Compositions Comprising Modified Circular Polyribonucleotides and Uses Thereof

Abstract

The present invention relates generally to pharmaceutical compositions and the preparation of modified cyclic polyribonucleotides and their uses.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 823,573, filed March 25, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

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

[0003] The present disclosure provides a pharmaceutical composition comprising a cyclic polyribonucleotide comprising a pharma- ceutically acceptable carrier or excipient and a first portion of contiguous unmodified nucleotides.In some embodiments, the cyclic polyribonucleotide comprises at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides.In some embodiments, the modified cyclic polyribonucleotide is delivered to a subject.

[0004] The present disclosure provides a method of reducing or decreasing the immunogenicity of a cyclic polyribonucleotide in a subject, the method comprising the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified nucleotide and a first portion comprising at least about 5-1000 contiguous nucleotides having 5% or less modified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining a reduced immunogenicity for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In one embodiment, the method of reducing or decreasing the immunogenicity of a cyclic polyribonucleotide in a subject comprises the steps of providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining a reduced or decreased immunogenicity for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In some embodiments, the first portion comprises an IRES. In some embodiments, the first portion comprises at least about 5-1000 contiguous unmodified nucleotides. In some embodiments, the first portion lacks 5'-methylcytidine pseudouridine or N1-methyl-pseudouridine. In some embodiments, 5% or less of the nucleotides in the first portion are modified nucleotides. In some embodiments, 5% or less of the nucleotides in the IRES of the first portion are modified nucleotides.

[0005] The present disclosure provides a method of expressing one or more expression sequences in a subject, comprising the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least about 5-1000 contiguous nucleotides having at least one modified nucleotide and 5% or less modified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject, and obtaining increased expression of the one or more expression sequences compared to expression of the one or more expression sequences in a fully modified cyclic polyribonucleotide equivalent in a cell or tissue of the subject. In an embodiment, the method of expressing one or more expression sequences in a subject comprises providing a hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide, a first portion of contiguous unmodified nucleotides, and one or more expression sequences; administering the hybrid modified cyclic polyribonucleotide to the subject, and obtaining increased expression of the one or more expression sequences compared to expression of the one or more expression sequences in a fully modified cyclic polyribonucleotide equivalent in a cell or tissue of the subject. In an embodiment, the first portion comprises an IRES. In some embodiments, the first portion comprises at least about 5-1000 contiguous unmodified nucleotides. In some embodiments, the first portion lacks 5'-methylcytidine, pseudouridine, or N1-methyl-pseudouridine. In some embodiments, 5% or less of the nucleotides in the first portion are modified nucleotides. In some embodiments, 5% or less of the nucleotides in the IRES of the first portion are modified nucleotides.

[0006] The present disclosure provides a method for increasing stability of a cyclic polyribonucleotide in a subject, comprising the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified nucleotide and a first portion comprising at least about 5-1000 contiguous nucleotides having 5% or less modified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject, and obtaining increased stability for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In an embodiment, the method for increasing stability of a cyclic polyribonucleotide in a subject comprises the steps of providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject, and obtaining increased stability for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In an embodiment, the first portion comprises an IRES. In some embodiments, the first portion comprises at least about 5-1000 contiguous unmodified nucleotides. In some embodiments, the first portion lacks 5'-methylcytidine, pseudouridine, or N1-methyl-pseudouridine. In some embodiments, 5% or less of the nucleotides in the first portion are modified nucleotides. In some embodiments, 5% or less of the nucleotides in the IRES of the first portion are modified nucleotides.

[0007] In one aspect, a method of reducing the immunogenicity of a cyclic polyribonucleotide in a subject comprises the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least one modified nucleotide and at least about 5-1000 contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining reduced immunogenicity for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject.

[0008] In one aspect, a method of reducing immunogenicity of a cyclic polyribonucleotide in a subject comprises the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least one modified nucleotide and at least about 5-1000 contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining reduced immunogenicity for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject.

[0009] In some embodiments, the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleotides. In some embodiments, the first portion lacks 5'-methylcytidine or pseudouridine. In some embodiments, 5% or less of the nucleotides in the first portion are modified nucleotides. In some embodiments, the cyclic polyribonucleotide is translatable. In some embodiments, the hybrid modified cyclic polyribonucleotide has a) at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher expression than the corresponding unmodified cyclic polyribonucleotide; b) at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times longer half-life than the corresponding unmodified cyclic polyribonucleotide; ) has a longer half-life than the corresponding unmodified cyclic polyribonucleotide; or d) has at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times less immunogenic than the corresponding unmodified cyclic polyribonucleotide as assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β.In certain embodiments, the at least one modified nucleotide is selected from the group consisting of a) N(6) methyl adenosine (m6A), 5'-methyl cytidine, and pseudouridine; and b) 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP). , TO-Dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-Methylacetamide (2'-O-NMA), Locked Nucleic Acid (LNA), Ethylene Nucleic Acid (ENA), Peptide Nucleic Acid (PNA), 1',5'-Anhydrohexitol Nucleic Acid (HNA), Morpholino, Methylphosphonate Nucleotides, Thiophosphonate Nucleotides, and 2'-Fluoro N3-P5'-Phosphoramidites; or c) any modified nucleotide from Table 2. In certain embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the nucleotides of the hybrid modified cyclic polyribonucleotide are modified nucleotides. In certain embodiments, the hybrid modified cyclic polyribonucleotide comprises a binding site configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, which is comprised of unmodified nucleotides. In some embodiments, the hybrid modified circular polyribonucleotide comprises one or more expression sequences. In some embodiments, the first portion comprises an IRES consisting of unmodified nucleotides.In certain embodiments, one or more expressed sequences of hybrid modified circular polyribonucleotides have a) a higher translation efficiency than a fully modified circular polyribonucleotide equivalent; b) at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher translation efficiency than a fully modified circular polyribonucleotide equivalent; c) a higher translation efficiency than a corresponding unmodified circular polyribonucleotide; or d) at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher translation efficiency than a corresponding unmodified circular polyribonucleotide.

[0010] In one embodiment, a method for expressing one or more expressible sequences in a subject comprises the steps of: providing a hybrid modified cyclic polyribonucleotide comprising one or more expressible sequences, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least one modified nucleotide and at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining increased expression of the one or more expressible sequences compared to the expression of the corresponding one or more expressible sequences in a fully modified cyclic polyribonucleotide equivalent in a cell or tissue of the subject.

[0011] In one embodiment, a method for increasing stability of a cyclic polyribonucleotide in a subject, the method comprising the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a modified cyclic polyribonucleotide and a first portion comprising at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining increased stability for the hybrid modified cyclic polyribonucleotide compared to the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject.

[0012] In one embodiment, the first portion comprises an IRES.

[0013] In one aspect, the disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or excipient and a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified nucleotide and a first portion, the first portion comprising at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous unmodified nucleotides. In certain embodiments of this aspect, the first portion comprises 5% or less modified nucleotides.

[0014] In another aspect, the disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or excipient and a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified nucleotide and a first portion, the first portion comprising at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides, the first portion lacking 5'-methylcytidine or pseudouridine. In certain embodiments of this aspect, the first portion comprises 5% or less modified nucleotides.

[0015] In one embodiment, the pharmaceutical composition comprises a hybrid modified cyclic polyribonucleotide, wherein the hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding portion of a first target, e.g., an RNA, DNA, protein, or cellular target (wherein the first binding portion is a first cyclic polyribonucleotide (cyclic RNA) binding motif composed of unmodified nucleotides); and wherein the first target and the hybrid modified cyclic polyribonucleotide form a complex.

[0016] In one embodiment, the pharmaceutical composition comprises a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding portion of a first target, e.g., an RNA, DNA, protein, or cellular target, wherein the first binding portion is a first cyclic polyribonucleotide (cyclic RNA) binding motif comprised of unmodified nucleotides; and a second binding portion configured to bind to a second binding portion of a second target, wherein the second binding portion is a second cyclic RNA binding motif, wherein the first binding portion is different from the second binding portion, and the first target, the second target, and the hybrid modified cyclic polyribonucleotide form a complex, and wherein the first target or the second target is not a microRNA.

[0017] In one embodiment, a pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding portion of a first target (wherein the first binding portion is a first cyclic polyribonucleotide (cyclic RNA) binding motif); and a second binding portion configured to bind to a second binding portion of a second target (wherein the second binding portion is a second cyclic RNA binding motif), the first binding portion being different from the second binding portion, and both the first target and the second target being microRNA. In one embodiment, the hybrid modified cyclic polyribonucleotide has a lower immunogenicity than the corresponding unmodified cyclic polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotide has a half-life that is at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times longer than the corresponding unmodified cyclic polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotide has a half-life that is longer than the corresponding unmodified cyclic polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotide has at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times less immunogenic than the corresponding unmodified cyclic polyribonucleotide as assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β. In some embodiments, the hybrid modified cyclic polyribonucleotide has a longer half-life than the corresponding unmodified cyclic polyribonucleotide.In certain embodiments, the at least one modified nucleotide is selected from the group consisting of N(6) methyladenosine (m6A), 5'-methylcytidine, and pseudouridine. In certain embodiments, the at least one modified nucleic acid is selected from the group consisting of 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, and 2'-fluoro N3-P5'-phosphoramidites. In certain embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the nucleotides of the hybrid modified cyclic polyribonucleotide are modified nucleotides. In certain embodiments, the modified cyclic polyribonucleotide comprises a binding site configured to bind to a peptide, protein, biomolecule, DNA, RNA, or cellular target, which is comprised of unmodified nucleotides. In certain embodiments, the first portion comprises the binding site. In certain embodiments, the modified cyclic polyribonucleotide comprises an internal ribosome entry site (IRES) which is comprised of unmodified nucleotides. In certain embodiments, the first portion comprises an IRES. In certain embodiments, the IRES comprises 5% or less modified nucleotides.

