron-exon sequence from the T4 bacteriophage gene td and the Tetrahymena intervening sequence (IVS) rRNA. .

[0191] Other Cyclization Methods

[0276] In some embodiments, a linear circular polyribonucleotide may contain complementary sequences, including repetitive or non-repetitive nucleic acid sequences, within individual introns or across adjacent introns. The repetitive nucleic acid sequence is a sequence present within a segment of the circular polyribonucleotide. In some embodiments, the circular polyribonucleotide contains a repetitive nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence comprises a poly-CA or poly-UG sequence. In some embodiments, the circular polyribonucleotide contains at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the circular polyribonucleotide, and the hybridized segment forms an internal duplex. In some embodiments, the repetitive nucleic acid sequence and the complementary repetitive nucleic acid sequence from two separate circular polyribonucleotides hybridize to generate a single circularized polyribonucleotide, and the hybridized segment forms an internal duplex. In some embodiments, the complementary sequences are found at the 5' and 3' ends of the linear circular polyribonucleotide. In certain embodiments, the complementary sequence comprises about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more paired nucleotides.

[0192]

[0277] In certain embodiments, chemical methods of cyclization can be used to generate cyclic polyribonucleotides, including, but not limited to, click chemistry (e.g., alkyne and azide-based methods or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.

[0193]

[0278] In some embodiments, enzymatic methods of circularization can be used to generate circular polyribonucleotides. In some embodiments, a ligation enzyme, such as a DNA or RNA ligase, can be used to generate a circular polyribonuclease or a complementary template, a complementary strand of a circular polyribonuclease, or a circular polyribonuclease.

[0194]

[0279] Circularization of cyclic polyribonucleotides can be carried out by methods known in the art, such as "RNA circularization strategies in vivo and in vitro" by Petkovic and Muller, Nucleic Acids Res, 2015, 43(4):2454-2465, and RNA circularization methods. This can be done as described in "In vitro circularization of RNA" by Muller and Appel in Biol, 2017, 14(8):1018-1027.

[0195] duplicate element

[0280] Circular polyribonucleotides can encode sequences and / or motifs useful for replication. Replication of circular polyribonucleotides can occur by generating complementary circular polyribonucleotides. In some embodiments, circular polyribonucleotides contain motifs for initiating transcription, where transcription is driven by either endogenous cellular machinery (DNA-dependent RNA polymerase) or RNA-dependent RNA polymerase encoded by the circular polyribonucleotide. The product of a rolling circle transcription event can be cleaved by a ribozyme to generate either a complementary or propagated circular polyribonucleotide in a unit length. The ribozyme can be encoded by the circular polyribonucleotide, its complement, or an RNA sequence in trans. In some embodiments, the encoded ribozyme can contain a sequence or motif that modulates (inhibits or promotes) the activity of the ribozyme to control circular RNA propagation. In some embodiments, the unit length sequence can be ligated into a circular form by an intracellular RNA ligase. In some embodiments, the circular polyribonucleotide contains a replication element useful for self-amplification. Examples of such replication elements include, but are not limited to, the HDV replication domain described elsewhere herein, the RNA promoter of Potato Spindle Tuber Viroid (see, e.g., Kolonko 2005 Virology), and the antigenome 5'-CGGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGACGCACGUCCACUCGGAUGGCUAAGGGAGAGCCA-3' or the genome 5'-CGGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGACGCACGUCCACUCGGAUGGCUAAGGGAGAGCCA-3'. '-UGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCGGAGGGGACCGUCCCCUCGGUAAUGGCGAAUGGGACCCA-3' and the like.

[0196]

[0281] In some embodiments, the cyclic polyribonucleotide comprises at least one stagger element described herein to promote replication. The stagger element in the cyclic polyribonucleotide can cleave a long transcript replicated from the cyclic polyribonucleotide to a specific length, which is then circularized to form a complement to the cyclic polyribonucleotide.

[0197]

[0282] In another embodiment, the circular polyribonucleotide contains at least one ribozyme sequence to cleave a long transcript replicated from the circular polyribonucleotide to a specific length, where a separate encoded ribozyme cleaves the transcript at the ribozyme sequence. Circularization forms a complement to the circular polyribonucleotide.

[0198]

[0283] In certain embodiments, the cyclic polyribonucleotide is substantially resistant to degradation, eg, by exonucleases.

[0284] In some embodiments, cyclic polyribonucleotides replicate within cells. In some embodiments, cyclic polyribonucleotides replicate within cells at a rate of about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 99%, or any percentage therebetween. In some embodiments, cyclic polyribonucleotides are replicated within cells and delivered to daughter cells. In some embodiments, cells deliver at least one cyclic polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, cells undergoing meiosis deliver cyclic polyribonucleotides to daughter cells with at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% efficiency. In some embodiments, cells undergoing mitosis deliver cyclic polyribonucleotides to daughter cells with at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% efficiency.

[0199]

[0285] In certain embodiments, the cyclic polyribonucleotide replicates within a host cell. In one embodiment, the cyclic polyribonucleotide is capable of replicating within a mammalian cell, such as a human cell.

[0200]

[0286] In some embodiments, the cyclic polyribonucleotide replicates within the host cell, but the cyclic polyribonucleotide is not integrated into the host's genome, for example, by the host's chromosome. In some embodiments, the cyclic polyribonucleotide has a negligible recombination frequency, for example, with the host's chromosome. In some embodiments, the cyclic polyribonucleotide has a recombination frequency, for example, with the host's chromosome, of 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, or 0.1 cM / Mb.

[0201] Other arrays

[0287] In some embodiments, the cyclic polyribonucleotide further comprises another nucleic acid sequence. In some embodiments, the cyclic polyribonucleotide may comprise other sequences, including DNA, RNA, or artificial nucleic acids. The other sequences 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 cyclic polyribonucleotide comprises an siRNA to target a different locus of the same gene expression product as the cyclic polyribonucleotide. In one embodiment, the cyclic polyribonucleotide comprises an siRNA to target a different gene expression product than the cyclic polyribonucleotide.

[0202]

[0288] In some embodiments, the cyclic polyribonucleotide lacks a 5'-UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3'-UTR. In some embodiments, the cyclic polyribonucleotide lacks a poly-A sequence. In some embodiments, the cyclic polyribonucleotide lacks a termination element. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks susceptibility to exonuclease degradation. In some embodiments, the lack of susceptibility to exonuclease degradation can mean that the cyclic polyribonucleotide is not degraded by exonucleases, or is degraded only in the presence of exonucleases to a limited extent that is equal to or similar to that in the absence of exonucleases. In some embodiments, the cyclic polyribonucleotide lacks exonuclease degradation. In some embodiments, the cyclic polyribonucleotide has reduced degradation when exposed to exonucleases. In some embodiments, the cyclic polyribonucleotide lacks a linkage to a cap-binding protein. In some embodiments, the cyclic polyribonucleotide lacks a 5' cap.

[0203]

[0289] In some embodiments, the cyclic polyribonucleotide lacks a 5'-UTR and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic polyribonucleotide lacks a 3'-UTR and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic polyribonucleotide lacks a poly-A sequence and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic polyribonucleotide lacks a termination element and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic polyribonucleotide lacks a cap and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic polyribonucleotide lacks a 5'-UTR, a 3'-UTR, and an IRES and is capable of expressing a protein from one or more expression sequences thereof. In certain embodiments, the circular polyribonucleotide comprises one or more of the following sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding an exogenous gene, a sequence encoding a therapeutic agent, a regulatory element (e.g., a translation modulator, such as a translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids targeting an endogenous gene (siRNA, lncRNAs, shRNA), and a sequence encoding a therapeutic mRNA or protein.

[0204]

[0290] Other sequences may have a length of about 2 to about 10,000 nt, about 2 to about 5,000 nt, about 10 to about 100 nt, about 50 to about 150 nt, about 100 to about 200 nt, about 150 to about 250 nt, about 200 to about 300 nt, about 250 to about 350 nt, about 300 to about 500 nt, about 10 to about 1,000 nt, about 50 to about 1,000 nt, about 100 to about 1,000 nt, about 1,000 to about 2,000 nt, about 2,000 to about 3,000 nt, about 3,000 to about 4,000 nt, about 4,000 to about 5,000 nt, or any range therebetween.

[0205]

[0291] As a result of its circularization, cyclic polyribonucleotide may contain some characteristics that distinguish it from linear RNA.For example, cyclic polyribonucleotide is less susceptible to degradation by exonucleases compared to linear RNA.Therefore, cyclic polyribonucleotide is more stable than linear RNA, especially when incubated in the presence of exonucleases.The increased stability of cyclic polyribonucleotide compared to linear RNA makes cyclic polyribonucleotide more useful as a cell transformation reagent for producing polypeptides, and can be stored more easily and for longer than linear RNA.The stability of exonuclease-treated cyclic polyribonucleotide can be tested using standard methods in the art (for example, by gel electrophoresis) to determine whether RNA degradation occurs.

[0206]

[0292] Furthermore, unlike linear RNA, circular polyribonucleotides are less susceptible to dephosphorylation when they are incubated with phosphatases, such as calf intestinal phosphatase.

[0207] Nucleotide spacer sequence

[0293] In certain embodiments, the cyclic polyribonucleotide comprises a spacer sequence.

[0208]

[0294] In some embodiments, the cyclic polyribonucleotide comprises at least one spacer sequence, hi some embodiments, the cyclic polyribonucleotide comprises 1, 2, 3, 4, 5, 6, 7 or more spacer sequences.

[0209]

[0295] In certain embodiments, the cyclic polyribonucleotide is 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 0.95:1, about 0.95:1, about 0.10:1, about 0.11:1, 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 0.95:1, about 0.95:1, about 0.105:1, about 0.115:1, about 0.116:1, about 0.117:1, about 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 of spacer sequences to non-spacer sequences of the circular polyribonucleotide, e.g., the expressed sequence.

[0210]

[0296] In certain embodiments, the spacer sequence is 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, approximately 1.5:1, approximately 1.6:1, approximately 1.7:1, approximately 1.8:1, approximately 1.9:1, approximately 1.95:1, approximately 1.975:1, approximately 1.98:1, approximately 2.1:1, approximately 2.2:1, approximately 2.3:1, approximately 2.4:1, approximately 2.5:1, approximately 2.6:1, approximately 2.7:1, approximately 2.8:1, approximately 2.9:1, approximately 3:1, approximately 3.1:1, approximately 3. The ratio of spacer sequence to non-spacer elements downstream of the circular polyribonucleotide (e.g., toward the 3' end of the spacer sequence) can be about 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 certain embodiments, the spacer sequence is 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, approximately 1.5:1, approximately 1.6:1, approximately 1.7:1, approximately 1.8:1, approximately 1.9:1, approximately 1.95:1, approximately 1.975:1, approximately 1.98:1, approximately 2.1:1, approximately 2.2:1, approximately 2.3:1, approximately 2.4:1, approximately 2.5:1, approximately 2.6:1, approximately 2.7:1, approximately 2.8:1, approximately 2.9:1, approximately 3:1, approximately 3.1:1, approximately 3. The ratio of spacer sequence to non-spacer elements upstream of the circular polyribonucleotide (e.g., toward the 5' end of the spacer sequence) can be about 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.

[0211]

[0297] In certain embodiments, the spacer sequence is at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides. The nucleic acid sequence may comprise a sequence of at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least about 200 ribonucleotides, at least about 250 ribonucleotides, at least about 300 ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 600 ribonucleotides, at least about 700 ribonucleotides, at least about 800 ribonucleotides, at least about 900 ribonucleotides, or at least about 100 ribonucleotides.