[0018] In some embodiments, the hybrid modified cyclic polyribonucleotide comprises one or more expression sequences. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises one or more expression sequences and an IRES, and the hybrid modified cyclic polyribonucleotide comprises 5'-methylcytidine, pseudouridine, or a combination thereof outside the IRES. In some embodiments, the one or more expression sequences of the hybrid modified cyclic polyribonucleotide have a higher translation efficiency than the corresponding fully modified cyclic polyribonucleotide. In some embodiments, the one or more expression sequences of the hybrid modified cyclic polyribonucleotide have at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher translation efficiency than the fully modified cyclic polyribonucleotide equivalent. In some embodiments, the fully modified cyclic polyribonucleotide equivalent comprises at least one modified nucleotide outside the first portion and more than 5% modified nucleotides in the first portion. In some embodiments, one or more expressed sequences of hybrid modified cyclic polyribonucleotides have a higher translation efficiency than the corresponding unmodified cyclic polyribonucleotides. In some embodiments, one or more expressed sequences of hybrid modified cyclic polyribonucleotides have a translation efficiency at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than the corresponding unmodified cyclic polyribonucleotides. In some embodiments, one or more expressed sequences of hybrid modified cyclic polyribonucleotides have a higher translation efficiency than the fully modified cyclic polyribonucleotide equivalent. In some embodiments, one or more expressed sequences of hybrid modified cyclic polyribonucleotides have a higher translation efficiency than the fully modified cyclic polyribonucleotides having a first portion that includes more than 10% modified nucleotides. In some embodiments, one or more expressed sequences of hybrid modified cyclic polyribonucleotides have a higher translation efficiency than the fully modified cyclic polyribonucleotides having a first portion that includes 100% modified pseudouridine or 5' methylcytosine.In some embodiments, one or more expression sequences of hybrid modified cyclic polyribonucleotides have at least about 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times higher translation efficiency than a fully modified cyclic polyribonucleotide equivalent having a first portion comprising modified nucleotides. In some embodiments, the translation efficiency is measured in either a cell comprising a hybrid modified cyclic polyribonucleotide or a fully modified cyclic polyribonucleotide equivalent, or in an in vitro translation system (e.g., rabbit reticulocyte lysate). In some embodiments, the hybrid modified cyclic polyribonucleotide is capable of rolling circle type translation.

[0019] In some embodiments, each of the one or more expressed sequences is separated from the subsequent expressed sequence by a stagger element in the hybrid modified circular polyribonucleotide, and rolling circle translation of the one or more expressed sequences produces at least two polypeptide molecules. In some embodiments, the pharma- ceutically acceptable carrier or excipient is ethanol. In some embodiments, the stagger element prevents the production of a single polypeptide from (a) two translations of a single expressed sequence, or (b) one or more translations of two or more expressed sequences. In some embodiments, the stagger element is a sequence separated from one or more expressed sequences. In some embodiments, the stagger element comprises a portion of an expressed sequence of one or more expressed sequences. In certain embodiments, the hybrid modified circular polyribonucleotide is capable of rolling circle translation, and the hybrid modified circular polyribonucleotide is configured such that at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the total polypeptides produced during rolling circle translation of the hybrid modified circular polyribonucleotide are distinct polypeptides, each of the distinct polypeptides being produced from a single translation or less than a single translation of one or more expressed sequences. In some embodiments, the hybrid modified cyclic polyribonucleotide is configured such that at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the total polypeptides (mol / mol) produced during rolling circle translation of the hybrid modified cyclic polyribonucleotide are distinct polypeptides, and 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 comprises rabbit reticulocyte lysate.In some embodiments, the stagger element is at the 3' end of at least one of the one or more expressed sequences, and the stagger element is configured to stall the ribosome during rolling circle translation of the hybrid modified circular polyribonucleotide. In some embodiments, the stagger element encodes a peptide sequence selected from the group consisting of a 2A sequence and a 2A-like sequence. In some embodiments, the stagger element encodes a sequence having a C-terminal sequence that is GP. In some embodiments, the stagger element encodes a sequence having a C-terminal consensus sequence that is D(V / I)ExNPGP, where x=any amino acid. In some embodiments, the stagger element encodes a sequence selected from the group consisting of GDVESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, GDVEQNPGP, IESNPGP, GDIELNPGP, HDIETPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP. In some embodiments, the stagger element is at the 3' end of each of the one or more expressed sequences. In some embodiments, the stagger element of the first expressed sequence in the hybrid modified circular polyribonucleotide is upstream (5' end) of the first translation initiation sequence of the expressed sequence following the first expressed sequence in the hybrid modified 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 of a first expressed sequence in a hybrid modified circular polyribonucleotide is upstream (5' end) of a first translation start sequence of an expressed sequence following a first expression in the hybrid modified circular polyribonucleotide, and the hybrid modified circular polyribonucleotide is translated contiguously, and a corresponding hybrid modified circular polyribonucleotide including a second stagger element upstream of a second translation start sequence of a second expressed sequence in the corresponding hybrid modified circular polyribonucleotide is not translated contiguously, and the second stagger element in the corresponding hybrid modified circular polyribonucleotide is at a distance that is, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more greater than the distance between the second translation start sequence, the stagger element in the hybrid modified circular polyribonucleotide, and the first translation start. In certain embodiments, the distance between the stagger element and the first translation start is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or more. In some embodiments, the distance between the second staggered element and the second translation start is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or more, greater than the distance between the staggered element and the first translation start. In some embodiments, the expression sequence following the first expression sequence in the hybrid modified circular polyribonucleotide is an expression sequence other than the first expression sequence. In some embodiments, the expression sequence following the first expression sequence in the hybrid modified circular polyribonucleotide is the first expression sequence.

[0020] In some embodiments, the hybrid modified circular polyribonucleotide comprises at least one structural element selected from a) an encryptogen; b) a staggered element; c) a regulatory element; d) a replication element; and f) a pseudo-double-stranded secondary structure. In some embodiments, the hybrid modified circular polyribonucleotide comprises 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. In some embodiments, the hybrid modified circular polyribonucleotide has 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 higher translation efficiency than its linear counterpart. In some embodiments, the hybrid modified circular polyribonucleotide has at least 5-fold higher translation efficiency than its linear counterpart. In some embodiments, the hybrid modified circular polyribonucleotide lacks at least one of: a) 5'-UTR; b) 3'-UTR; c) poly-A sequence; d) 5'-cap; e) termination element; f) susceptibility to exonuclease degradation; and g) binding to a cap-binding protein. In some embodiments, the one or more expressed sequences include a Kozak initiation sequence. In some embodiments, the pseudohelical structure includes at least one double-stranded RNA segment with at least one non-double-stranded segment. In some embodiments, the pseudohelical structure includes a first sequence and a second sequence linked by a repeat sequence, e.g., an A-rich sequence. In some embodiments, the encryptogen includes a splicing element. In some embodiments, the encryptogen includes a protein binding site, e.g., a ribonucleotide binding protein.In some embodiments, the encryptogen comprises an immune protein binding site, for example, to avoid an immune response, for example, a CTL response. In some embodiments, the hybrid modified cyclic polyribonucleotide has at least 2-fold less immunogenicity than a counterpart lacking the encryptogen, for example, as assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β. In some embodiments, the hybrid modified cyclic polyribonucleotide further comprises a riboswitch. In some embodiments, the hybrid modified cyclic polyribonucleotide further comprises an aptazyme. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises a non-canonical translation initiation sequence, for example, a GUG, CUG initiation codon, for example, a translation initiation sequence that initiates expression under stress conditions. In some embodiments, the one or more expression sequences encode a peptide. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises a regulatory nucleic acid, for example, a non-coding RNA. In some embodiments, the hybrid modified cyclic polyribonucleotide has a size ranging from about 20 bases to about 20 kb. In some embodiments, the hybrid modified cyclic polyribonucleotide is synthesized by cyclization of a linear polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises multiple expression sequences having either the same nucleotide sequence or different nucleotide sequences. In some embodiments, the hybrid modified cyclic polyribonucleotide is substantially resistant to degradation, e.g., exonucleases.

[0021] In one embodiment, the hybrid modified cyclic polyribonucleotide comprises a modified cyclic polyribonucleotide comprising a first binding site configured to bind to a first binding moiety of a first target, e.g., RNA, DNA, protein, cell membrane, etc., where the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif; and a second binding site configured to bind to a second binding moiety of a second target, where the second binding moiety is a second cyclic RNA binding motif, wherein the first binding moiety is different from the second binding moiety, and the first target, the second target, and the hybrid modified cyclic polyribonucleotide form a complex, and wherein the first target or the second target is not a microRNA.

[0022] In one embodiment, the hybrid modified cyclic polyribonucleotide comprises a hybrid modified cyclic polyribonucleotide comprising a first binding site configured to bind to a first binding moiety of a first target (wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif); and a second binding site configured to bind to a second binding moiety of a second target (wherein the second binding moiety is a second cyclic RNA binding motif), wherein the first binding moiety is different from the second binding moiety, and both the first target and the second target are microRNAs.

[0023] In some embodiments, the first and second targets interact with each other. In some embodiments, the complex regulates a cellular process. In some embodiments, the first and second targets are the same and the first and second binding sites bind to different moieties. In some embodiments, the first and second targets are different. In some embodiments, the hybrid modified cyclic polyribonucleotide further comprises one or more additional binding sites configured to bind to a third or more binding moieties. In some embodiments, the one or more targets are the same and the one or more binding sites are configured to bind to different moieties. In some embodiments, the formation of the complex regulates a cellular process. In some embodiments, the hybrid modified cyclic polyribonucleotide, when contacted with the first and second targets, regulates a cellular process associated with the first or second target. In some embodiments, the first and second targets interact with each other in the complex. In some embodiments, the cellular process is associated with the development of a disease or pathology. In some embodiments, the cellular process is different from the translation of the hybrid modified circular polyribonucleic acid. In some embodiments, the cellular process is associated with the development of a disease or pathology. In some embodiments, the first target comprises a deoxyribonucleic acid (DNA) molecule and the second target comprises a protein. In some embodiments, the complex regulates directed transcription of a DNA molecule, epigenetic remodeling of a DNA molecule, or degradation of a DNA molecule. In some embodiments, the first target comprises a first protein and the second target comprises a second protein. In some embodiments, the complex regulates degradation of a first protein, translocation of a first protein, or signal transduction, or regulates a native protein function, or inhibits the formation of a complex formed by direct interaction between a first and a second protein. In some embodiments, the first target comprises a first ribonucleic acid (RNA) molecule and the second target comprises a second RNA molecule. In some embodiments, the complex regulates degradation of a first RNA molecule. In some embodiments, the first target comprises a protein and the second target comprises an RNA molecule.In some embodiments, the complex modulates the translocation of a protein or inhibits the formation of a complex formed by direct interaction between a protein and an RNA molecule. In some embodiments, the first binding moiety comprises a receptor and the second binding moiety comprises a substrate of the receptor. In some embodiments, the complex inhibits the activation of the receptor. In some embodiments, the modified cyclic polyribonucleotide comprises a binding site configured to bind to a binding moiety of a target, where the binding moiety is a ribonucleic acid (RNA) binding motif, the hybrid modified cyclic polyribonucleotide is translation-deficient or translation-deficient, and the target is not a microRNA. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises a binding site configured to bind to a binding moiety of a target, where the binding moiety is a ribonucleic acid (RNA) binding motif, the hybrid modified cyclic polyribonucleotide is translation-deficient or translation-deficient, and the target is a microRNA. In some embodiments, the target comprises a DNA molecule. In some embodiments, the binding of the binding moiety to the hybrid modified cyclic polyribonucleotide modulates the interference with the transcription of the DNA molecule. In some embodiments, the target comprises a protein. In some embodiments, the binding of the binding moiety to the hybrid modified cyclic polyribonucleotide inhibits the interaction of the protein with other molecules. In some embodiments, the protein is a receptor, and the binding of the first binding moiety to the modified cyclic polyribonucleotide activates the receptor. In some embodiments, the protein is a first enzyme, and the hybrid modified cyclic polyribonucleotide further comprises a second binding site configured to bind to a second enzyme, and the binding of the first and second enzymes to the hybrid modified cyclic polyribonucleotide modulates the enzymatic activity of the first and second enzymes. In some embodiments, the target comprises a messenger RNA (mRNA) molecule. In some embodiments, the binding of the binding moiety to the hybrid modified cyclic polyribonucleotide modulates the interference with the translation of an mRNA molecule. In some embodiments, the target comprises a ribosome. In some embodiments, the binding of the binding moiety to the hybrid modified cyclic polyribonucleotide modulates the interference with the translation process.In some embodiments, the target comprises a circular RNA molecule. In some embodiments, the binding of the binding moiety to the hybrid modified cyclic polyribonucleotide sequesters the circular RNA molecule. In some embodiments, the binding of the binding moiety to the hybrid modified cyclic polyribonucleotide sequesters the microRNA molecule. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises a binding site configured to bind to a binding moiety on a membrane of a cellular target; wherein the binding moiety is a ribonucleic acid (RNA) binding motif. In some embodiments, the hybrid modified cyclic polyribonucleotide further comprises a second binding site configured to bind to a second binding moiety on a second cellular target, wherein the second binding moiety is a second RNA binding motif. In some embodiments, the hybrid modified cyclic polyribonucleotide is configured to bind to both targets. In some embodiments, the hybrid modified cyclic polyribonucleotide further comprises a second binding site configured to bind to a second binding moiety, wherein the binding of both targets to the hybrid modified cyclic polyribonucleotide induces a conformational change in the first target, thereby inducing downstream signaling of the targets.

[0024] In certain embodiments, the present disclosure provides a composition described herein formulated in a carrier, such as a membrane or lipid bilayer.

[0025] In one aspect, the disclosure provides a method of treatment comprising administering to a subject having a disease or condition a pharmaceutical composition described herein.

[0026] In one aspect, the disclosure provides a method of making a pharmaceutical composition, the method comprising producing a hybrid modified circular polyribonucleotide as described herein.

[0027] In one aspect, the disclosure provides a method of making a hybrid modified circular polyribonucleotide as described herein, the method comprising circularizing a linear polyribonucleotide having a nucleic acid sequence as a hybrid modified circular polyribonucleotide.

[0028] In one aspect, the disclosure provides an engineered cell comprising a composition described herein.

[0029] definition 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.

[0030] The term "pharmaceutical composition" is also intended to disclose that the cyclic polyribonucleotide contained within the pharmaceutical composition can be used for the treatment of the human or animal body by therapy, and is therefore intended to be equivalent to "cyclic polyribonucleotide for therapeutic use."

[0031] The circular polyribonucleotides, compositions comprising such circular polyribonucleotides, methods of using such circular polyribonucleotides, etc. described herein are based in part on examples showing how circular polyribonucleotide effectors comprising different elements, such as replication elements, expression sequences, stagger elements and encryptogens (see, for example, Example 10) or expression sequences, stagger elements and regulatory elements (see, for example, Examples 32 and 40), can be used to achieve different technical effects (e.g., increased translation efficiency over linear equivalents in Examples 10 and 40 and increased half-life over linear equivalents in Example 40). It is based on these examples in particular that the following description contemplates various variations of the specific findings and combinations contemplated in the examples.

[0032] As used herein, the terms "circRNA" or "circular polyribonucleotide" or "circular RNA" are used interchangeably and refer to a polyribonucleotide molecule having a structure with no free ends (i.e., no free 3' and / or 5' ends), e.g., a polyribonucleotide that forms a circular or endless structure via covalent or non-covalent bonds.

[0033] As used herein, the terms "modified circular polyribonucleotide" or "modified circular RNA" or "modified circRNA" are used interchangeably and refer to a circular polyribonucleotide that contains at least one modified nucleotide. A modified circular RNA may or may not be uniformly modified along the entire length of the molecule.

[0034] As used herein, the terms "hybrid modified circular polyribonucleotide equivalent" or "hybrid modified circular polyribonucleotide" or "hybrid modified circRNA" or "hybrid modified circular RNA" are used interchangeably and refer to a modified circular polyribonucleotide having the same nucleotide sequence as a control modified circular polyribonucleotide and a first portion of contiguous nucleotides that contains 5% or less of the modified nucleotides described herein. In some embodiments, the first portion of contiguous nucleotides contains unmodified nucleotides (i.e., contains no modified nucleotides or contains only unmodified nucleotides). For example, the first portion of contiguous unmodified nucleotides contains an IRES. The hybrid modified circular RNA may or may not be modified along its entire length. For example, in certain embodiments, the hybrid modified circular RNA is [(all cytosines = methylcytosines) + (all uridines = pseudouridines) + (unmodified IRES)]. In another example, the modified hybrid is [(all adenosines = m6a) + unmodified IRES].

[0035] As used herein, the terms "fully modified cyclic polyribonucleotide equivalent" or "completely modified cyclic polyribonucleotide equivalent" or "full length modified cyclic polyribonucleotide" or "fully modified cyclic RNA" are used interchangeably and refer to a modified cyclic polyribonucleotide having the same nucleotide sequence as a control hybrid modified cyclic polyribonucleotide and a first portion containing 5% or less modified nucleotides corresponding to the first portion of the control hybrid cyclic polyribonucleotide. For example, the first portion contains an IRES with 5% or less modified nucleotides (i.e., a modified IRES). A fully modified cyclic RNA may or may not be uniformly modified along the entire length of the molecule. For example, a fully modified cyclic polyribonucleotide contains a first portion containing an IRES with more than 5% modified nucleotides, and 50% of the nucleotides outside the first portion are modified nucleotides (e.g., 50% of the uridines outside the first portion are pseudouridines). In certain embodiments, the fully modified circular polyribonucleotide is [(all cytosines = methylcytosines) + (all uridines = pseudouridines) + modified IRES]. In another example, the fully modified circular polyribonucleotide is [(all adenosines = m6a) + modified IRES].

[0036] As used herein, the term "modified ribonucleotide" refers to any ribonucleotide analog or derivative having one or more chemical modifications relative to the chemical composition of an unmodified natural ribonucleotide, such as the natural unmodified nucleotides adenosine (A), uridine (U), guanine (G), cytidine (C), and monophosphate, as shown by the chemical formulas in Table 1 below. In certain embodiments, the chemical modification of the modified ribonucleotide is a modification to any one or more functional groups of the ribonucleotide, such as the sugar, the nucleobase, or the internucleoside linkage (e.g., to the linking phosphate / phosphodiester bond / phosphodiester backbone).

[0037] As used herein, the term "linear equivalent" refers to a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence similarity therebetween) as a circular polyribonucleotide and has two free ends (i.e., the non-circularized form (and fragments thereof) of a circularized polyribonucleotide). In some embodiments, a linear equivalent (e.g., pre-circularized form) is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence similarity therebetween) as a circular polyribonucleotide and the same or similar nucleic acid modifications and has two free ends (i.e., the non-circularized form (and fragments thereof) of a circularized polyribonucleotide). In some embodiments, a linear equivalent is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage of sequence similarity therebetween) and a different or no nucleic acid modification, and has two free ends (i.e., the non-circularized form (and fragments thereof) of a circularized polyribonucleotide). In some embodiments, a fragment of a polyribonucleotide molecule that is a linear equivalent is any portion of the linear equivalent polyribonucleotide molecule that is shorter than the linear equivalent polyribonucleotide molecule. In some embodiments, the linear equivalent further comprises a 5' cap. In some embodiments, the linear equivalent further comprises a polyadenosine tail. In some embodiments, the linear equivalent further comprises a 3' UTR. In some embodiments, the linear equivalent further comprises a 5' UTR.

[0038] As used herein, the term "fragment" refers to any portion of a nucleotide molecule that is at least one nucleotide shorter than the nucleotide molecule. For example, the nucleotide molecule can be a circular polyribonucleotide molecule, and the fragment can be a polyribonucleotide or any number of consecutive polyribonucleotides that are part of the circular polyribonucleotide molecule. As another example, the nucleotide molecule can be a linear polyribonucleotide molecule, and the fragment can be a monoribonucleotide or any number of consecutive polyribonucleotides that are part of the linear polyribonucleotide molecule.

[0039] As used herein, the term "encryptogen" refers to a nucleic acid sequence or structure of a circular polyribonucleotide that serves to reduce, avoid, and / or evade detection by immune cells and / or reduce the induction of an immune response against a circular polyribonucleotide.

[0040] As used herein, the term "expressed sequence" is a nucleic acid sequence that codes for a product, such as a peptide or polypeptide, or a regulatory nucleic acid. An exemplary expressed sequence that codes for a peptide or polypeptide can include multiple nucleotide triplets, each of which can code for an amino acid, and are referred to as a "codon."

[0041] As used herein, the term "immunity protein binding site" is 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" is any protein or peptide that is associated with an immune response to, for example, 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] As used herein, the phrase "quasi-helical structure" refers to a higher order structure of a circular polyribonucleotide in which at least a portion of the circular polyribonucleotide folds into a helical structure.