[0212]

[0298] In certain embodiments, the spacer sequence spans the entire 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, such as 65%, 60%, 55%, 50%, 55%, 50%, 45%, 40%, 39%, 38%, 39%, 40%, 41%, 41%, 42%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, The spacer sequence may have a low GC content of less than 7%, 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%. In certain embodiments, the spacer sequence may contain 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 one embodiment, the spacer sequence comprises at least five or more consecutive adenine ribonucleotides.In certain embodiments, the spacer sequence is at least 6 consecutive adenine ribonucleotides, at least 7 consecutive adenine ribonucleotides, at least 8 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. nucleotides, 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.

[0213]

[0299] In some embodiments, a spacer sequence is positioned between one or more elements. In some embodiments, the spacer sequence provides conformational flexibility between the 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, -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 may comprise a nucleic acid such as DNA or RNA.

[0214]

[0300] In certain embodiments, the spacer sequence may encode an RNA sequence and preferably a protein or peptide sequence including a secretory signal peptide.

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

[0215]

[0302] In certain embodiments, the spacer is operably linked to another sequence described herein.

[0216] Non-nucleic acid linkers

[0303] The cyclic polyribonucleotides described herein may also include non-nucleic acid linkers. In some embodiments, the cyclic polyribonucleotides described herein have non-nucleic acid linkers between one or more of the sequences or elements described herein. In one embodiment, one or more of the sequences or elements described herein are linked using 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 or protein linker. Such linkers can be 2 to 30 amino acids or more. Linkers include flexible, rigid, or cleavable linkers described herein.

[0217]

[0304] The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linkers). Flexible linkers can be useful for joining domains that require some degree of movement or interaction and can contain small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the protein moiety.

[0218]

[0305] Rigid linkers are useful for maintaining a certain distance between domains and maintaining their independent functions. Rigid linkers may also be useful when spatial separation of domains is important for maintaining the stability or biological activity of one or more components during fusion. Rigid linkers include α-helical structures or Pro-rich sequences, (XP) n where X represents any amino acid, preferably Ala, Lys or Glu.

[0219]

[0306] A cleavable linker can release a free functional domain in vivo. In some embodiments, the linker can be cleaved under certain conditions, such as in the presence of a reducing agent or a protease. In vivo cleavable linkers can utilize the reversibility of disulfide bonds. One example includes a thrombin-sensitive sequence (e.g., PRS) between two Cys residues. 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 are described, for example, in Chen et al. 2013. Fusion Protein Linker: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10):1357-1369. In vivo cleavage of the fusion linker can also be achieved by proteases expressed in vivo in specific cells or tissues under pathological conditions (e.g., cancer or inflammation) or restricted to specific intracellular compartments. The specificity of many proteases provides for slower cleavage of the linker in restricted compartments.

[0220]

[0307] Examples of linking molecules include hydrophobic linkers, such as negatively charged sulfonate groups; lipids, such as poly(-CH2-) hydrocarbon chains, e.g., polyethylene glycol (PEG) groups, including unsaturated, hydroxylated, amidated, or other N-containing forms; non-carbon linkers; carbohydrate linkers; phosphodiester linkers; or other molecules capable of covalently linking two or more polypeptides. Also included are non-covalent linkers, such as hydrophobic lipid globules to which polypeptides are linked via hydrophobic regions or hydrophobic extensions of the polypeptides, e.g., leucine, isoleucine, valine, or possibly alanine, phenylalanine, or a series of residues rich in tyrosine, methionine, glycine, or other hydrophobic residues. Polypeptides can also be linked using charge-based chemistry, such that a positively charged portion of a polypeptide is linked to the negative charge of another polypeptide or nucleic acid.

[0221] Stability / Half-life

[0308] In some embodiments, the cyclic polyribonucleotide provided herein has an increased half-life compared to a control, for example, a linear polyribonucleotide (linear equivalent) that has the same nucleotide sequence but is not circular.In some embodiments, the cyclic polyribonucleotide is substantially resistant to degradation, for example, exonuclease.In some embodiments, the cyclic polyribonucleotide is resistant to autolysis.In some embodiments, the cyclic polyribonucleotide lacks an enzyme cleavage site, for example, a Dicer cleavage site. In certain embodiments, a cyclic 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 10,000% longer than a control, e.g., a linear counterpart.

[0222]

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

[0223] qualification

[0310] A cyclic polyribonucleotide may contain one or more substitutions, insertions and / or additions, deletions and covalent modifications relative to the reference sequence, particularly the parent polyribonucleotide, and is included within the scope of the present invention.

[0224]

[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). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than 100 different nucleoside modifications identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27:196-197). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In certain embodiments, the mRNA is selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5- The compound comprises at least one nucleoside selected from the group consisting of 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 certain embodiments, the mRNA is selected from the group consisting of 5-aza-cytidine, pseudocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio and at least one nucleoside selected from the group consisting of 1-methyl-1-deaza-pseudocytidine, 1-methyl-1-deaza-pseudocytidine, 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-pseudocytidine, and 4-methoxy-1-methyl-pseudocytidine. In one embodiment, the mRNA is 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-glycinylcarbamoyl adenosine, and N6-threonylcarbamoyl. It comprises at least one nucleoside selected from the group consisting of adenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In certain embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 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.

[0225]

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

[0226]

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

[0227]

[0314] In certain embodiments, the modification may include a chemical modification or a cell-induced modification. For example, some non-limiting examples of intracellular RNA modifications are found in Nat. Reviews Mol. Described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions" in Cell Biol, 2017, 18:202-210.

[0228]

[0315] In certain embodiments, chemical modifications to the ribonucleotides of cyclic polyribonucleotides can promote immune evasion. Cyclic polyribonucleotides can be synthesized and / or modified by methods well established in the art, such as those described in "Current Protocols in Nucleic Acid Chemistry," Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation (mono-, di-, and tri-phosphorylation), conjugation, inverted linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, inverted linkage, etc.), base modifications (e.g., substitution with stabilizing bases, destabilizing bases, or bases that base-pair with a wide range of partners), removal of bases (abasic nucleotides), or conjugated bases. Modified ribonucleotide bases can also include 5-methylcytidine and pseudouridine. In certain embodiments, base modifications can modulate expression, immune response, stability, and subcellular localization, to name a few functional effects of cyclic polyribonucleotides. In certain embodiments, modifications include bi-orthogonal nucleotides, For example, unnatural bases are included. See, for example, Kimoto et al., which is incorporated herein by reference. See et al, Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039 / c7cc06661a.

[0229]

[0316] In certain embodiments, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions of one or more ribonucleotides of a cyclic polyribonucleotide can include not only backbone modifications but also modifications or substitutions of phosphodiester linkages. Specific examples of cyclic polyribonucleotides include cyclic polyribonucleotides containing modified backbones or non-natural internucleoside linkages, such as internucleoside modifications, including, but not limited to, modifications or substitutions of phosphodiester linkages. Cyclic polyribonucleotides with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleosides. In certain embodiments, cyclic polyribonucleotides include ribonucleotides that have a phosphorus atom in their internucleoside backbone.

[0230]

[0317] Modified cyclic polyribonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and their usual 3'-5' linkages, boranophosphates with 2'-5' linkage analogs, and those with reverse polarity, where adjacent pairs of nucleoside units are linked (3'-5' to 5'-3' or 2'-5' to 5'-2').Various salts, mixed salts, and free acid forms are also included.In some embodiments, cyclic polyribonucleotides can be negatively or positively charged.

[0231]

[0318] Modified nucleotides that can be incorporated into cyclic polyribonucleotides can be modified on the internucleoside linkage (e.g., the phosphate backbone). In this specification, the terms "phosphate" and "phosphodiester" are used interchangeably with respect to the polynucleotide backbone. The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with various substituents. Furthermore, modified nucleosides and nucleotides can contain extensive replacement of the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, phosphorodiamidate, alkyl or aryl phosphonate, and phosphotriester. In phosphorodithioate, both non-linking oxygens are replaced with sulfur. Phosphate linkers can also be modified by replacement of the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene-phosphonates).

[0232]

[0319] The non-natural phosphorothioate backbone linkage provides a-thio-substituted phosphate moieties to provide stability to RNA and DNA polymers. Phosphorothioate DNA and RNA have increased nuclease resistance and therefore a longer half-life in the cellular environment. Phosphorothioates linked to cyclic polyribonucleotides are predicted to reduce innate immune responses by weaker binding / activation of cellular innate immune molecules.

[0233]

[0320] In certain embodiments, modified nucleosides include α-thio-nucleosides (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine).

[0234]

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

[0322] In certain embodiments, the cyclic polyribonucleotide can include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides can be incorporated into the cyclic polyribonucleotide, such as bifunctional modifications. Cytotoxic nucleosides include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), and 6-mercaptopurine. Further examples include fludarabine phosphate, N4-behenoyl-1-β-D-arabinofuranosylcytosine, N4-octadecyl-1-β-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidate).

[0235]

[0323] A cyclic polyribonucleotide may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., natural nucleotides, purines or pyrimidines, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in a cyclic polyribonucleotide or in a given predetermined sequence region thereof. In some embodiments, the cyclic polyribonucleotide contains pseudouridine. In some embodiments, the cyclic polyribonucleotide contains inosine, which may facilitate the immune system's characterization of the cyclic polyribonucleotide as endogenous to viral RNA. Incorporation of inosine may also mediate improved RNA stability / reduced degradation. See, for example, Yu, Z. et al. (2015) RNA editing by ADAR1, the entire contents of which are incorporated by reference. See marks dsRNA as "self". Cell Res. 25, 1283-1284.

[0236]

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

[0237]

[0325] Various sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in a cyclic polyribonucleotide. Those skilled in the art will understand that nucleotide analogs or other modifications can be placed at any position in a cyclic polyribonucleotide so that the function of the cyclic polyribonucleotide is not substantially impaired. Modifications can also be made in non-coding regions. Cyclic polyribonucleotides may contain from about 1% to about 100% modified nucleotides (either 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 percentage therebetween (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%).

[0238] structure

[0326] In some embodiments, cyclic polyribonucleotides comprise higher-order structures, such as secondary or tertiary structures. In some embodiments, complementary segments of cyclic polyribonucleotides are folded into double-stranded segments and connected by hydrogen bonds between pairs, such as AU and CG. In some embodiments, helices, also known as stems, are formed within the molecule, with double-stranded segments connected to terminal loops. In some embodiments, cyclic polyribonucleotides have at least one segment with a pseudo-double-stranded secondary structure. In some embodiments, a segment having a pseudo-double-stranded secondary structure has at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more paired nucleotides. In some embodiments, a cyclic polyribonucleotide has one or more segments (e.g., 2, 3, 4, 5, 6 or more) having a pseudo-double-stranded secondary structure. In some embodiments, the segments are separated by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides.

[0239]

[0327] In some embodiments, one or more sequences of the cyclic polyribonucleotide comprise substantially single-stranded to double-stranded regions. In some embodiments, the ratio of single-stranded to double-stranded regions can affect the functionality of the cyclic polyribonucleotide.

[0240]

[0328] In some embodiments, the cyclic polyribonucleotide sequence is one or more sequences that are substantially single-stranded. In some embodiments, the one or more sequences of the substantially single-stranded cyclic polyribonucleotide may contain a protein- or RNA-binding site. In some embodiments, the substantially single-stranded cyclic polyribonucleotide sequence may be conformationally flexible to allow for increased interaction. In some embodiments, the cyclic polyribonucleotide sequence is intentionally engineered to contain such secondary structures to bind or increase protein or nucleic acid binding.