[0043] As used herein, the phrase "quasi-double-stranded secondary structure" refers to a higher order structure of a circular polyribonucleotide in which at least a portion of the circular polyribonucleotide forms an internal duplex.

[0044] As used herein, the term "regulatory element" refers to a moiety, such as a nucleic acid sequence, that regulates the expression of an expression sequence within a circular polyribonucleotide.

[0045] As used herein, the term "repetitive nucleotide sequence" refers to a nucleic acid sequence that is repeated within a stretch of DNA or RNA or throughout a genome. In some embodiments, the repetitive nucleotide sequence comprises a polyCA or polyTG (UG) sequence. In some embodiments, the repetitive nucleotide sequence comprises a repeated sequence in the Alu family of introns.

[0046] As used herein, the term "replication elements" are sequences and / or motifs that are useful for the replication or initiation of transcription of a circular polyribonucleotide.

[0047] As used herein, the term "stagger element" is a moiety, such as a nucleotide sequence, that induces pausing of the ribosome during translation. In some embodiments, the stagger element is a non-conserved sequence of amino acids with strong alpha-helical propensity followed by the consensus sequence -D(V / I)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] As used herein, the term "substantially resistant" means having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% resistance compared to a control.

[0049] As used herein, the term "stoichiometric translation" refers to the substantially equal production of expression products translated from circular polyribonucleotide.For example, in a circular polyribonucleotide having two expression sequences, the stoichiometric translation of the circular polyribonucleotide can mean that the expression products of the two expression sequences can have substantially equal amounts, for example, the amount difference (e.g., molar difference) between the two expression sequences can be less than about 0 or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, or any percentage therebetween.

[0050] As used herein, the term "translation initiation sequence" is a nucleic acid sequence that initiates the translation of an expression sequence in a circular polyribonucleotide.

[0051] As used herein, the term "termination element" is a portion of a nucleic acid sequence or the like that stops the translation of an expressed sequence in a circular polyribonucleotide.

[0052] As used herein, the term "translation efficiency" refers to the rate or amount of protein or peptide production from a ribonucleotide transcript. In one embodiment, translation efficiency can be expressed as the amount of protein or peptide produced per a 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, in a given period of time.

[0053] As used herein, the term "circularization efficiency" refers to the measurement of the resulting circular polyribonucleotide compared to its starting material.

[0054] As used herein, the term "immunogenic" refers to the potential to induce an immune response to a substance. In some embodiments, when the immune system of an organism or a type of immune cell is exposed to an immunogenic substance, an immune response can be induced. The term "non-immunogenic" refers to the absence or absence of an immune response above a detectable threshold to a substance. In some embodiments, when the immune system of an organism or a type of immune cell is exposed to a non-immunogenic substance, no immune response is detected. 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 an immunogenicity assay is used to measure a natural immune response (e.g., to measure an inflammation marker) to a cyclic polyribonucleotide, the non-immunogenic polyribonucleotides provided herein can result in the production of a natural immune response at a level below a predetermined threshold. The predetermined threshold can be, for example, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or less than the level of the marker caused by the natural immune response to a control reference.

[0055] As used herein, the term "pharmaceutical acceptable" refers to an ingredient that is not biologically or otherwise objectionable, e.g., an ingredient may be incorporated into a pharmaceutical formulation of the present invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation in which it is included. In certain embodiments, when the term "pharmaceutical acceptable" is used to refer to an excipient, it indicates that the ingredient has met the necessary standards of toxicological and manufacturing testing, or that it is included in the inactive ingredient guide prepared by the US Food and Drug Administration.

[0056] As used herein, the term "carrier" refers to a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., cyclic polyribonucleotide) into a cell by covalent modification of the cyclic polyribonucleotide, partially or completely by an encapsulating agent, or a combination thereof. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles (e.g., nanoparticles that encapsulate or are covalently attached to cyclic polyribonucleotides), liposomes, fusosomes, ex vivo fractionated reticulocytes, exosomes, protein carriers (e.g., proteins covalently attached to cyclic polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents).

[0057] As used herein, the term "naked delivery" refers to a formulation for delivery to a cell that does not use a carrier and does not have covalent modifications to moieties that aid in delivery to the cell. A naked delivery formulation does not include any transfection reagent, cationic carrier, carbohydrate carrier, nanoparticle carrier, or protein carrier. For example, a naked delivery formulation of a cyclic polyribonucleotide is a formulation that includes a cyclic polyribonucleotide that does not have covalent modifications and does not include a carrier.

[0058] The term "diluent" refers to a vehicle comprising an inert solvent in which the compositions described herein (e.g., compositions comprising cyclic polyribonucleotides) can be diluted or dissolved. The diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof. The diluent can be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and 1,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, or powdered sugar.

[0059] Incorporation by Reference 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.

[0060] The following detailed description of the embodiments of the 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. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for reducing the immunogenicity of a cyclic polyribonucleotide in a subject, comprising: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least one modified nucleotide and at least about 5-1000 contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; obtaining reduced immunogenicity for said hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of said subject. (Item 2) 2. The method of claim 1, wherein the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleotides. (Item 3) 3. The method of claim 1 or 2, wherein the first portion lacks 5'-methylcytidine or pseudouridine. (Item 4) 4. The method according to any one of items 1 to 3, wherein the cyclic polyribonucleotide has translation ability. (Item 5) The hybrid modified cyclic polyribonucleotide is (a) has at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3 fold higher expression than the corresponding unmodified circular polyribonucleotide; (b) has a half-life that is at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times longer than a corresponding unmodified circular polyribonucleotide; (c) has a longer half-life than the corresponding unmodified circular polyribonucleotide; or (d) the method of any one of items 1 to 4, which has at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times less immunogenicity than a corresponding unmodified cyclic polyribonucleotide, as assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β. (Item 6) The at least one modified nucleotide is (a) N(6)-methyladenosine (m6A), 5'-methylcytidine, and pseudouridine; (b) 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, and 2'-fluoro N3-P5'-phosphoramidites; or (c) The method according to any one of items 1 to 5, wherein the modified nucleotide is selected from the group consisting of any modified nucleotide in Table 2. (Item 7) 7. The method of any one of items 1 to 6, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the nucleotides of the hybrid modified cyclic polyribonucleotide are modified nucleotides. (Item 8) 8. The method according to any one of items 1 to 7, wherein the hybrid modified cyclic polyribonucleotide comprises a binding site consisting of unmodified nucleotides and configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target. (Item 9) 8. The method according to any one of items 1 to 7, wherein the hybrid modified circular polyribonucleotide comprises one or more expression sequences. (Item 10) 10. The method of any one of items 1 to 7 or 9, wherein the first part comprises an IRES consisting of unmodified nucleotides. (Item 11) The one or more expression sequences of the hybrid modified circular polyribonucleotides are (a) Higher translation efficiency than fully modified circular polyribonucleotide equivalents; (b) a translation efficiency that is at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than a fully modified circular polyribonucleotide equivalent; (c) a higher translation efficiency than the corresponding unmodified circular polyribonucleotide; or (d) a translation efficiency that is at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than the corresponding unmodified circular polyribonucleotide 11. The method according to item 9 or 10, comprising the steps of: (Item 12) 1. A method for expressing one or more expression sequences in a subject, comprising: providing a hybrid modified cyclic polyribonucleotide comprising one or more expression sequences, said hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least one modified nucleotide and at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive unmodified nucleotides; administering the hybrid modified circular polyribonucleotide to the subject; and obtaining increased expression of the one or more expressed sequences compared to the expression of the corresponding one or more expressed sequences in a fully modified circular polyribonucleotide equivalent in the subject's cells or tissues. (Item 13) 1. A method for increasing stability of a cyclic polyribonucleotide in a subject, comprising: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising a modified cyclic polyribonucleotide and at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous unmodified nucleotides; administering the hybrid modified circular polyribonucleotide to the subject; obtaining increased stability for said hybrid modified cyclic polyribonucleotides as compared to corresponding unmodified cyclic polyribonucleotides in said subject's cells or tissues; The method includes: (Item 14) 14. The method of claim 12 or 13, wherein the first portion comprises an IRES. (Item 15) 1. A pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising: at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding moiety of a first target, e.g., an RNA, DNA, protein, or cellular target, wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif comprised of unmodified nucleotides; wherein said first target and said hybrid modified circular polyribonucleotide form a complex. (Item 16) 1. A pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising: at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding moiety of a first target, e.g., an RNA, DNA, protein, or cellular target, wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif comprised of unmodified nucleotides; and a second binding site configured to bind to a second binding moiety of a second target, wherein said second binding moiety is a second circular RNA binding motif; the first binding moiety is different from the second binding moiety; the first target, the second target, and the hybrid modified circular polyribonucleotide form a complex; The pharmaceutical composition, wherein the first target or the second target is not a microRNA. (Item 17) 1. A pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising: at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding moiety of a first target, wherein the first binding moiety is a first cyclic polyribonucleotide (cyclic RNA) binding motif; and a second binding site configured to bind to a second binding moiety of a second target, wherein said second binding moiety is a second circular RNA binding motif; the first binding moiety is different from the second binding moiety; The pharmaceutical composition, wherein both the first target and the second target are microRNAs. [Brief description of the drawings]