[0241]

[0329] In some embodiments, the cyclic polyribonucleotide sequence is substantially double-stranded. In some embodiments, one or more sequences of the substantially double-stranded cyclic polyribonucleotide may contain a conformational recognition site, such as a riboswitch or an aptazyme. In some embodiments, the substantially double-stranded cyclic polyribonucleotide sequence may be conformationally fixed. In some such cases, the conformationally fixed sequence may sterically shield the cyclic polyribonucleotide from protein or nucleic acid binding. In some embodiments, the sequence of the cyclic polyribonucleotide is intentionally engineered to include such secondary structures to avoid or reduce protein or nucleic acid binding.

[0242]

[0330] There are 16 possible base pairings, but only six of these (AU, GU, GC, UA, UG, CG) can form actual base pairs. The rest are called mismatches and exist at very low frequencies in the helix. In some embodiments, the structure of the cyclic polyribonucleotide cannot be easily disrupted without affecting its function and causing fatal consequences, thereby providing the option to maintain the secondary structure. In some embodiments, the primary structure of the stem (i.e., its nucleotide sequence) can still be changed while still maintaining the helical region. The nature of the bases is related to the higher order structure, and substitutions are possible as long as they maintain the secondary structure. In some embodiments, the cyclic polyribonucleotide has a pseudo-helical structure. In some embodiments, the cyclic polyribonucleotide has at least one segment with a pseudo-helical structure. In some embodiments, the segment having a pseudo-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 cyclic polyribonucleotide has one or more segments (e.g., 2, 3, 4, 5, 6 or more) having a pseudo-helical structure. In some embodiments, the segments are separated by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides. In some embodiments, the cyclic polyribonucleotide comprises at least one U-rich or A-rich sequence or a combination thereof. In some embodiments, the U-rich and / or A-rich sequence is arranged to generate a triple quasi-helical structure. In some embodiments, the cyclic polyribonucleotide has a double quasi-helical structure.In some embodiments, the cyclic polyribonucleotide has one or more segments (e.g., 2, 3, 4, 5, 6 or more) with a double pseudo-helical structure. In some embodiments, the cyclic polyribonucleotide includes at least one C-rich and / or G-rich sequence. In some embodiments, the C-rich and / or G-rich sequence is arranged to generate a triple pseudo-helical structure. In some embodiments, the cyclic polyribonucleotide has an intramolecular triple pseudo-helical structure that promotes stabilization.

[0243]

[0331] In one embodiment, the cyclic polyribonucleotide has two pseudo-helical structures (e.g., separated by a phosphodiester bond) whose terminal base pairs are stacked such that the pseudo-helical structures are collinear, resulting in a "coaxially stacked" substructure.

[0244]

[0332] In certain embodiments, the cyclic polyribonucleotide comprises a tertiary structure having one or more motifs, such as a pseudoknot, a G-quadruplex, a helix, and coaxial stacking.

[0245]

[0333] In certain embodiments, the cyclic polyribonucleotide has at least one binding site, such as at least one protein binding site, at least one miRNA binding site, at least one lncRNA 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.

[0246] delivery

[0334] The cyclic polyribonucleotides described herein can also be included in pharmaceutical compositions along with delivery vehicles.

[0247]

[0335] The pharmaceutical compositions described herein can be formulated with carriers such as, for example, pharmaceutical and / or polymeric carriers, e.g., liposomes, and can be delivered to a subject in need thereof (e.g., a human or 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 polymer, calcium phosphate, dendrimer); electroporation or other methods of membrane disruption (e.g., nucleofection); viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, particle bombardment ("gene gun"), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. Delivery methods are described, for example, in Gori et al., "Delivery and Specificity of CRISPR / Cas9 Genomes," in Editing Technologies for Human Gene Therapy.Human Gene Therapy.July 2015, 26(7):443-451.doi:10.1089 / hum.2015.074; and Zuris et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct 30;33(1):73-80.

[0248]

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

[0249]

[0337] In some embodiments, the cyclic polyribonucleotide is non-immunogenic in the host. In some embodiments, the cyclic polyribonucleotide elicits a reduced or incapable of generating a response by the host's immune system compared to a response elicited by a reference compound, such as a linear polynucleotide corresponding to the described cyclic polyribonucleotide or a cyclic polyribonucleotide lacking an encryptogen. 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).

[0250]

[0338] In certain embodiments, a host or host cell is contacted with (e.g., delivered or administered to) a cyclic polyribonucleotide. In certain embodiments, the host is a mammal, such as a human. The amount of cyclic 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 cyclic polyribonucleotide, the cyclic polyribonucleotide, or expression product, or both, is identified as effective in increasing or decreasing host growth.

[0251] How to generate

[0339] In certain embodiments, the cyclic polyribonucleotide comprises a deoxyribonucleic acid sequence that is non-naturally occurring and can be produced using recombinant techniques (e.g., in vitro using DNA plasmids, as described in detail below) or chemical synthesis.

[0252]

[0340] It is within the scope of the present invention that the DNA molecules used to generate the RNA circles may comprise DNA sequences encoding the original naturally occurring nucleic acid sequence, modified versions thereof, or synthetic polypeptides not normally found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules may 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 mixtures of oligonucleotides and ligation of mixtures to "assemble" mixtures of nucleic acid molecules, and combinations thereof.

[0253]

[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 concatemerized to produce the circular polyribonucleotides described herein. The circularization or concatemerization mechanism can be performed by methods such as, but not limited to, chemical, enzymatic, splint ligation, or ribozyme-catalyzed methods. The newly formed 5'- / 3'-bond can be an intramolecular bond or an intermolecular bond.

[0254]

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

[0255]

[0343] Various methods for synthesizing cyclic polyribonucleotides have also been described in the art (see, e.g., U.S. Pat. No. 6,210,931, U.S. Pat. No. 5,773,244, U.S. Pat. No. 5,766,903, U.S. Pat. No. 5,712,128, U.S. Pat. No. 5,426,180, U.S. Patent Application Publication No. 20100137407, WO 1992001813, and WO 2010084371, the contents of each of which are incorporated herein by reference in their entirety).

[0256]

[0344] In some embodiments, the cyclic polyribonucleotides may be cleaned up after production to remove production impurities, such as free ribonucleic acid, linear or nicked RNA, DNA, proteins, etc. In some embodiments, the cyclic polyribonucleotides may be purified by any known method commonly used in the art. Non-limiting examples of purification methods include column chromatography, gel exclusion, size exclusion, etc.

[0257] Pharmaceutical Composition

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

[0258]

[0346] Although the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and a veterinary pharmacologist of ordinary skill could design and / or make such modifications with only routine experimentation, if any. Subjects to which the pharmaceutical compositions are contemplated include, but are not limited to, humans and / or other primates; cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys; and / or mammals, including commercially relevant mammals such as birds.

[0259]

[0347] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparatory methods include the step of combining the active ingredient with an excipient and / or one or more other accessory ingredients, and then, as necessary and / or desired, portioning, shaping, and / or packaging the product.

[0260] Method of Expression

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

[0261]

[0349] In some embodiments, the methods for protein expression involve 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 a cyclic polyribonucleotide into a polypeptide. In some embodiments, the methods for protein expression involve translation of a cyclic polyribonucleotide into a polypeptide of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, 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 certain embodiments, the method for protein expression comprises translation of a circular polyribonucleotide into a polypeptide of about 5, 10, 15, 20, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 amino acids. In certain embodiments, the method comprises translation of a circular polyribonucleotide into a contiguous polypeptide provided herein, a separate polypeptide provided herein, or both.

[0262]

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

[0263]

[0351] In one aspect, the present disclosure provides a method for in vivo expression of one or more expression sequences in a subject, the method comprising: administering a cyclic polyribonucleotide comprising one or more expression sequences to cells of the subject; and expressing the one or more expression sequences from the cyclic polyribonucleotide in the cells. In one embodiment, the cyclic polyribonucleotide is configured so that expression of the one or more expression sequences in the cells at a later time point is equal to or greater than that at an earlier time point. In one embodiment, the cyclic polyribonucleotide is configured so that expression of the one or more expression sequences in the cells does not decrease by more than about 40% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or more. In one embodiment, the cyclic polyribonucleotide is configured so that expression of the one or more expression sequences in the cells is maintained at a level that does not change by more than about 40% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or more. In one embodiment, the cyclic polyribonucleotide is administered using any of the delivery methods described herein. In some embodiments, the cyclic polyribonucleotide is administered to a subject by intravenous injection. In some embodiments, administration of the cyclic polyribonucleotide includes, but is not limited to, prenatal administration, neonatal administration, postnatal administration, oral administration, injection (e.g., intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous, and intramuscular), intraocular administration, and intranasal administration.

[0264]

[0352] In some embodiments, methods for protein expression include modification, folding, or other post-translational modifications of the translation product, hi some embodiments, methods for protein expression include post-translational modifications in vivo, for example, via cellular machinery.

[0265]

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

[0354] The above-described embodiments can be combined to obtain the above-described functional properties. This is also illustrated by the following examples, which describe exemplary combinations and the functional properties that can be obtained. Table 1 provides an exemplary overview showing how the various elements described above can be combined and the functional properties that can be observed.

[0266] [Table 1-1]

[0267] [Table 1-2] [Example]

[0268]

[0355] The following examples are provided to further illustrate certain embodiments of the present invention, but are not intended to limit the scope of the invention; by their illustrative nature, it will be understood that other procedures, methods, or techniques known to those skilled in the art may be substituted.

[0269] Example 1: In vitro circular RNA generation

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

[0270]

[0357] As shown in Figure 2, circular RNAs are designed with a start codon (SEQ ID NO: 1), ORF (SEQ ID NO: 2), stagger element (SEQ ID NO: 3), encryptogen (SEQ ID NO: 4), and IRES (SEQ ID NO: 5). Circularization allows for rolling circle translation, multiple open reading frames (ORFs) with alternating stagger elements for separate ORF expression and controlled protein stoichiometry, and an optional IRES to target the RNA for ribosome entry without encryptogen and poly-A sequences to attenuate or mitigate RNA immunogenicity.

[0271]

[0358] In this example, circular RNA is generated as follows: Unmodified linear RNA is synthesized by in vitro transcription using T7 RNA polymerase from a DNA segment containing an ORF encoding GFP linked to the 5'- and 3'-ZKSCAN1 introns and 2A sequences. The transcribed RNA is 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 the RNA purification system.

[0272]

[0359] Splint-ligated circular RNA is generated by treatment of transcribed linear RNA and DNA splints with T4 DNA ligase (New England Bio, Inc., M0202M), and circular RNA is isolated after enrichment by RNase R treatment. RNA quality is assessed by agarose gel or automated electrophoresis (Agilent).

[0273] Example 2: In vivo circular RNA production, cell culture

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

[0274]

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

[0275]

[0362] 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% CO. 1 μg of the above expression plasmid is transfected using a lipid transfection reagent (Life Technologies), and total RNA from transfected cells is isolated 1 h to 20 days post-transfection using a phenol-based RNA isolation reagent (Life Technologies) according to the manufacturer's instructions.

[0276]

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

[0277]

[0364] Further RNA measurements are performed using Northern blot analysis. Briefly, whole cell extracts are obtained using a phenol-based reagent (TRIzol), or nuclear and cytoplasmic protein extracts are obtained by cell fractionation using a commercially available kit (CelLytic NuCLEAR Extraction Kit, Sigma). To inhibit RNA polymerase II transcription, cells are treated with flavopiridol (1 mM final concentration; Sigma) for 0–6 h at 37 °C. For RNase R treatment, 10 mg of total RNA is treated with 20 U of RNase R (Epicentre) for 1 h at 37 °C.