[0061] [Figure 1-1] 1A, 1B, and 1C show that the modified circular RNA was translated intracellularly. [Figure 1-2] 1A, 1B, and 1C show that the modified circular RNA was translated intracellularly. [Figure 1-3]1A, 1B, and 1C show that the modified circular RNA was translated intracellularly. [Figure 2-1] Figures 2A, 2B, and 2C show that the modified circular RNA has reduced immunogenicity compared to unmodified circular RNA to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Figure 2-2] Figures 2A, 2B, and 2C show that the modified circular RNA has reduced immunogenicity compared to unmodified circular RNA to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Figure 2-3] Figures 2A, 2B, and 2C show that the modified circular RNA has reduced immunogenicity compared to unmodified circular RNA to cells, as assessed by MDA5, OAS, and IFN-β expression in transfected cells. [Diagram 3] 1 shows that the hybrid modified circular RNA has reduced immunogenicity compared to unmodified circular RNA as assessed by intracellular RIG-I, MDA5, IFN-β, and OAS expression. [Figure 4] FIG. 1 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). [Diagram 5] FIG. 1 shows a schematic diagram of an exemplary in vivo generation process of circular RNA. [Figure 6] 1 shows the design of an exemplary circular RNA containing a start codon, an ORF encoding GFP, a stagger element (2A) and an encryptogen. [Figure 7] FIG. 1 is a schematic showing in vivo stoichiometric protein expression of two different circular RNAs. [Figure 8] FIG. 1 is a schematic showing in vivo protein expression in a mouse model from an exemplary circular RNA. [Figure 9]FIG. 1 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 10] FIG. 1 shows a schematic diagram of an exemplary circular RNA with a pseudohelical structure (HDVmin), which can be subjected to SHAPE analysis for its structural information. [Figure 11] FIG. 1 shows a schematic diagram of an exemplary circular RNA with a functional pseudohelical structure (HDVmin) that exhibits HDAg binding activity. [Figure 12] FIG. 1 is a schematic diagram showing transcription, self-cleavage and ligation of an exemplary self-replicating circular RNA. [Figure 13] 1 is an image of a denaturing PAGE gel showing in vitro generation of various exemplary circular RNAs. [Figure 14] 1 is a graph summarizing the circularization efficiency of various exemplary circular RNAs. [Figure 15] 1 is a denaturing PAGE gel image showing the reduced degradation susceptibility of an exemplary circular RNA compared to its linear counterpart. [Figure 16] 1 is a denaturing PAGE gel image showing an exemplary circular RNA after an exemplary purification process. [Figure 17] 1 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 18] 1 is a Western blot image showing rolling circle translation of an exemplary circular RNA. [Figure 19] 1 shows Western blot images illustrating the production of distinct proteins or long contiguous peptides from various exemplary circular RNAs with and without an exemplary stagger element. [Figure 20A] 1 is a Western blot image showing a comparison of protein expression between various exemplary circular RNAs with exemplary stagger or termination elements (stop codons). [Figure 20B] 1 is a graph summarizing signal intensities from Western blot analysis of protein products translated from two exemplary circular RNAs. [Figure 21] 1 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 22] 1 is a graph summarizing RNA amounts at different collection time points in a time course experiment examining the half-life of an exemplary circular RNA. [Figure 23A] FIG. 13 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. [Figure 23B] FIG. 13 is a graph showing qRT-PCR analysis of linear and circular RNA levels using primers specific for circular RNA. [Figure 24] 1 is an image showing blots of cell lysates from circular and linear RNA probed for EGF protein and β-tubulin loading control. [Diagram 25] FIG. 1 is a graph showing qRT-PCR analysis of immune-related genes from 293T cells transfected with circular or linear RNA. [Figure 26] 1 is a graph showing luciferase activity of proteins expressed from circular RNA by rolling circle translation. [Figure 27] 1 is a graph showing the luciferase activity of proteins expressed from circular or linear RNA. [Figure 28] 1 is a graph showing the luciferase activity of proteins expressed from linear or circular RNA by rolling circle translation. [Figure 29] 1 is a graph showing luciferase activity of protein expressed from circular RNA via IRES translation initiation. [Diagram 30] FIG. 13 is a graph showing luciferase activity of proteins expressed from circular RNA by IRES-initiated and rolling circle translation. [Diagram 31] 1 is an image showing a protein blot of expression products from circular or linear RNA. [Diagram 32] 1 is an image showing a protein blot of expression products from circular or linear RNA. [Diagram 33] 1 shows the predicted structure of an exemplary circular RNA with a pseudo-double-stranded structure. [Diagram 34] 1 shows the predicted structure of an exemplary circular RNA with pseudohelical structure. [Diagram 35] 1 shows the predicted structure of an exemplary circular RNA with a pseudohelical structure linked to repeat sequences. [Diagram 36] 1 presents experimental data showing that degradation of an exemplary circular RNA with RNAse H produced nucleic acid degradation products consistent with circular RNA but not concatemeric RNA. [Figure 37] Electrophoretic images of DNA of various lengths generated for the formation of various RNA lengths are shown. [Figure 38] Experimental data are shown confirming RNA circularization using RNAse R treatment and qPCR analysis for circular junctions of various lengths. [Figure 39] 1 shows the generation of an exemplary circular RNA with a protein binding site. [Diagram 40] 1 shows the generation of an exemplary circular RNA with a miRNA binding site. [Diagram 41] 1 shows the generation of an exemplary circular RNA by self-splicing. [Diagram 42] Figure 1 shows experimental data demonstrating higher stability of circular RNA in dividing cells compared to linear controls. [Diagram 43] 1 shows experimental data demonstrating protein expression from an exemplary circular RNA with multiple expressed sequences and rolling circle translation of an exemplary circular RNA with multiple expressed sequences. [Diagram 44] It is shown that after injection into mice, circular RNA was detected at higher levels than linear RNA in mouse liver at 3, 4 and 7 days after injection. [Figure 45A]FIG. 1 shows that after injection of mice with circular or linear RNA expressing Gaussia luciferase, Gaussia luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23 days after administration of circular RNA, whereas activity was detected in plasma only at 1 and 2 days after administration of modified linear RNA. [Figure 45B] FIG. 1 shows that after injection of mice with circular or linear RNA expressing Gaussia luciferase, Gaussia luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23 days after administration of circular RNA, whereas activity was detected in plasma only at 1 and 2 days after administration of modified linear RNA. [Diagram 46] 1 shows that after injection of RNA, circular RNA, but not linear RNA, was detected in the liver and spleen at 16 days after administration of RNA. [Figure 47] Figure 2 shows that after RNA injection, linear RNA, but not circular RNA, demonstrated immunogenicity as assessed by RIG-I, MDA-5, IFN-B and OAS. [Figure 48] Various exemplary cyclization methods are shown. [Figure 49] We show that circular RNA containing modified nucleotides has reduced immunogenicity in vivo compared to modified mRNA and compared to circular RNA produced with only unmodified nucleotides. [Figure 50] We show that circular RNAs containing modified nucleotides have increased stability in vivo over their fully unmodified counterparts and over modified mRNAs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The present invention generally relates to pharmaceutical compositions and formulations of cyclic polyribonucleotides and their use.In some embodiments, the cyclic polyribonucleotide comprises at least one modified polyribonucleotide and a first portion of consecutive unmodified nucleotides.In some embodiments, the modified cyclic polyribonucleotide is delivered to a subject.

[0063] The present disclosure provides a method of reducing or decreasing the immunogenicity of a cyclic polyribonucleotide in a subject, comprising the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide and a first portion of contiguous nucleotides having 5% or less modified nucleotides; administering the hybrid modified cyclic polyribonucleotide to a subject; and obtaining a reduced immunogenicity for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In one embodiment, the method of reducing or decreasing the immunogenicity of a cyclic polyribonucleotide in a subject comprises the steps of providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least about 5-1000 contiguous nucleotides having at least one modified nucleotide and 5% or less modified nucleotides; administering the hybrid modified cyclic polyribonucleotide to a subject; and obtaining a reduced immunogenicity for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In one embodiment, a method for reducing or decreasing the immunogenicity of a cyclic polyribonucleotide in a subject includes providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides, administering the hybrid modified cyclic polyribonucleotide to the subject, and obtaining reduced immunogenicity for the hybrid modified cyclic polyribonucleotide compared to the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In one embodiment, the first portion comprises an IRES. In one embodiment, the first portion comprises at least about 5-1000 contiguous unmodified nucleotides. In one embodiment, the first portion lacks 5'-methylcytidine or pseudouridine. In one embodiment, 5% or less of the nucleotides in the first portion are modified nucleotides.

[0064] The present disclosure provides a method of expressing one or more expression sequences in a subject, comprising the steps of providing a hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide, a first portion of contiguous nucleotides having 5% or less modified nucleotides, and one or more expression sequences, administering the hybrid modified cyclic polyribonucleotide to the subject, and obtaining increased expression of the one or more expression sequences compared to the expression of the corresponding one or more expression sequences in a fully modified cyclic polyribonucleotide in a cell or tissue of the subject. In one embodiment, the method of expressing one or more expression sequences in a subject comprises the steps of providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least one modified nucleotide and at least about 5-1000 contiguous nucleotides having 5% or less modified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject, and obtaining increased expression of the one or more expression sequences compared to the expression of the corresponding one or more expression sequences in a fully modified cyclic polyribonucleotide in a cell or tissue of the subject. In some embodiments, a method for expressing one or more expressible sequences in a subject includes providing a hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide, a first portion of contiguous unmodified nucleotides, and one or more expressible sequences, administering the hybrid modified cyclic polyribonucleotide to a subject, and obtaining increased expression of the one or more expressible sequences compared to expression of the corresponding one or more expressible sequences in a cell or tissue of the subject in a fully modified cyclic polyribonucleotide. In some embodiments, the first portion comprises an IRES. In some embodiments, the first portion comprises at least about 5-1000 contiguous unmodified nucleotides. In some embodiments, the first portion lacks 5'-methylcytidine or pseudouridine. In some embodiments, 5% or less of the nucleotides in the first portion are modified nucleotides.

[0065] The present disclosure provides a method for increasing stability of a cyclic polyribonucleotide in a subject, comprising the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide and a first portion of contiguous nucleotides having 5% or less modified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining increased stability for the hybrid modified cyclic polyribonucleotide compared to the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In one embodiment, the method for increasing stability of a cyclic polyribonucleotide in a subject comprises the steps of providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion comprising at least about 5-1000 contiguous nucleotides having at least one modified nucleotide and 5% or less modified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining increased stability for the hybrid modified cyclic polyribonucleotide compared to the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In one embodiment, a method for increasing stability of a cyclic polyribonucleotide in a subject includes providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to the subject; and obtaining increased stability for the hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In one embodiment, the first portion comprises an IRES. In one embodiment, the first portion comprises at least about 5-1000 contiguous unmodified nucleotides. In one embodiment, the first portion lacks 5'-methylcytidine or pseudouridine. In one embodiment, 5% or less of the nucleotides in the first portion are modified nucleotides.

[0066] Methods of Using Modified Circular Polyribonucleotides In some aspects, the invention described herein includes methods of using compositions of hybrid modified cyclic polyribonucleotides and delivery of hybrid modified cyclic polyribonucleotides. In some embodiments, the hybrid modified cyclic polyribonucleotides are delivered to a subject. Compared to the corresponding unmodified cyclic polyribonucleotides, administration of the hybrid modified cyclic polyribonucleotides described herein to a subject may result in reduced or decreased immunogenicity, increased translation efficiency (e.g., increased expression of one or more expression sequences in the hybrid modified cyclic polyribonucleotides), or increased stability in the subject's cells or tissues. Compared to the fully modified cyclic polyribonucleotide equivalents, administration of the hybrid modified cyclic polyribonucleotides described herein to a subject may result in increased translation efficiency (e.g., increased expression of one or more expression sequences in the hybrid modified cyclic polyribonucleotides) in the subject's cells or tissues. In some embodiments, the hybrid modified cyclic polyribonucleotides include at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides. In some embodiments, the present disclosure provides a method of using a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified nucleotide and a first portion, the first portion comprising at least about 5 consecutive unmodified nucleotides. In some embodiments, the hybrid cyclic polyribonucleotide comprises one or more expression sequences.

[0067] In some embodiments, the first portion comprises at least about 5-1000 contiguous nucleotides having 5% or less modified nucleotides. In some embodiments, the first portion comprises at least about 5-1000 contiguous unmodified nucleotides. In some embodiments, the first portion lacks 5'-methylcytidine, pseudouridine, or N1-methyl-pseudouridine. In some embodiments, 5% or less of the nucleotides in the first portion are modified. In some embodiments, 0%, 1%, 2%, 3%, 4%, or 5% or less of the nucleotides in the first portion are modified. In some embodiments, no nucleotides in the first portion are modified. In some embodiments, the first portion is an IRES. In some embodiments, the first portion is an IRES comprising 5% or less modified nucleotides. In some embodiments, the first portion is an IRES comprising no modified nucleotides (e.g., comprising only unmodified nucleotides). In some embodiments, the first portion is an IRES consisting of unmodified nucleotides. In some embodiments, the first portion comprises a binding site configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, which is comprised of unmodified nucleotides. In some embodiments, the hybrid modified cyclic polyribonucleotide is translation competent. In some embodiments, the hybrid modified cyclic polyribonucleotide is in a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier or excipient.

[0068] The hybrid modified cyclic polyribonucleotide may include a first portion that includes at least one modified nucleotide and contiguous unmodified nucleotide. The modified nucleotide is outside the first portion. The modified polyribonucleotide of the hybrid modified cyclic polyribonucleotide may be an analog or derivative having one or more chemical modifications to the chemical composition of unmodified natural ribonucleotide, such as the natural unmodified nucleotides adenosine (A), uridine (U), guanine (G), cytidine (C), and monophosphate, as shown by the chemical formula in Table 1. The chemical modification of the modified ribonucleotide may be a modification to any one or more functional groups of the ribonucleotide, such as the sugar, nucleobase, or internucleoside bond (e.g., to the linking phosphate / phosphodiester bond / phosphodiester backbone). In some embodiments, the modified nucleotide of the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid cyclic polyribonucleotide) can be any modification known by those skilled in the art, such as those specified in Gilbert, WV, et al. Science. 2016 Jun17; 352 (6292): 1408-1412, which is incorporated herein by reference, or those in this document. For example, the modification can be as described in Table 2.

[0069] [Table 1]

[0070] [Table 2]

[0071] In certain embodiments, the modified nucleotides are N(6) methyl adenosine (m6A), 5'-methyl cytidine (5mC), pseudouridine, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl ... O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotide, thiophosphonate nucleotide, and 2'-fluoro N3-P5'-phosphoramidite. In some embodiments, the modified nucleotide is any modified nucleotide known by those of skill in the art, such as those identified in Gilbert, WV, et al. Science. 2016 Jun17;352(6292):1408-1412, which is incorporated herein by reference, or in this document.

[0072] The first portion of the hybrid modified cyclic polyribonucleotide described herein comprises at least about 5-1000 contiguous nucleotides. In some embodiments, the first portion comprises at least about 5-1000, 10-1000, 20-1000, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000, or 900-2000 contiguous nucleotides. The first portion of the hybrid modified cyclic polyribonucleotide described herein may comprise contiguous nucleotides having 5% or less modified nucleotides. In some embodiments, the first portion comprises contiguous nucleotides having 0%, 1%, 2%, 3%, 4%, or 5% or less modified nucleotides. In some embodiments, the first portion is an IRES. In some embodiments, the first portion is an IRES that contains 5% or less modified nucleotides. In some embodiments, the first portion is an IRES that does not contain modified nucleotides (e.g., contains only unmodified nucleotides). In some embodiments, the first portion is an IRES that consists of unmodified nucleotides. The first portion of the hybrid modified circular polyribonucleotide described herein may contain consecutive unmodified nucleotides. The first portion may contain at least about 5 consecutive unmodified nucleotides. In some embodiments, the first portion contains at least about 5-1000 consecutive unmodified nucleotides. In some embodiments, the first portion comprises at least about 5-1000, 10-1000, 20-1000, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000, or 900-2000 consecutive unmodified nucleotides. In some embodiments, the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000, or any number therebetween.In some embodiments, the first portion comprises 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or any number therebetween of contiguous unmodified nucleotides. The first portion may comprise an IRES. In some embodiments, the first portion lacks 5'-methylcytidine, pseudouridine, or N1-methyl-pseudouridine. In certain embodiments, the first moiety is selected from the group consisting of N(6) methyl adenosine (m6A), 5'-methyl cytidine (5mC), pseudouridine, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), and the like. ), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, and 2'-fluoroN3-P5'-phosphoramidites. In some embodiments, the first portion lacks a nucleotide modification known by those of skill in the art, such as those identified in Gilbert, WV, et al. Science. 2016 Jun17;352(6292):1408-1412, which is incorporated herein by reference, or in this document.

[0073] The hybrid modified cyclic polyribonucleotides described herein may include, outside the first portion, 5'-methylcytidine, pseudouridine, or combinations thereof. The hybrid modified cyclic polyribonucleotides may include, outside the first portion, N(6) methyladenosine (m6A), 5'-methylcytidine (5mC), pseudouridine, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO ... The modified nucleotide may include a modification selected from the group consisting of aminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotide, thiophosphonate nucleotide, and 2'-fluoroN3-P5'-phosphoramidite, where the modified nucleotide is outside the first portion. In some embodiments, the modified nucleotide outside the first portion is any modified nucleotide known by those of skill in the art, such as those identified in Gilbert, WV, et al. Science. 2016 Jun17;352(6292):1408-1412, which is incorporated herein by reference, or in this document.

[0074] Reduced immunogenicity A method of reducing or decreasing the immunogenicity of a cyclic polyribonucleotide in a subject may include providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides, administering the hybrid modified cyclic polyribonucleotide to a subject, and obtaining a reduced or decreased immunogenicity for the modified cyclic polyribonucleotide compared to the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In an embodiment, the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous unmodified nucleotides. In an embodiment, 5% or less of the nucleotides in the first portion are modified. In an embodiment, 1%, 2%, 3%, 4%, or 5% or less of the nucleotides in the first portion are modified. In an embodiment, no nucleotides in the first portion are modified. In an embodiment, the first portion is an IRES. In some embodiments, the first portion comprises a binding site configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, which is comprised of unmodified nucleotides. In some embodiments, the first portion is an IRES that comprises 5% or less modified nucleotides. In some embodiments, the first portion is an IRES that does not comprise modified nucleotides. In some embodiments, the first portion is an IRES that comprises unmodified nucleotides. In some embodiments, the reduced or decreased immunogenicity for the modified cyclic polyribonucleotide is at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times lower compared to the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of a subject.

[0075] In some embodiments, the hybrid modified cyclic polyribonucleotides disclosed herein have reduced or decreased immunogenicity after administration to a subject, as compared to the corresponding unmodified cyclic polyribonucleotides. In some embodiments, the hybrid modified cyclic polyribonucleotides disclosed herein have at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times less immunogenic than the corresponding unmodified cyclic polyribonucleotides after administration to a subject. In one embodiment, the immunogenicity described herein is assessed by the level of expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β following administration of a hybrid modified circular polyribonucleotide to a subject.

[0076] Increased translation efficiency The present disclosure provides a method of expressing one or more expression sequences in a subject, comprising providing a hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide, a first portion of contiguous unmodified nucleotides, and one or more expression sequences, administering the hybrid modified cyclic polyribonucleotide to a subject, and obtaining increased expression of the one or more expression sequences compared to the expression of the corresponding one or more expression sequences of the fully modified cyclic polyribonucleotide equivalent in a cell or tissue of the subject. In an embodiment, the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous unmodified nucleotides. In an embodiment, 5% or less of the nucleotides in the first portion are modified. In an embodiment, 1%, 2%, 3%, 4%, or 5% or less of the nucleotides in the first portion are modified. In an embodiment, no nucleotides in the first portion are modified. In an embodiment, the first portion is an IRES. In some embodiments, the first portion comprises a binding site configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, which is comprised of unmodified nucleotides. In some embodiments, the first portion is an IRES that comprises 5% or less modified nucleotides. In some embodiments, the first portion is an IRES that does not comprise modified nucleotides. In some embodiments, the first portion is an IRES that comprises unmodified nucleotides. In some embodiments, the hybrid modified circular polyribonucleotide comprises one or more expression sequences.

[0077] In some embodiments, the increased expression of one or more expressed sequences of the hybrid modified cyclic polyribonucleotide is similar to or higher than the one or more expressed sequences of the fully modified cyclic polyribonucleotide equivalent. In some embodiments, the increased expression of one or more expressed sequences of the hybrid modified cyclic polyribonucleotide is at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than the one or more expressed sequences of the fully modified cyclic polyribonucleotide equivalent. In some embodiments, the increased expression of one or more expressed sequences of the hybrid modified cyclic polyribonucleotide is similar to or higher than the corresponding unmodified cyclic polyribonucleotide. In certain embodiments, the increased expression of one or more expression sequences of the hybrid modified circular polyribonucleotide is at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than the corresponding unmodified circular polyribonucleotide.