[0278]

[0365] Northern blots using oligonucleotide probes are performed as follows: Oligonucleotide probes and PCR primers are designed using standard primer design tools. A T7 promoter sequence is added to the reverse primer to generate an antisense probe for in vitro transcription reactions. In vitro transcription is performed using T7 RNA polymerase with DIG-RNA labeling mix according to the manufacturer's instructions. The DNA template is removed by DNA I digestion, and the RNA probe is purified by phenol-chloroform extraction and subsequent precipitation. The probe is used at 50 ng / ml. Total RNA (2 μg–10 μg) is denatured using glyoxal loading dye (Ambion) and resolved on a 1.2% agarose gel in MOPS buffer. The gel is immersed in 1x TBE for 20 minutes and transferred to a Hybond-N+ membrane (GE Healthcare) for 1 hour (15 V) using a semi-dry blotting system (Bio-Rad). The membrane is dried and subjected to a 120,000 μJ / cm 2 The membranes were then UV-crosslinked (at 265 nm) once at 68°C. Prehybridization was performed for 1 hour at 68°C, followed by overnight hybridization with a DIG-labeled in vitro transcribed RNA probe. The membranes were washed three times for 30 minutes at 68°C with 2x SSC, 0.1% SDS, followed by three washes for 30 minutes at 68°C with 0.2x SSC, 0.1% SDS. Immunodetection was performed using anti-DIG directly conjugated to an alkaline phosphatase antibody. Immunoreactive beads were visualized using a chemiluminescent alkaline phosphatase substrate (CDP star reagent) and an imaging detection and quantification system (LAS-4000 detection system).

[0279] Example 3: Preparation of circular RNA and in vitro translation

[0366] This example demonstrates gene expression and detection of a gene product from circular RNA.

[0280]

[0367] In this example, a circular RNA is designed with a start codon (SEQ ID NO: 1), a GFP ORF (SEQ ID NO: 2), a stagger element (SEQ ID NO: 3), a human-derived encryptogen (SEQ ID NO: 4), and with or without an IRES (SEQ ID NO: 5) (see Figure 4). In this example, the circular RNA is generated in vitro or intracellularly as described in Examples 1 and 2.

[0281]

[0368] The circular RNA was incubated in rabbit reticulocyte lysate (Promega, Fitzburgh, WI, USA) for 5 hours or overnight at 30°C. The final composition of the reaction mixture was 70% rabbit reticulocyte lysate, 10 μM methionine and leucine, 20 μM amino acids other than methionine and leucine, and 0.8 U / μL RNase inhibitor (Toyobo, Osaka). An aliquot was taken from the mixture and separated on a 10-20% gradient polyacrylamide / sodium dodecyl sulfate (SDS) gel (Atto, Tokyo). 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. Hemoglobin protein was removed during this process, while non-hemoglobin proteins were enriched.

[0282]

[0369] After centrifugation at 1,400 × g for 5 min, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel. Commercially available standards (Bio-Rad) were used as size markers. After electrophoretic transfer to polyvinylidene fluoride (PVDF) membranes (Millipore) using the semi-dry method, the blots were visualized using a chemiluminescence kit (Rockland).

[0283]

[0370] GFP protein is visualized in cell lysates and is expected to be detected in greater amounts in circular than linear RNA as a result of rolling circle translation.

[0284] Example 4: Stoichiometric protein expression from circular RNA

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

[0285]

[0372] In this example, one circular RNA is designed to contain an encryptogen (SEQ ID NO: 4) and an ORF encoding GFP (SEQ ID NO: 2) and an ORF encoding RFP (SEQ ID NO: 8), with staggered elements (SEQ ID NO: 3) flanking the GFP and RFP ORFs (see Figure 5). Another circular RNA is similarly designed, but instead of flanking 2A sequences, it has a stop and start codon between the GFP and RFP ORFs. Circular RNAs are generated in vitro or in cells as described in Examples 1 and 2.

[0286]

[0373] The circular RNA was incubated in rabbit reticulocyte lysate (Promega, Fitzburgh, WI, USA) for 5 hours or overnight at 30°C. The final composition of the reaction mixture was 70% rabbit reticulocyte lysate, 10 μM methionine and leucine, 20 μM amino acids other than methionine and leucine, and 0.8 U / μL RNase inhibitor (Toyobo, Osaka). An aliquot was taken from the mixture and separated on a 10-20% gradient polyacrylamide / sodium dodecyl sulfate (SDS) gel (Atto, Tokyo). 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. Hemoglobin protein was removed during this process, while non-hemoglobin proteins were enriched.

[0287]

[0374] After centrifugation at 1,400 × g for 5 min, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel. Commercially available standards (Bio-Rad) were used as size markers. After electrophoretic transfer to polyvinylidene fluoride (PVDF) membranes (Millipore) using the semi-dry method, the blots were visualized using a chemiluminescence kit (Rockland).

[0288]

[0375] Circular RNAs in which the GFP and RFP ORFs are not separated by a stop and start codon are predicted to have equal amounts of both proteins, whereas cells treated with circular RNAs containing a start and stop codon between the ORFs are predicted to have different amounts of both proteins.

[0289] Example 5: Non-immunogenicity in cell culture

[0376] This example demonstrates the in vivo evaluation of the immunogenicity of circular RNA following cell infection.

[0290]

[0377] In this example, circular RNAs are designed to contain an encryptogen, e.g., a ZKSCAN1 intron, and a GFP ORF. Additionally, control circular RNAs are designed to contain the GFP ORF, with or without an intron (see Figure 6). Circular RNAs are generated in vitro or intracellularly as described in Examples 1 and 2. HeLa cells are transfected with 500 ng of circular RNA.

[0291]

[0378] Transfection of circular RNA 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); (4) cationic polymers (PEI, polybrene, DEAE-dextran) (Turbofect); (4) virus-like particles (L1 from HPV, VP1 from polyomavirus) (Tonges et al., 2006); (5) exosomes (Exo-Fect from SBI); (6) nanostructured calcium phosphate (nanoCaP) (Olton et al., 2006); (6) peptide transduction domains (TAT, polyR, SP, pVEC, SynB1, etc.) (Zhang et al., 2009); (7) vesicles (VSV-G, TAMEL) (Liu et al., 2015). (2017); (8) cell squeezing; (SQZ Biotechnologies); (9) nanoparticles (Neuhaus et al., 2016); and / or (10) magnetofection (Mair et al., 2009). The transfection method is performed in cell culture medium (DMEM 10% FBS), and the cells are then cultured for 24–48 hours.

[0292]

[0379] Two to 48 hours after transfection, the medium is removed and the relative expression of the indicated RNAs and transfected RNAs is measured by qRT-PCR.

[0380] For qRT-PCR analysis, total RNA was isolated from cells using phenol-based RNA isolation solution (TRIzol) and RNA (Qiagen) according to the manufacturer's instructions. qRT-PCR analysis was performed in triplicate using a PCR master mix (Brilliant II SYBR Green qRT-PCR Master Mix) and a PCR cycler (LightCycler 480). The mRNA levels of well-known innate immune regulators, such as RIG-I, MDA5, OAS, OASL, and PKR, were quantified and normalized to actin, GAPDH, or HPRT levels. The relative expression of the indicated RNA genes for circular RNA transfections was normalized by the level of transfected RNA and compared to the expression levels of cells transfected with circular RNA containing no encryptogen.

[0293]

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

[0382] GFP-positive cells containing the encryptogen are predicted to exhibit an attenuated immunogenic response.

[0294]

[0383] Furthermore, (1) primary mouse dendritic cells; (2) human embryonic kidney 293 cells (InvivoGen) stably expressing TLR-7, 8, or 9; (3) monocyte-derived dendritic cells (AllCells); or (4) Raw 264.7 cells were transfected with DNA plasmids containing the ZKSCAN1 or td intron that generate circular RNA encoding GFP as described above. Six to 48 hours after transfection, cell culture supernatants were collected, and cytokine expression was measured using ELISA. When cell culture supernatants were collected, cells were harvested for Northern blot, gene expression array, and FACS analysis.

[0295]

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

[0296]

[0385] For Northern blot analysis, samples are processed and analyzed as described above. Probes are derived from plasmids specific for the coding regions of human IFN-α 13, IFN-β (Open Biosystems), TNF-α, or GAPDH (ATCC). Cells transfected with circular RNA containing the encryptogen are expected to have reduced cytokine expression compared to control-transfected cells.

[0297]

[0386] For gene expression arrays, RNA is isolated using a phenol-based solution (TRIzol) and / or an RNA isolation kit (RNeasy, Qiagen). The RNA is amplified and analyzed (e.g., on an Illumina human HT12v4 chip in an Illumina BeadStation 500GX). The levels in mock-control treated cells are used as a baseline for calculating fold increases. Cells transfected with circular RNA containing the encryptogen are expected to have reduced cytokine expression compared to control-transfected cells.

[0298]

[0387] For FACS analysis, cells are stained with directly conjugated antibodies against CD83 (Research Diagnostics Inc.), HLA-DR, CD80, or CD86 and analyzed in a flow cytometer. Cells transfected with encryptogen-containing circular RNA are expected to show reduced expression of these markers compared to control-transfected cells.

[0299] Example 6: Riboswitches for selective expression

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

[0300]

[0389] In this example, a circular RNA is designed to contain an encryptogen (SEQ ID NO: 4), a synthetic riboswitch (SEQ ID NO: 9) that controls expression of an ORF encoding GFP (SEQ ID NO: 2), along with staggered elements (2A sequences) (SEQ ID NO: 3) flanking the GFP ORF (see Figure 7). Circular RNAs are generated in vitro or intracellularly as described in Examples 1 and 2.

[0301]

[0390] Theophylline induces activation of the riboswitch, resulting in an off-switch of gene expression (as described by Auslander et al., 2010). The riboswitch is predicted to control GFP expression from the circular RNA. Observing GFP expression in the presence of theophylline was unexpected.

[0302]

[0391] HeLa cells are 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 evaluate selective expression. The transfection method is as described in Example 5.

[0303]

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

[0304] Example 7: In vivo expression

[0393] This example demonstrates the ability to express protein from circular RNA in vivo.

[0305]

[0394] In this example, a circular RNA is designed to contain an encryptogen (SEQ ID NO:4) and an ORF encoding GFP (SEQ ID NO:2) or RFP (SEQ ID NO:8) or luciferase (SEQ ID NO:10), with staggered elements (SEQ ID NO:3) flanking the GFP, RFP, or luciferase ORFs (see Figure 8). The circular RNA is generated in vitro or in cells as described in Examples 1 and 2.

[0306]

[0395] Administer 300 mg / kg (6 mg) of circular RNA (50 µL volume) to 6-8 week-old male BALB / c mice via intradermal (ID), intramuscular (IM), oral (PO), intraperitoneal (IP), or intravenous (IV) administration along with the GFP, RFP, or luciferase ORF described herein or linear RNA as a control. Animals are given a single dose or three injections (on days 1, 3, and 5).

[0307]

[0396] Blood, heart, lungs, spleen, kidneys, liver, and skin injection site are collected from untreated control mice and at 2, 4, 8, 24, 48, 72, 96, 120, 168, and 264 hours post-dose (n=4 mice / time point). Blood samples are collected by jugular vein puncture at the end of the study.

[0308]

[0397] Circular RNA quantification for both serum and tissues is performed using branched DNA (bDNA) quantification (Panomics / Affymetrix). A standard curve on each plate of known amounts of RNA (added to untreated tissue samples) is used to quantify RNA in treated tissues. The amount, calculated in picograms (pg), is normalized to the amount of tissue weighed in the lysate applied to the plate. Protein expression (RFP or GFP) is assessed by FACS or Western blot in each tissue, as described in the previous example.

[0309]

[0398] Another group of mice administered luciferase circular RNA was injected with 3 mg of luciferin at 6, 24, 48, 72, and 96 hours after administration, and the animals were imaged in an in vivo imaging system (IVIS Spectrum, PerkinElmer). Six hours after administration, three animals were sacrificed and dissected, and the muscle, skin, draining lymph nodes, liver, and spleen were imaged ex vivo.

[0310]

[0399] The mice are expected to express GFP, RFP or luciferase in the treated tissues.

[0311] Example 8: In vivo biodistribution

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

[0312]

[0401] In this example, mice are treated with circular RNA encoding luciferase, as described in Example 9. Briefly, the circular RNA is designed to contain an encryptogen (SEQ ID NO: 4) and an ORF encoding luciferase (SEQ ID NO: 10), with staggered elements (SEQ ID NO: 3) flanking the luciferase ORF (see Figure 9). The circular RNA is generated in vitro or intracellularly, as described in Examples 1 and 2.

[0313]

[0402] Mice were administered luciferase circular RNA and injected with 3 mg of luciferin at 6, 24, 48, 72, and 96 hours after administration, and the animals were imaged in an in vivo imaging system (IVIS Spectrum, PerkinElmer). Six hours after administration, three animals were sacrificed and dissected, and the muscle, skin, draining lymph nodes, liver, and spleen were imaged ex vivo.

[0314]

[0403] Circular RNA quantification for both serum and tissue is performed using branched DNA (bDNA) quantification (Panomics / Affymetrix). A standard curve on each plate of known amounts of RNA (added to untreated tissue samples) is used to quantify RNA in treated tissue. The amount, calculated in picograms (pg), is normalized to the amount of tissue weighed in the lysate applied to the plate.

[0315]

[0404] Separate groups of 6-8 week-old male BALB / c mice were administered luciferase circular RNA intramuscularly (IM) or intradermally (ID) at four dose levels: 10, 2, 0.4, and 0.08 mg (n = 6 per group). At 6, 24, 48, 72, and 96 hours post-administration, the animals were injected with 3 mg of luciferin and imaged in an in vivo imaging system (IVIS Spectrum, PerkinElmer). Six hours post-administration, three animals were sacrificed, dissected, and muscle, skin, draining lymph nodes, liver, and spleen were imaged ex vivo. Tissues from the mice were also evaluated for luciferase expression and the tissue distribution of this expression was analyzed, as described in Example 9.

[0316]

[0405] Mice are expected to exhibit luciferase expression in treated tissues.

[0317] Example 9: Non-immunogenic in vivo

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

[0318]

[0407] This example describes the quantification and comparison of immune responses following administration of encryptogen-bearing circular RNAs (see Figure 10). In one embodiment, any of the encryptogen-bearing circular RNAs reduces the immunogenic response following one or more administrations of the circular RNA compared to a control (e.g., compared to administration of a control RNA).

[0319]

[0408] An indicator of immunogenicity for circular RNA is cytokine levels in serum.

[0409] In this example, cytokine serum concentrations are examined after one or more administrations of circular RNA. Circular RNA from any one of the previous 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: mice injected systemically and / or locally with circular RNA containing an encryptogen and with circular RNA without an encryptogen.

[0320]

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

[0321]

[0411] In addition to serum cytokine levels, the expression of inflammatory markers is another indicator of immunogenicity. In this example, spleen tissues from mice treated with vehicle (no circular RNA), linear RNA, or circular RNA were collected 1, 4, and 24 hours after administration. Samples were analyzed using the following techniques: qRT-PCR analysis, Northern blot, or FACS analysis.

[0322]

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

[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.

[0323]

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

[0324]

[0415] In one embodiment, the encryptogen-containing circular RNA will reduce cytokine levels (as measured by ELISA, Northern blot, FACS and / or qRT-PCR) after one or more administrations compared to control RNA.

[0325] Example 10: Circular RNA contains at least one double-stranded RNA segment

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

[0326]

[0417] In this example, circular RNA is synthesized to contain a GFP ORF and an IRES by one of the methods described above (see Figure 11). A dot blot assay with J2 and K1 monoclonal antibodies is used to measure double-stranded RNA structures of at least 40 bp in length. Circular RNA (200 ng) is blotted onto a nylon membrane (supercharged Nytran), dried, and then resuspended in TBS-T buffer. The membranes were blocked with 5% nonfat dry milk in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4, and incubated with dsRNA-specific mAb J2 or K1 (English & Scientific Consulting) for 60 minutes. The membranes were 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 chemiluminescence Western blot detection reagents (Amersham).

[0327]

[0418] Circular RNAs are predicted to generate internal pseudo-double-stranded RNA segments.

[0328]

[0419] Example 11: Circular RNA contains a pseudo-double-stranded structure

[0420] This example demonstrates that circular RNA contains a pseudo-double-stranded structure.

[0329]

[0421] In this example, circular RNA is synthesized by one of the methods described above, with and without the addition of HDVmin expression (Griffin et al., 2014). This RNA sequence forms a pseudohelical structure (see Figure 12) and is used as a positive control (as shown by Griffin et al., 2014).

[0330]

[0422] To test whether the circular RNA structure contains a functional pseudoduplex, we determine the secondary structure using selective 2'OH acylation analyzed by primer extension (SHAPE). SHAPE assesses the local backbone flexibility of RNA at single-nucleotide resolution. The reactivity of base positions to SHAPE electrophiles is related to the secondary structure: base-paired positions are less reactive, and unpaired positions are more reactive.

[0331]

[0423] SHAPE was performed on circular RNA, HDVmin, and linear RNA. SHAPE was performed using N-methylisatoic anhydride (NMIA) or benzoyl cyanide (BzCN) essentially as described by Wilkinson et al. (2006) and Griffin et al. (2014), respectively. Briefly, for SHAPE using BzCN, 1 μl of 800 mM BzCN in dimethyl sulfoxide (DMSO) was added to a 20 μl reaction mixture containing 3–6 pmol of RNA in 160 mM Tris, pH 8.0, and 1 U / L of RNAse inhibitor (e.g., SuperaseI RNase Inhibitor) and incubated at 37°C for 1 min. A control reaction mixture contained 1 μl of DMSO without BzCN. After incubation with BzCN, the RNA is extracted with phenol-chloroform, purified as directed by the manufacturer (e.g., using the RNA Clean & Concentrator-5 kit), and resuspended in 6 μl of 10 mM Tris, pH 8.0. A single-dye system is used to detect BzCN adducts. The RNA is annealed with a primer labeled with 6-carboxyfluorescein (6-FAM). Primer extension is performed using reverse transcriptase (SuperScript III, Invitrogen) according to the manufacturer's recommendations, with the following modifications to the incubation conditions: 42°C for 5 minutes, 55°C for 30 minutes, 65°C for 25 minutes, and 75°C for 15 minutes. Two sequencing ladders are generated using either 0.5 mM ddATP or 0.5 mM ddCTP in the primer extension reaction. Primer extension products are precipitated with ethanol, washed to remove excess salt, and resolved by capillary electrophoresis alongside commercially available size standards (eg, Liz size standards, Genewiz Fragment Analysis Service).

[0332]

[0424] The intact electropherograms are analyzed using a primary fragment analysis tool (e.g., PeakScanner, Applied Biosystems). Peaks at each position on the electropherogram are then integrated. For each RNA analyzed, y-axis scaling to correct for loading error is performed so that the background for each primer extension reaction is normalized to the background of a negative control reaction performed on RNA not treated with BzCN. Signal decay correction is performed for each reaction. The data is applied. Peaks are aligned to a ladder generated from two sequencing reactions. At each position, the peak area of ​​the negative control is subtracted from the peak area in the BzCN-treated sample; these values ​​are then converted to normalized SHAPE reactivity by dividing the subtracted peak area by the average of the top 2%–10% of subtracted peak areas.

[0333]

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

[0334]

[0426] Circular RNAs are predicted to have a pseudo-double-stranded structure.

[0335] Example 12: Circular RNA contains a functional pseudohelical structure

[0427] This example demonstrates that circular RNA contains a functional pseudohelical structure.

[0336]

[0428] In this example, circular RNA is synthesized by one of the methods described above (Defenbaugh et al., 2009), with the addition of expression of 395L. This RNA sequence is folded into a pseudohelical structure (as shown above by the RNA secondary structure folding algorithm mfold and Defenbaugh et al., 2009), Figure 13. This structure is essential for complex formation with the hepatitis D antigen (HDAg).

[0337]

[0429] Therefore, to test whether the circular RNA structure contains a functional pseudostructure, we incubated circular and linear RNA with HDAg-160 or HDAg-195 and analyzed binding using an EMSA assay. Binding reactions were performed in 25 μl of 10 mM Tris-HCl (pH 7.0), 25 mM KCl, 10 mM NaCl, 0.1 g / L bovine serum albumin (New England Biolabs), 5% glycerol, 0.5 mM DTT, 0.2 U / L RNase inhibitor (Applied Biosystems), and 1 mM phenylmethylsulfonyl fluoride. The circular RNA was incubated with HDAg protein (obtained as described by Defenbaugh et al., 2009) at concentrations ranging from 0 to 110 nM. The reaction mixture is collected on ice, incubated at 37°C for 1 hour, and electrophoresed on a 6% native polyacrylamide gel in 0.5% Tris-borate-EDTA at 240V for 2.5 hours. The levels of free and bound RNA are determined using a nucleic acid stain (e.g., Gel Red). Binding is calculated as the intensity of unbound RNA relative to the intensity of all lanes minus background.

[0338]

[0430] Circular RNAs are predicted to have a functional pseudohelical structure.

[0339] Example 13: Self-transcription / replication

[0431] In this example, a circular RNA is synthesized by one of the methods described above, with the addition of expression of an HDV replication domain (as described by Beeharry et al., 2014), an antigenome replication-competent ribozyme, and a nuclear localization signal. These RNA sequences allow the circular RNA to localize to the nucleus, where host RNA polymerase binds to and transcribes the RNA. This RNA is then self-cleaved using the ribozyme. The RNA is then ligated and self-replicated again (see Figure 14).

[0340]

[0432] Circular RNA (1-5 μg) is transfected into HeLa cells using the techniques described above. HeLa cells are grown at 37°C and 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) containing high glucose (Life Technologies) supplemented with penicillin-streptomycin and 10% fetal bovine serum. After transfection, HeLa cells are cultured for an additional 4-72 hours. Total RNA from transfected cells is then isolated 1 hour to 20 days posttransfection using a phenol-based RNA isolation reagent (Life Technologies) according to the manufacturer's instructions. The total amount of circular RNA encoding the HDV domain is determined and compared to control circular RNA using qPCR as described herein.

[0341] Example 14: Circular RNA stability / half-life

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

[0342]

[0434] Human fibroblasts (e.g., IMR-90) are grown to confluence in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C under 5% CO2 on tissue culture-treated plates. When fibroblasts reach confluence, they cease division due to contact inhibition (Leontieva et al., 2014). Lipid transfection reagent (2 μL; Invitrogen) is added to a mixture of 1 μg of circular or linear RNA (described above) and 145 μL of serum-reduced 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 × 10 cells suspended in DMEM containing 10% FBS are transfected. 5 Add the cells to the circular RNA solution (described above).