[0078] In certain embodiments, the increased expression of one or more sequences of the hybrid modified circular polyribonucleotide 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 fully modified circular polyribonucleotide equivalent. In some embodiments, the increased expression of one or more sequences of the hybrid modified circular polyribonucleotide is at least about 10% higher than the fully modified circular polyribonucleotide equivalent. In some embodiments, the increased expression of one or more sequences of the hybrid modified circular polyribonucleotide is at least about 20% higher than the fully modified circular polyribonucleotide equivalent. In some embodiments, the increased expression of one or more sequences of the hybrid modified circular polyribonucleotide is at least about 50% higher than the fully modified circular polyribonucleotide equivalent. In certain embodiments, the increased expression of one or more sequences of the hybrid modified cyclic polyribonucleotide 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 that of the corresponding unmodified cyclic polyribonucleotide. In some embodiments, the increased expression of one or more sequences of the hybrid modified cyclic polyribonucleotide is at least about 10% higher than that of the corresponding unmodified cyclic polyribonucleotide. In some embodiments, the increased expression of one or more sequences of the hybrid modified cyclic polyribonucleotide is at least about 20% higher than that of the corresponding unmodified cyclic polyribonucleotide.In some embodiments, the increased expression of one or more sequences of the hybrid modified cyclic polyribonucleotide is at least about 50% higher than that of the corresponding unmodified cyclic polyribonucleotide. In some embodiments, the increased expression of one or more sequences of the hybrid modified cyclic polyribonucleotide is at 1 day, 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, 28 days or more, or any days therebetween, after administration to a subject.

[0079] In some embodiments, the expression of one or more expressed sequences of a hybrid modified cyclic polyribonucleotide has a translation efficiency similar to or higher than that of one or more expressed sequences of a fully modified cyclic polyribonucleotide equivalent after administration to a subject. In some embodiments, the expression of one or more expressed sequences of a hybrid modified cyclic polyribonucleotide has a translation efficiency at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than that of one or more expressed sequences of a fully modified cyclic polyribonucleotide equivalent after administration to a subject. In some embodiments, the expression of one or more expressed sequences of a hybrid modified cyclic polyribonucleotide has a translation efficiency similar to or higher than that of the corresponding unmodified cyclic polyribonucleotide after administration to a subject. In certain embodiments, one or more expressed sequences of a hybrid modified circular polyribonucleotide have a translation efficiency that is at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than the corresponding unmodified circular polyribonucleotide after administration to a subject.

[0080] In certain embodiments, expression of one or more expressed sequences of a hybrid modified circular polyribonucleotide has a translation efficiency similar to or greater than that of one or more expressed sequences of a fully modified circular polyribonucleotide equivalent at 1 day, 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, 28 days or more, or any days in between, after administration to a subject. In some embodiments, the one or more expressed sequences of the hybrid modified cyclic polyribonucleotide have a translation efficiency at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times higher than one or more expressed sequences of a fully modified cyclic polyribonucleotide equivalent at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28 days or more, or any days in between, after administration to a subject. In some embodiments, the expression of one or more expressed sequences of the hybrid modified cyclic polyribonucleotide has a translation efficiency similar to or higher than the corresponding unmodified cyclic polyribonucleotide after administration to a subject. In certain embodiments, one or more expression sequences of a hybrid modified circular polyribonucleotide have a translation efficiency that is at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, or 3 times greater than a corresponding unmodified circular polyribonucleotide at 1 day, 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, 28 days, or more, or any days in between, after administration to a subject.

[0081] In certain embodiments, the increased expression of one or more expression sequences of the hybrid modified cyclic polyribonucleotide is similar to or greater than a fully modified cyclic polyribonucleotide equivalent having a first portion comprising more than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleotides at 1 day, 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, 28 days, or more, or any days in between, after administration to a subject. In certain embodiments, the increased expression of one or more expression sequences of the hybrid modified circular polyribonucleotide is greater than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, or more, or any day therebetween, after administration to the subject. at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times higher than a fully modified circular polyribonucleotide equivalent having a first portion comprising 60%, 70%, 80%, 90%, or 100% modified nucleotides.

[0082] In certain embodiments, the increased expression of one or more expression sequences of a hybrid modified circular polyribonucleotide is similar to or higher than a fully modified circular polyribonucleotide equivalent having a first portion comprising more than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleotides. In certain embodiments, the increased expression of one or more expression sequences of the hybrid modified circular polyribonucleotide is greater than 5%, or at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times higher than a fully modified circular polyribonucleotide equivalent having a first portion comprising more than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleotides.

[0083] In certain embodiments, the increased expression of one or more expression sequences of the hybrid modified cyclic polyribonucleotide is similar to or greater than a fully modified cyclic polyribonucleotide equivalent having a first portion comprising more than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleotides at 1 day, 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, 28 days, or more, or any days in between, after administration to a subject. In certain embodiments, the increased expression of one or more expression sequences of the hybrid modified circular polyribonucleotide is greater than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, or more, or any day therebetween, after administration to the subject. at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times higher than a fully modified circular polyribonucleotide equivalent having a first portion comprising 60%, 70%, 80%, 90%, or 100% modified nucleotides.

[0084] In certain embodiments, the increased expression of one or more sequences of a hybrid modified circular polyribonucleotide comprises more than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleotides at 1 day, 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, 28 days, or more, or any days in between, after administration to a subject. 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 that of a fully modified circular polyribonucleotide equivalent having a first portion. In certain embodiments, the increased expression of one or more sequences of the hybrid modified cyclic polyribonucleotide is at least about 10% higher than that of a fully modified cyclic polyribonucleotide equivalent having a first portion comprising more than 5% or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% modified nucleotides at 1 day, 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, 28 days or more, or any days in between, after administration to a subject.In certain embodiments, the increased expression of one or more sequences of the hybrid modified cyclic polyribonucleotide is at least about 20% higher than that of a fully modified cyclic polyribonucleotide equivalent having a first portion comprising more than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleotides at 1 day, 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, 28 days or more, or any days in between, after administration to a subject. In certain embodiments, the increased expression of one or more sequences of the hybrid modified cyclic polyribonucleotide is at least about 50% higher than that of a fully modified cyclic polyribonucleotide equivalent having a first portion comprising more than 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleotides at 1 day, 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, 28 days or more, or any days in between, after administration to a subject.

[0085] As described herein, in certain embodiments, translation efficiency or increased expression is measured in either cells containing hybrid modified circular polyribonucleotides or the corresponding unmodified or fully modified circular polyribonucleotide equivalents, or in an in vitro translation system (e.g., rabbit reticulocyte lysate).

[0086] Increased stability The present disclosure provides a method for increasing stability of a cyclic polyribonucleotide in a subject, comprising the steps of: providing a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides; administering the hybrid modified cyclic polyribonucleotide to a subject; and obtaining increased stability for the hybrid modified cyclic polyribonucleotide compared to the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of the subject. In some embodiments, the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous unmodified nucleotides. In some embodiments, 5% or less of the nucleotides in the first portion are modified. In some embodiments, 1%, 2%, 3%, 4%, or 5% or less of the nucleotides in the first portion are modified. In some embodiments, no nucleotides in the first portion are modified. In some embodiments, the first portion is an IRES. In some embodiments, the first portion comprises a binding site configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, which is comprised of unmodified nucleotides. In some embodiments, the first portion is an IRES that comprises 5% or less modified nucleotides. In some embodiments, the first portion is an IRES that does not comprise modified nucleotides. In some embodiments, the first portion is an IRES that comprises unmodified nucleotides. In some embodiments, the increased stability of the hybrid modified cyclic polyribonucleotide is at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times higher than the corresponding unmodified cyclic polyribonucleotide in a cell or tissue of a subject.

[0087] In some embodiments, the hybrid modified cyclic polyribonucleotides disclosed herein have increased stability after administration to a subject, as compared to the corresponding unmodified cyclic polyribonucleotides. In some embodiments, the hybrid modified cyclic polyribonucleotides disclosed herein have increased stability after administration to a subject, at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times greater than the corresponding unmodified cyclic polyribonucleotides.

[0088] In some embodiments, the hybrid modified cyclic polyribonucleotides disclosed herein have increased stability compared to the corresponding unmodified cyclic polyribonucleotides at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28 days or more, or any days in between, after administration to a subject. In some embodiments, the hybrid modified cyclic polyribonucleotides disclosed herein have increased stability compared to the corresponding unmodified cyclic polyribonucleotides at 14 days after administration to a subject. In certain embodiments, the disclosed hybrid modified cyclic polyribonucleotides have increased stability of at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times greater than a corresponding unmodified cyclic polyribonucleotide at 1 day, 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, 28 days or more, or any days in between, after administration to a subject. In certain embodiments, the disclosed hybrid modified cyclic polyribonucleotides have increased stability of at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times greater than the corresponding unmodified cyclic polyribonucleotide at 14 days after administration to a subject.

[0089] In some embodiments, the hybrid modified cyclic polyribonucleotide has a longer half-life than the corresponding unmodified cyclic polyribonucleotide after administration to a subject. In some embodiments, the hybrid modified cyclic polyribonucleotide has a half-life that is at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times longer than the corresponding unmodified cyclic polyribonucleotide after administration to a subject. In some embodiments, the half-life is measured by introducing the hybrid modified cyclic polyribonucleotide or the corresponding unmodified cyclic polyribonucleotide into a cell and measuring the level of the hybrid modified cyclic polyribonucleotide or the corresponding unmodified cyclic polyribonucleotide introduced inside the cell.

[0090] In some embodiments, the hybrid modified cyclic polyribonucleotide has a half-life after administration to a subject that is at least that of the corresponding unmodified cyclic polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotide has a half-life after administration to a subject that is increased over that of the corresponding unmodified cyclic polyribonucleotide. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more after administration to a subject. In certain embodiments, the hybrid modified cyclic polyribonucleotide has an intracellular half-life or persistence 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, following administration to a subject. In certain embodiments, the hybrid modified cyclic polyribonucleotide has a half-life or persistence in cells 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, after administration to a subject. In some embodiments, the hybrid modified cyclic polyribonucleotide has a half-life or persistence in cells during cell division after administration to a subject. In some embodiments, the hybrid modified cyclic polyribonucleotide has a half-life or persistence in cells after administration to a subject and after division.In certain embodiments, the hybrid modified cyclic polyribonucleotide has a half-life or persistence within 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, following administration to a subject.

[0091] In some embodiments, the hybrid modified cyclic polyribonucleotide persists in the cell during cell division after administration to a subject. In some embodiments, the hybrid modified cyclic polyribonucleotide persists in daughter cells after mitosis after administration to a subject. In some embodiments, the hybrid modified cyclic polyribonucleotide replicates in the cell and is passed on to daughter cells after administration to a subject. In some embodiments, the cell passes on at least one hybrid modified cyclic polyribonucleotide to daughter cells with at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% efficiency after administration to a subject. In some embodiments, the cell undergoing meiosis passes on the hybrid modified cyclic polyribonucleotide to daughter cells with at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% efficiency after administration to a subject. In certain embodiments, cells undergoing mitosis pass on the hybrid modified circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% following administration to a subject.