[0343]

[0435] Cells are cultured and then harvested at 1, 2, 3, 4, 5, 10, 20, and 30 days after circular RNA transfection. Cells are isolated for q-rt-PCR and a separate subset for FACS analysis. To measure GFP circular RNA and mRNA levels, qPCR reverse transcription using random hexamers is performed as described in Example 2. Cells are analyzed using GFP antibodies and FACS as described herein.

[0344]

[0436] The circular RNAs are expected to persist in cells for at least several days and retain functional expression of the GFP protein.

[0345] Example 15: Circular RNA preservation in daughter cells

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

[0346]

[0438] Human fibroblasts (e.g., IMR-90) are grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C under 5% CO on tissue culture-treated plates. Cells are passaged periodically to maintain exponential growth. Lipid transfection reagent (2 μL; Invitrogen) is added to a mixture of 1 μg of circular or linear RNA (described above) and 145 μL of serum-reduced medium (Opti-MEM I solution) in one well of a 12-well tissue culture-treated plate. After 15 minutes of incubation at room temperature, 1 × 10 cells suspended in DMEM containing 10% FBS are transfected. 5 HeLa cells are added to the circular RNA solution (described above). After 24 hours of incubation at 37°C and 5% CO2, the cells are pulsed with BrdU (e.g., Sigma-Aldrich). The BrdU labeling period is optimized for each cell type according to the doubling time of their specific population; for example, IMR-90 human fibroblasts, which have a doubling time of 27 hours, are pulsed for 8-9 hours, as described by Elabd et al. (2013).

[0347]

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

[0348]

[0440] The circular RNA persists in the daughter cells, which are predicted to express the GFP protein.

[0349] Example 16: Circular RNA circularization

[0441] This example demonstrates the in vitro generation of circular RNA using splint ligation.

[0350]

[0442] Non-natural circular RNAs can be engineered to contain one or more desirable properties and can be produced using recombinant DNA techniques. As shown in the examples below, splint ligation circularized linear RNA.

[0351]

[0443] Circular RNA1 was designed to encode triple FLAG-tagged EGF without a stop codon (264 nt). It has a Kozak sequence (SEQ ID NO: 11) at the start codon for translation initiation. Circular RNA2 has the same sequence as circular RNA1, except that it has a stop element (triple stop codon) (273 nt, SEQ ID NO: 12). Circular RNA3 was designed to encode triple FLAG-tagged EGF without a stop element (stop codon) (330 nt) and flanked by stagger elements (2A sequence, SEQ ID NO: 13). Circular RNA4 has the same sequence as circular RNA3, except that it has a stop element (triple stop codon) (339 nt).

[0352]

[0444] In this example, circular RNA was generated as follows. DNA templates for in vitro transcription were amplified from gBlocks gene fragments containing the corresponding sequence (IDT) with a forward primer containing a T7 promoter and a reverse primer containing 2-O-methylated nucleotides. The amplified DNA templates were gel-purified using a DNA gel purification kit (Qiagen). 250–500 ng of purified DNA template was subjected to in vitro transcription. Linear, 5′-monophosphorylated in vitro transcripts were generated from each DNA template containing the corresponding sequence using T7 RNA polymerase 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. Approximately 40 μg of linear RNA was generated in each reaction. After incubation, each reaction was treated with DNase to remove the DNA template. In vitro transcribed RNA was precipitated with ethanol in the presence of 2.5 M ammonium acetate to remove unincorporated monomers.

[0353]

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

[0354]

[0446] Circularization of linear RNA was monitored by separating the RNA on a 6% denaturing PAGE. The slower-migrating RNA bands are more consistent with circular RNA than linear RNA in denaturing polyacrylamide gels due to their circular structure. As can be seen in Figure 17, the addition of ligase to the RNA mixture (+ lane) generated a new band that appeared to exceed the linear RNA band present in the mixture lacking ligase (- lane). The slower-migrating band was seen in all RNA mixtures, indicating successful splint ligations (e.g., circularization) performed on multiple constructs compared to the negative control.

[0355] Example 17: RNA circularization efficiency

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

[0356]

[0448] Non-natural circular RNAs engineered to contain one or more desirable properties can be generated using splint-mediated circularization. As shown in the examples below, splint ligation circularized linear RNAs with higher efficiency than controls.

[0357]

[0449] Circular RNA1, Circular RNA2, Circular RNA3, and Circular RNA4 described in Example 1 were also used here. Circular RNA5 was designed to encode FLAG-tagged EGF flanked by 2A sequences, followed by FLAG-tagged nanoluciferase (873 nt, SEQ ID NO: 17). Circular RNA6 has the same sequence as circular RNA5, except that it contained 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).

[0358]

[0450] In this example, to measure the efficiency of RNA circularization, six different sizes of linear RNA (264 nt, 273 nt, 330 nt, 339 nt, 873 nt, and 762 nt) were generated and circularized as described in Example 1. The circular RNAs were resolved by 6% denaturing PAGE, and the corresponding RNA bands in the gel for linear or circular RNA were excised for purification. The excised RNA gel bands were crushed, and the RNA was eluted overnight in 800 μl of 300 mM NaCl. The gel debris was removed by centrifugation filtering, and the RNA was precipitated with ethanol in the presence of 0.3 M sodium acetate.

[0359]

[0451] Circularization efficiency was calculated as follows: the amount of eluted circular RNA divided by the amount of total RNA eluted (circular + linear RNA), and the results are shown as a graph in FIG.

[0452] Ligation of linear RNA with T4 RNAse ligase 2 produced circular RNA at a higher efficiency rate than the control. Trend data showed that more constructs were circularized at a higher rate.

[0360] Example 18: Circular RNA lacking sensitivity to degradation

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

[0361]

[0454] Circular RNAs are more resistant to exonuclease degradation than linear RNAs due to the lack of 5' and 3' ends. As shown in the examples below, circular RNAs are less susceptible to degradation than their linear RNA counterparts.

[0362]

[0455] Circular RNA5 was generated and circularized as described in Example 2 for use in the assays described herein.

[0456] To test for circularization of circular RNA5, 20 ng / μl of linear or circular RNA5 was incubated for 30 min at 37°C with 2 U / μl of RNAse R, a 3'-5' exoribonuclease that digests linear RNA but not lariat or circular RNA structures. After incubation, the reaction mixture was analyzed by 6% denaturing PAGE.

[0363]

[0457] The linear RNA band present in the lane lacking exonuclease is a circular RNA. It was absent in lane A5 (see FIG. 19), indicating that circular RNA5 exhibited higher resistance to exonuclease treatment compared to the linear RNA control.

[0364] Example 19: Isolation and purification of circular RNA

[0458] This example demonstrates circular RNA purification.

[0365]

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

[0366]

[0460] Circular RNA1, circular RNA2, circular RNA3, circular RNA4, circular RNA5 and circular RNA6 described in Example 2 were isolated as described herein.

[0461] In this example, linear and circular RNAs were generated as described. To purify the circular RNAs, the ligation mixture was resolved on a 6% denaturing PAGE, and the RNA bands corresponding to each circular RNA were excised. The excised RNA gel fragments were crushed, and the RNA was eluted overnight in 800 μl 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. The eluted circular RNAs were analyzed by 6% denaturing PAGE (see Figure 20).

[0367]

[0462] A single band was visualized by PAGE for circular RNAs with variable sizes.

[0368] Example 20: Detection of protein expression

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

[0369]

[0464] Protein expression is the process of producing a specific protein from mRNA. This process involves transcription of DNA into messenger RNA (mRNA), followed by translation of the mRNA into a polypeptide chain that is ultimately folded into a functional protein and can be targeted to a specific intracellular or extracellular location.

[0370]

[0465] As shown in the examples below, proteins were expressed in vitro from circular RNA sequences.

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

[0371]

[0467] Linear or circular RNA was incubated in rabbit reticulocyte lysate in a volume of 25 μl 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 of linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 × g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μl of 2× SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 × g for 5 minutes, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel.

[0372]

[0468] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, 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 the Western blot band intensity was measured using ImageJ.

[0373]

[0469] Fluorescence was detected, indicating the presence of an expression product, thus demonstrating that the circular RNA drives protein expression.

[0374] Example 21: IRES-independent expression

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

[0375]

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

[0376]

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

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

[0377]

[0474] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, 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 the Western blot band intensity was measured using ImageJ.

[0378]

[0475] Expression product was detected in the circular RNA reaction mixture even in the absence of an IRES.

[0379] Example 22: Cap-independent expression

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

[0380]

[0477] A cap is a specifically modified nucleotide at the 5' end of an mRNA. The 5' cap is useful for stabilizing linear mRNA and for translation initiation. Circular RNAs drive product expression in the absence of a cap.

[0381]

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

[0479] Linear or circular RNA was incubated in rabbit reticulocyte lysate in a volume of 25 μl 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 of linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 × g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μl of 2× SDS sample buffer for 15 minutes at 70°C. After centrifugation at 1400 × g for 5 minutes, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel.

[0382]

[0480] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, 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 the Western blot band intensity was measured using ImageJ.

[0383]

[0481] Expression products were detected in the circular RNA reaction mixture even in the absence of a cap.

[0384] Example 23: Expression without 5'-UTR

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

[0385]

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

[0484] As shown in the Examples below, the 5'-untranslated region in the circular RNA sequence was not required for in vitro protein expression.

[0386]

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

[0387]

[0486] Linear or circular RNA was incubated in rabbit reticulocyte lysate in a volume of 25 μl 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 of linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 × g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μl of 2× SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 × g for 5 minutes, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel.

[0388]

[0487] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, 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 the Western blot band intensity was measured using ImageJ.

[0389]

[0488] The expression product was detected in the circular RNA reaction mixture even in the absence of the 5'UTR.

[0390] Example 24: Expression without 3'-UTR

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

[0391]

[0490] The 3'-untranslated region (3'-UTR) is the region immediately downstream of the translation termination codon and contains regulatory regions that can post-transcriptionally influence gene expression. The 3'-untranslated region may also play a role in gene expression by influencing mRNA localization, stability, transport, and translation efficiency. Additionally, the structural characteristics of the 3'-UTR and its use of alternative polyadenylation may play a role in gene expression.

[0392]

[0491] As shown in the Examples below, the 3'-UTR in the circular RNA sequence was not required for in vitro protein expression.

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

[0393]

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

[0394]

[0494] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, 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 the Western blot band intensity was measured using ImageJ.

[0395]

[0495] The expression product was detected in the circular RNA reaction mixture even in the absence of the 3'UTR.

[0396] Example 25: Expression without a stop codon

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

[0397]

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

[0398]

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

[0399]

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

[0400]

[0500] Linear or circular RNA was incubated in rabbit reticulocyte lysate in a volume of 25 μl 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 of linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 × g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μl of 2× SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 × g for 5 minutes, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel.

[0401]

[0501] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, the blot was incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blot was visualized using an ECL kit (see Figure 22), and the Western blot band intensity was measured using ImageJ.

[0402]

[0502] Expression products were detected in the circular RNA reaction mixture even in the absence of a stop codon.

[0403] Example 26: Expression of distinct proteins without termination elements (stop codons)

[0503] This example demonstrates the production of distinct protein products translated from circular RNA lacking a termination element (stop codon).

[0404]

[0504] Stagger elements, such as 2A peptides, can contain short amino acid sequences of approximately 20 aa, allowing for the production of multiple genes at equimolar levels from a single mRNA. Stagger elements can function by causing the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element, resulting in a separation between the end of the 2A sequence and the next peptide downstream. The separation occurs between the glycine and proline residues found on the C-terminus, with the upstream cistron having several additional residues added to its end, while the downstream cistron starts with a proline.

[0405]

[0505] As shown in the Examples below, circular RNA lacking a termination element (stop codon) generated large polypeptide polymers (left panel of Figure 23: No Stagger - Circular RNA lane), and inclusion of a 2A sequence at the 3' end of the coding region resulted in the production of a distinct protein of comparable size to that generated by the equivalent linear RNA construct (right panel of Figure 23: Stagger - Circular RNA lane).

[0406]

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

[0407]

[0507] Linear or circular RNA was incubated in rabbit reticulocyte lysate in a volume of 25 μl 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 of linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 × g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μl of 2× SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 × g for 5 minutes, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel.

[0408]

[0508] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, the blot was incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blot was visualized using an ECL kit (see Figure 23), and the Western blot band intensity was measured using ImageJ.

[0409]

[0509] Distinct expression products were detected, indicating that the circular RNA containing the staggered element drove the expression of individual proteins even in the absence of a termination element (stop codon).

[0410] Example 27: Rolling circle translation

[0510] This example demonstrates the increased in vitro biosynthesis of protein from circular RNA using staggered elements.

[0411]

[0511] Non-natural circular RNAs were engineered to contain staggered elements to compare protein expression with circular RNAs lacking the staggered elements. As shown in the Examples below, the staggered elements overexpressed proteins compared to otherwise identical circular RNAs lacking such sequences.

[0412]

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

[0413]

[0513] Linear or circular RNA was incubated in rabbit reticulocyte lysate in a volume of 25 μl 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 of linear or circular RNA. After incubation, hemoglobin protein was removed by adding acetic acid (0.32 μl) and water (300 μl) to the reaction mixture (16 μl) and centrifuging at 20,817 × g for 10 minutes at 15°C. The supernatant was removed, and the pellet was dissolved in 30 μl of 2× SDS sample buffer and incubated at 70°C for 15 minutes. After centrifugation at 1400 × g for 5 minutes, the supernatant was analyzed on a 10-20% gradient polyacrylamide / SDS gel.

[0414]

[0514] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, the blot was incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blot was visualized using an ECL kit (see Figure 24), and the Western blot band intensity was measured using ImageJ.

[0415]

[0515] Distinct expression products were detected, indicating that the circular RNA containing the staggered element drove the expression of individual proteins even in the absence of a termination element (stop codon).

[0416] Example 28: Expression of biologically active proteins in vitro

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

[0417]

[0517] Non-natural circular RNAs have been engineered to express bioactive therapeutic proteins. As shown in the examples below, bioactive proteins have been expressed from circular RNAs in reticulocyte lysates.

[0418]

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

[0419]

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

[0420]

[0520] As shown in Figure 25, much higher fluorescence was detected for both circular and linear RNA than for the control vehicle RNA, indicating that an expression product was present, thus demonstrating that the circular RNA expressed a biologically active protein.

[0421] Example 29: Circular RNAs with longer half-lives than their linear RNA counterparts

[0521] This example demonstrates that circular RNAs can be engineered to have extended half-lives compared to linear RNAs.

[0422]

[0522] Circular RNAs encoding therapeutic proteins conferred upon recipient cells the ability to produce higher levels of the encoded protein due to an extended biological half-life compared to, for example, linear RNAs. As shown in the Examples below, circular RNAs had a longer half-life in reticulocyte lysates than their linear RNA counterparts.

[0423]

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

[0424]

[0524] In this example, a time course experiment was performed to monitor RNA stability. 100 ng of linear or circular RNA was incubated with rabbit reticulocyte lysate, and samples were collected at 1, 5, 18, 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 mix (Bio-Rad).

[0425]

[0525] As shown in Figure 26, higher concentrations of circular RNA were detected at later time points than linear RNA, thus, circular RNA was more stable or had an increased half-life compared to its linear counterpart.

[0426] Example 30: Circular RNA demonstrated a longer half-life than linear RNA in cells

[0526] This example demonstrates that circular RNA can be delivered intracellularly and has an increased half-life within the cell compared to linear RNA.

[0427]

[0527] Non-natural circular RNAs have been engineered to express bioactive therapeutic proteins. As shown in the following examples, circular RNAs are present at higher levels than their linear RNA counterparts, demonstrating the longer half-life of circular RNAs.

[0428]

[0528] In this example, circular and linear RNAs were designed to encode EGF, termination, or non-termination sequences (SEQ ID NOs: 11, 19, 20, 21) flanked by Kozak, 2A. To monitor the half-life of the RNA in cells, 0.1 x 10 6 Cells were plated onto each well of a 12-well plate. One day later, 1 μg of linear or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). 24 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). The primer sequences were as follows: primers for linear or circular RNA, F: ACGACGGTGTGTGCATGTAT, R: TTCCCACCACTTCAGGTCTC; primers for circular RNA, F: TACGCCTGCAACTGTGTTGT, R: TCGATGATCTTGTCGTCGTC.

[0429]

[0529] Circular RNAs, like their linear counterparts, were successfully transfected into 293T cells. After 24 hours, the retained circular and linear RNAs were measured using qPCR. As shown in Figures 27A and 27B, circular RNAs were shown to have a longer half-life in cells compared to linear RNAs.

[0430] Example 31: Synthetic circular RNA was translated in cells, and the synthetic circular RNA was translated by rolling circle translation

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

[0431]

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

[0432]

[0532] In this example, to monitor the translation efficiency of linear or circular RNA in cells, 0.1 x 10 6 Cells were plated in each well of a 12-well plate. One day later, 1 μg of linear or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). 24 h 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. After electrophoretic transfer to a nitrocellulose membrane using the dry transfer method, the blot was incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. An anti-β-tubulin antibody was used as a loading control. The blot was visualized using an enhanced chemiluminescence (ECL) kit. Western blot band intensity was measured using ImageJ.

[0433]

[0533] Circular RNA was successfully transfected into 293T cells, similar to its linear counterpart. However, Figure 28 shows that 24 hours after transfection, EGF protein was detected in circular RNA-transfected cells, but not in linear RNA-transfected cells. Thus, circular RNA was translated in cells by rolling circle translation, compared to linear RNA.

[0434] Example 32: Synthetic circular RNA demonstrated reduced immunogenic gene expression in cells

[0534] This example demonstrates that circular RNAs can be engineered to have reduced immunogenicity compared to linear RNAs.

[0435]

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

[0436]

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

[0437]

[0537] In this example, 0.1×10 6 The levels of innate immune response genes were monitored intracellularly by plating 100 cells in each well of a 12-well plate. One day later, 1 μg of linear or circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). 24 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).

[0438]

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

[0439]

[0539] As shown in Figure 29, qRT-PCR levels of immune-related genes from circular RNA-transfected 293T cells showed 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 decreased in circular RNA-transfected cells compared to linear RNA-transfected cells.

[0440] Example 33: Increased expression from synthetic circular RNAs by rolling circle translation in cells

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

[0441]

[0541] Circular RNAs were designed to contain an IRES without a termination element (stop codon) along with the nanoluciferase gene or the EGF negative control gene. Cells were transfected with EGF negative control (SEQ ID NO: 22); nLUC stop (SEQ ID NO: 23): EMCV IRES, staggered sequence (2A sequence), 3x FLAG-tagged nLUC sequence, staggered sequence (2A sequence), and a stop codon; or nLUC stagger (SEQ ID NO: 24): EMCV IRES, staggered sequence (2A sequence), 3x FLAG-tagged nLUC sequence, and staggered sequence (2A sequence). As shown in Figure 30, both circular RNAs produced translation products with functional luciferase activity.

[0442]

[0542] In this example, translation of circular RNA was monitored in cells. 6 Cells were plated in 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 μl of RIPA buffer. Nanoluciferase activity in the lysates was measured using a luciferase assay system according to the manufacturer's protocol (Promega).

[0443]

[0543] As shown in Figure 30, both circular RNAs expressed proteins in cells. However, the circular RNA containing a stagger element, e.g., a 2A sequence, and lacking a termination element (stop codon) produced higher levels of protein product with functional luciferase activity than the circular RNA with a termination element (stop codon).

[0444] Example 34: Synthetic circular RNA translated in cells

[0544] This example demonstrates the translation of synthetic circular RNA in cells. Furthermore, this example shows that circular RNA produced more expression product than its linear counterpart.

[0445]

[0545] Circular RNAs, like their linear counterparts, were successfully transfected into 293T cells. As a negative control, cells were transfected with 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. As shown in Figure 31, the circular RNAs were translated into nanoluciferase in cells.

[0446]

[0546] Linear or circular RNA translation was monitored in cells.6 Cells were plated in each well of a 12-well plate. One day later, 300 ng of linear 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. Nanoluciferase activity in the lysates was measured using a luciferase assay system according to the manufacturer's protocol (Promega).

[0447]

[0547] As shown in Figure 31, circular RNA translation products were detected in the cells. Notably, circular RNAs had higher levels of luciferase activity or increased protein produced compared to their linear RNA counterparts.

[0448] Example 35: Rolling circle translation from synthetic circular RNA produced functional protein products in cells

[0548] This example demonstrates rolling circle translation of a functional protein product from a synthetic circular RNA lacking a termination element (stop codon), e.g., lacking a termination element (stop codon) and containing a staggered element, in cells. Furthermore, this example shows that the circular RNA containing the staggered element expressed more functional protein product than its linear counterpart.

[0449]

[0549] Circular RNA, like its linear counterpart, was successfully transfected into 293T cells. Cells were transfected with 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, circular RNA was translated into nanoluciferase in cells.

[0450]

[0550] Linear or circular RNA translation was monitored in cells. 6Cells were plated in each well of a 12-well plate. One day later, 300 ng of linear 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. Nanoluciferase activity in the lysates was measured using a luciferase assay system according to the manufacturer's protocol (Promega).

[0451]

[0551] As shown in Figure 32, circular RNA translation products were detected in the cells. Notably, circular RNAs lacking termination elements (stop codons) produced higher levels of protein products with functional luciferase activity than their linear RNA counterparts.

[0452] Example 36: Synthetic circular RNA is translated by IRES initiation in cells

[0552] This example demonstrates IRES-directed translation initiation of synthetic circular RNA in cells.

[0453]

[0553] Circular RNAs were designed to contain a Kozak sequence or IRES along with the nanoluciferase gene or the EGF negative control gene. Cells were transfected with EGF negative control (SEQ ID NO: 22), nLUC Kozak (SEQ ID NO: 25): Kozak sequence, 1x FLAG-tagged EGF sequence, staggered sequence (T2A sequence), 1x FLAG-tagged nLUC, staggered sequence (P2A sequence), and a 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 a stop codon. As shown in Figure 33, circular RNAs with IRESs showed higher levels of luciferase activity, corresponding to higher protein levels, compared to circular RNAs with Kozak sequences.

[0454]

[0554] In this example, translation of circular RNA was monitored in cells. 6Cells were plated in 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 μl of RIPA buffer. Nanoluciferase activity in the lysates was measured using a luciferase assay system according to the manufacturer's protocol (Promega).

[0455]

[0555] As shown in Figure 33, circular RNA primed protein expression through an IRES produced higher levels of protein product with functional luciferase activity than circular RNA primed protein expression with Kozak.

[0456] Example 37: Rolling circle translation of synthetic circular RNA in cells

[0556] This example demonstrates increased protein production by rolling circle translation of synthetic circular RNA in cells primed with an IRES.

[0457]

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

[0458]

[0558] In this example, translation of circular RNA was monitored in cells. 6 Cells were plated in 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 μl of RIPA buffer. Nanoluciferase activity in the lysates was measured using a luciferase assay system according to the manufacturer's protocol (Promega).

[0459]

[0559] As shown in Figure 34, the circular RNAs were translated into proteins in cells by the rolling circle method provided by both circular RNAs, but lacking a termination element (stop codon). However, rolling circle translation of the circular RNA initiated more protein production at the IRES, generating more protein product with functional luciferase activity compared to the circular RNA with the termination element Kozak translation initiation.

[0460] Example 38: Increased protein expression from circular RNA

[0560] This example demonstrates the translation of synthetic circular RNA in cells. Furthermore, this example shows that circular RNA produced a larger expression product of the appropriate molecular weight than its linear counterpart.

[0461]

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

[0462]

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

[0463]

[0563] After electrophoretic transfer to nitrocellulose membrane using the dry transfer method, the blot was incubated with anti-FLAG antibody and anti-mouse IgG peroxidase. The blot was visualized using an ECL kit, and the Western blot band intensity was measured using ImageJ.

[0464]

[0564] As shown in Figure 35, the circular RNA was translated into protein within the cells. Notably, the circular RNA produced higher levels of protein with the appropriate molecular weight compared to its linear RNA counterpart.

[0465] Example 39: Rolling circle translation of synthetic circular RNAs produced distinct protein products in cells

[0565] This example demonstrates that distinct protein products were translated by rolling circle translation from synthetic circular RNAs lacking a termination element (stop codon), e.g., in cells, but containing a stagger element instead of a termination element (stop codon). Furthermore, this example shows that the circular RNAs containing the stagger element expressed more protein products of the appropriate molecular weight than their linear counterparts.

[0466]

[0566] Circular RNA was designed to contain the nanoluciferase gene with a staggered element instead of a termination element (stop codon). Cells were transfected with 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). As shown in Figure 36, circular RNA produced higher levels of protein with the appropriate molecular weight compared to linear RNA.

[0467]

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

[0468]

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

[0469]

[0569] As shown in Figure 36, circular RNA translation products were detected in cells. Notably, circular RNAs lacking termination elements (stop codons) produced higher levels of distinct protein products with appropriate molecular weights than their linear RNA counterparts.

[0470] Example 40: Preparation of circular RNA with pseudo-double-stranded, helical structure

[0570] This example demonstrates that the circular RNA had both a quasi-double-stranded and helical structure.

[0471]

[0571] Non-natural circular RNAs have been engineered to adopt pseudo-double-stranded, helical structures, similar to those shown to be involved in the condensation of natural circular RNAs with inherently long in vivo half-lives (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).

[0472]

[0572] In this example, a circular RNA was designed to encode the EMCV IRES, 3XFLAG-tagged Nluc as the ORF, and a staggered sequence (EMCV 2A 3XFLAG Nluc 2A no stop). A thermodynamic RNA structure prediction tool (RNAfold) was used to assess the RNA secondary structure (Vienna RNA). Furthermore, the RNA tertiary structure was analyzed using an RNA modeling algorithm.

[0473]

[0573] As shown in Figures 37 and 38, circular RNA is modeled to adopt a pseudo-double-stranded, helical structure.

[0474] Example 41: Preparation of circular RNA with pseudohelical structure linked to repeat sequences

[0574] This example demonstrates that circular RNAs can be designed to have pseudohelical structures linked to repeat sequences.

[0475]

[0575] Non-natural circular RNAs have been engineered to adopt pseudohelical structures linked to repeat sequences. Similar structures have been shown to be involved in the condensation of natural circular RNAs, which have inherently long in vivo half-lives (Griffin et al. 2014; Guedj et al. 2015). 2014).

[0476]

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

[0477]

[0577] As shown in Figure 39, circular RNA is modeled to adopt a pseudohelical structure.

[0478] Example 42: Circularized RNA is circular and not concatemeric

[0578] This example demonstrates that degradation of circular RNA by RNAse H produced nucleolytic degradation products consistent with circular RNA and inconsistent with concatemeric RNA.

[0479]

[0579] When RNA is incubated with ligase, it either does not react or is unable to form intramolecular or intermolecular bonds, which produces circular (no free ends) or concatemeric RNA, respectively. Treatment of each type of RNA with a complementary DNA primer and RNAse H, a nonspecific endonuclease that recognizes DNA / RNA duplexes, is expected to produce a characteristic number of degradation products of specific sizes, depending on the starting RNA material.

[0480]

[0580] As shown in the Examples below, the ligated RNA was shown to be circular and not concatemeric based on the number and size of RNAs generated by RNAse H degradation.

[0481]

[0581] Circular and linear RNAs containing EMCV T2A 3XFLAG-Nluc P2A were generated.

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

[0482]

[0583] For the linear RNA used as described above, after binding of the DNA primer and subsequent cleavage by RNAse H, two cleavage products are predicted to be obtained at 1041 nt and 258 nt. Concatamers are predicted to produce three cleavage products of 258, 1041, and 1299 nt. Circularization is predicted to produce a single 1299 nt cleavage product.

[0483]

[0584] The number of bands in the linear RNA lane incubated with RNAse endonuclease produced two bands of 1041 nt and 258 nt as expected, while a single band of 1299 nt was produced in the circular RNA lane (see Figure 40), indicating that the circular RNA was indeed circular and not concatemeric.

[0484] Example 43: Preparation of large circular RNA

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

[0485]

[0586] Non-natural circular RNAs engineered to contain one or more desirable properties have been generated in a range of sizes depending on the desired function. As shown in the examples below, linear RNAs up to 6200 nt in size have been circularized.

[0486]

[0587] The plasmid pCDNA3.1 / CAT (6.2 kb) was used here. Primers were designed to anneal to pCDNA3.1 / CAT at regular intervals to generate DNA oligonucleotides corresponding to 500 nt, 1000 nt, 2000 nt, 4000 nt, 5000 nt, and 6200 nt. In vitro transcription of the indicated DNA oligonucleotides was performed to generate linear RNAs of the corresponding sizes. Circular RNAs were generated from these RNA oligonucleotides using splint DNA.

[0487]

[0588] To measure the efficiency of RNA circularization, linear RNAs of six different sizes (500 nt, 1000 nt, 2000 nt, 4000 nt, 5000 nt, and 6200 nt) were generated and circularized using a DNA splint and T4 DNA ligase 2. As a control, one reaction was performed without T4 RNA ligase. Half of the circularized sample was treated with RNAse R to remove linear RNA.

[0488]

[0589] To monitor circularization efficiency, each sample was analyzed using qPCR. Circular RNAs were generated from various DNAs of different lengths, as shown in Figure 41. Circularization of RNA was confirmed using RNAse R treatment and qPCR analysis of the circular junction, as shown in Figure 42. This example demonstrates the generation of circular RNAs of various lengths.

[0489] Example 44: Circular RNA engineered with protein binding sites

[0590] This example demonstrates the generation of circular RNAs with protein binding sites.

[0490]

[0591] In this example, one circular RNA is designed to contain the CVB3 IRES (SEQ ID NO: 56) and an ORF (SEQ ID NO: 57) encoding Gaussia luciferase (Gluc), followed by at least one protein binding site. In a specific example, the HuR-binding sequence (SEQ ID NO: 52) from the Sindbis virus 3'UTR is used to test the effect of protein binding on circular RNA immunogenicity. The HuR-binding sequence contains two elements: a U-rich element (URE; SEQ ID NO: 50) and a conserved sequence element (CSE; SEQ ID NO: 51). Circular RNAs without the HuR-binding sequence or with the URE are used as controls. A portion of the Anabaena autocatalytic intron and exon sequence is placed in front of the CVB3 IRES (SEQ ID NO: 56). Circular RNAs are generated in vitro as described. As shown in Figure 45, circular RNAs containing a HuR-binding site were generated.

[0491]

[0592] To monitor the effect of RNA-binding proteins on circular RNA immunogenicity, cells were plated in each well of a 96-well plate. One day later, 500 ng of circular RNA was transfected into each well using a lipid-based transfection reagent (Invitrogen). Translation efficiency, RNA stability, and immunogenicity were monitored daily for up to 72 hours. Culture medium was collected to monitor Gluc activity. Cell lysates for measuring RNA levels were prepared using a kit that allows for the measurement of relative gene expression by real-time RT-PCR (Invitrogen).

[0492]

[0593] Monitor translation efficiency by measuring Gluc activity using a Gaussia luciferase flash assay kit according to the manufacturer's instructions (Pierce).

[0594] For qRT-PCR analysis, cDNA was generated using a cell lysate preparation kit according to the manufacturer's instructions (Invitrogen). qRT-PCR analysis was performed using a PCR master mix (Brilliant II SYBR Green qRT-PCR). PCR was performed in triplicate using a PCR Master Mix and a PCR cycler (LightCycler 480). Circular RNA stability was measured using primers for Nluc. mRNA levels of well-known innate immune regulators, such as RIG-I, MDA5, OAS, OASL, and PKR, were quantified and normalized to actin values.

[0493] 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.

[0494]

[0596] Different cell types have their own nucleic acid regulatory mechanisms that target specific RNA sequences. Encoding these specific sequences in circular RNA can give different cell types their unique characteristics. As shown in the following examples, circular RNAs have been engineered to encode microRNA binding sites.

[0495]

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

[0496]

[0598] Circular RNA was generated in vitro. Unmodified linear RNA was transcribed in vitro from a DNA template containing all of the motifs listed above, in addition to a T7 RNA polymerase promoter driving transcription. The transcribed RNA was purified using an RNA cleanup kit (New England Biolabs, T2050), treated with RNA 5'-phosphohydrolase (RppH) (New England Biolabs, M0356) according to the manufacturer's instructions, and purified again using an RNA purification column. The RppH-treated RNA was circularized using splint DNA (GGCTATTCCCAATAGCCGTT) and T4 RNA Ligase II (New England Biolabs, M0239). Circular RNA was purified by urea-PAGE (Figure 43), eluted with buffer (0.5 M sodium acetate, 0.1% SDS, 1 mM EDTA), precipitated with ethanol, and resuspended in RNase-free water.

[0497]

[0599] As shown in Figure 43, circular RNAs were generated with miRNA binding sites.

[0498] Example 46: Self-splicing of circular RNA

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

[0499]

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

[0500]

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

[0501]

[0603] FIG. 44 shows circular RNA generated by self-splicing.

[0502] Example 47: Circular RNA with splicing elements containing encryptogens

[0604] This example demonstrates that circular RNAs can be engineered to have reduced immunogenicity.

[0503]

[0605] In this example, the circular RNA contains the CVB3 IRES, an ORF encoding Gaussia luciferase (GLuc), and two spacer elements flanking the IRES-ORF, which contain splicing elements that are part of the Anabaena autocatalytic intron and exon sequence (SEQ ID NO: 59).

[0504]

[0606] Circular RNA is generated in vitro.

[0607] In this example, the levels of innate immune response genes are monitored intracellularly by plating cells in each well of a 12-well plate. One day later, 1 μg of linear or circular RNA is transfected into each well using a lipid-based transfection reagent (Invitrogen). 24 hours after transfection, total RNA is isolated from the cells using a phenol-based extraction reagent (Invitrogen). Total RNA (500 ng) is subjected to reverse transcription to generate cDNA. qRT-PCR analysis is performed using a dye-based quantitative PCR mix (Bio-Rad).

[0505]

[0608] qRT-PCR levels of immune-related genes from BJ cells transfected with circular RNA containing splicing elements are p...

Claims

[Claim 1] The invention described in the specification.

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