[0092] Modified cyclic polyribonucleotides Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) are used in the methods described herein. In some embodiments, the hybrid modified cyclic polyribonucleotide comprises at least one modified polyribonucleotide and a first portion of contiguous unmodified nucleotides. In some embodiments, the hybrid modified cyclic polyribonucleotides described herein comprise at least one modified nucleotide and a first portion, wherein the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous unmodified nucleotides. In some embodiments, 5% or less of the nucleotides in the first portion are modified. In some embodiments, 0%, 1%, 2%, 3%, 4%, or 5% or less of the nucleotides in the first portion are modified. In some embodiments, no nucleotides in the first portion are modified. In some embodiments, the first portion is an IRES. In some embodiments, the first portion is an IRES comprising 5% or less modified nucleotides. In some embodiments, the first portion is an IRES that does not include modified nucleotides. In some embodiments, the first portion is an IRES that is composed of unmodified nucleotides. In some embodiments, the first portion comprises a binding site that is composed of unmodified nucleotides and is configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target. In some embodiments, the first portion lacks 5'-methylcytidine, pseudouridine, or N1-methyl-pseudouridine. In some embodiments, the hybrid modified cyclic polyribonucleotide is in a pharmaceutical composition that further comprises a pharma- ceutically acceptable carrier or excipient. In some embodiments, the hybrid modified cyclic polyribonucleotide is delivered to a subject. The hybrid modified cyclic polyribonucleotides described herein may have reduced or decreased immunogenicity, increased translation efficiency (e.g., increased expression of one or more expression sequences in the hybrid modified cyclic polyribonucleotide), or increased stability compared to their fully modified cyclic polyribonucleotide counterparts.

[0093] The first portion comprises consecutive nucleotides having 5% or less modified nucleotides in the hybrid modified cyclic polyribonucleotide. In some embodiments, 0%, 1%, 2%, 3%, 4%, or 5% or less consecutive nucleotides in the first portion are modified. The first portion comprises consecutive unmodified nucleotides in the hybrid modified cyclic polyribonucleotide. The first portion may comprise at least about 5 consecutive unmodified nucleotides. In some embodiments, the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 consecutive nucleotides having 0%, 1%, 2%, 3%, 4%, or 5% or less modified nucleotides. In some embodiments, the first portion comprises at least about 5-1000 consecutive nucleotides having 0%, 1%, 2%, 3%, 4%, or 5% or less modified nucleotides. In some embodiments, the first portion comprises at least about 5-1000, 10-1000, 20-1000, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000, or 900-2000 contiguous nucleotides having 0%, 1%, 2%, 3%, 4%, or 5% or less modified nucleotides. In some embodiments, the first portion comprises at least about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous unmodified nucleotides. In some embodiments, the first portion comprises at least about 5-1000 contiguous unmodified nucleotides. In some embodiments, the first portion comprises at least about 5-1000, 10-1000, 20-1000, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, 900-1000, or 900-2000 contiguous unmodified nucleotides. In some embodiments, the first portion comprises 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or any number therebetween contiguous unmodified nucleotides. The first portion may comprise an IRES.In certain embodiments, the first portion comprises a binding site. In certain embodiments, the first portion comprises a binding site configured to bind to a peptide, a protein, a biomolecule, DNA, RNA, or a cellular target.

[0094] In some embodiments, the hybrid modified cyclic polyribonucleotide has modified nucleotides, such as 5' methylcytidine and pseudouridine, throughout the entire cyclic polyribonucleotide, except for the IRES element or the binding site configured to bind to a protein, DNA, RNA, or cellular target. In these cases, the hybrid modified cyclic polyribonucleotide has a lower immunogenicity compared to the corresponding unmodified cyclic polyribonucleotide. In these cases, the hybrid modified cyclic polyribonucleotide has a lower immunogenicity compared to the corresponding cyclic polyribonucleotide that does not include 5' methylcytidine and pseudouridine. In certain embodiments, the hybrid modified cyclic polyribonucleotide has at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times less immunogenic than the corresponding unmodified cyclic polyribonucleotide. In certain embodiments, the immunogenicity described herein is assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β. In some embodiments, the hybrid modified cyclic polyribonucleotide has a longer half-life than the corresponding unmodified cyclic polyribonucleotide, e.g., a corresponding cyclic polyribonucleotide that does not include 5' methylcytidine and pseudouridine. In some embodiments, the hybrid modified cyclic polyribonucleotide has a longer half-life that is at least about 1.1, 1.2, 1.3, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.3, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times longer than the corresponding unmodified cyclic polyribonucleotide.In one embodiment, the half-life is measured by introducing a hybrid modified cyclic polyribonucleotide or a corresponding cyclic polyribonucleotide into a cell and measuring the level of the hybrid modified cyclic polyribonucleotide or the corresponding cyclic polyribonucleotide introduced inside the cell.

[0095] The modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may include at least one modified nucleotide. The hybrid modified cyclic polyribonucleotide described herein may include a first portion that includes consecutive unmodified nucleotides and at least one modified nucleotide. The modified nucleotide is outside the first portion. The modified polyribonucleotide of the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may be an analog or derivative having one or more chemical modifications to the chemical composition of unmodified natural ribonucleotides, such as the natural unmodified nucleotides adenosine (A), uridine (U), guanine (G), cytidine (C) shown as described herein. The chemical modification of the modified ribonucleotide may be a modification to any one or more functional groups of the ribonucleotide, such as the sugar, nucleobase, or internucleoside bond (e.g., to the linking phosphate / phosphodiester bond / phosphodiester backbone).

[0096] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more post-transcriptional modifications (e.g., capping, truncation, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the 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 certain embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) is selected from the group consisting of pyridin-4-one ribonucleosides, 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- The nucleoside comprises at least one nucleoside selected from the group consisting of uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.In certain embodiments, the modified cyclic polyribonucleotide is 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-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 certain embodiments, the modified cyclic polyribonucleotide 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-isopentenyl adenosine, N6-(cis-hydroxyisopentenyl ) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 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.

[0097] In certain embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) is selected from the group consisting of N(6) methyl adenosine (m6A), 5'-methylcytidine (5mC), pseudouridine, or N1-methyl-pseudouridine, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMA ... The modified cyclic polyribonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',5'-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotide, thiophosphonate nucleotide, and 2'-fluoro N3-P5'-phosphoramidite. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises nucleotide modifications known by those of skill in the art, such as those identified in Gilbert, WV, et al. Science. 2016 Jun17;352(6292):1408-1412, which is incorporated herein by reference, or those in this document.

[0098] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can include any useful modification to the sugar, nucleobase, or internucleoside linkage (e.g., to the linking phosphate / phosphodiester linkage / 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, the modification (e.g., one or more modifications) is present in each of the sugar and the internucleoside linkage. The modification can be a modification 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.

[0099] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises at least one N(6) methyl adenosine (m6A) modification to enhance translation efficiency. In some embodiments, the N(6) methyl adenosine (m6A) modification may reduce or decrease the immunogenicity of the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide).

[0100] In some embodiments, the modification may include chemical modification or cell-induced modification. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions" from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

[0101] In another embodiment, "pseudouridine" is1 acp 3 In another embodiment, the term refers to m 1 In another embodiment, the term refers to Ψ (1-methylpseudouridine). In another embodiment, the term refers to Ψm (2'-O-methylpseudouridine). In another embodiment, the term refers to m5D (5-methyldihydrouridine). In another embodiment, the term refers to m 3 Ψ (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the pseudouridines above. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

[0102] In some embodiments, chemical modifications to the ribonucleotides of the cyclic polyribonucleotides may promote immune evasion. Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified 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 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., replacement with stabilizing bases, destabilizing bases, or bases that base-pair with a wide repertoire of partners), removal of bases (abasic nucleotides), or conjugated bases. Modified ribonucleotide bases can also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications can regulate the expression, immune response, stability, and intracellular localization of cyclic polyribonucleotides, to name a few functional effects. In some embodiments, modifications include bi-orthogonal nucleotides, such as unnatural bases. See, for example, Kimoto et al, Chem Commun (Camb), 2017, 53:12309, DOI:10.1039 / c7cc06661a, which is incorporated herein by reference.

[0103] In some embodiments, the sugar modification (e.g., at the 2' or 4' position) or sugar substitution of one or more ribonucleotides of the cyclic polyribonucleotide, as well as the backbone modification, include modification or substitution of a phosphodiester bond. Specific examples of modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) include cyclic polyribonucleotides that include modified backbones or non-natural internucleoside linkages, such as internucleoside modifications, including, but not limited to, modification or substitution of a phosphodiester bond. Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone may also be considered to be oligonucleosides. In certain embodiments, the modified cyclic polyribonucleotide (eg, a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) comprises ribonucleotides having a phosphorus atom in their internucleoside backbone.

[0104] Modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thiono phosphoramidates, thiono alkyl phosphonates, thiono alkyl phosphotriesters, and their normal 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 certain embodiments, the cyclic polyribonucleotide can be negatively or positively charged.

[0105] Modified nucleotides that can be incorporated into modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be modified on the internucleoside linkage (e.g., phosphate backbone). In this specification, the terms "phosphate" and "phosphodiester" are used interchangeably with respect to polynucleotide backbones. The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with various substituents. In addition, modified nucleosides and nucleotides can include extensive replacement of unmodified phosphate moieties with alternative internucleoside linkages as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linked oxygens 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).

[0106] Non-natural phosphorothioate backbone linkages provide a-thio-substituted phosphate moieties to provide stability to RNA and DNA polymers. Phosphorothioate DNA and RNA have increased nuclease resistance and therefore longer half-life in cellular environments. Phosphorothioate linked to circular polyribonucleotides is predicted to reduce innate immune response by weaker binding / activation of cellular innate immune molecules.

[0107] In certain embodiments, modified nucleosides include α-thio-nucleosides (e.g., 5'-0-(1-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine (a-thio-cytidine), 5'-0-(1-thiophosphate)-guanosine, 5'-0-(1-thiophosphate)-uridine, or 5'-0-(1-thiophosphate)-pseudouridine).

[0108] 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.

[0109] In some embodiments, a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) can include one or more cytotoxic nucleosides. For example, a cytotoxic nucleoside can be incorporated into a cyclic polyribonucleotide, such as a bifunctional modification. 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(lH,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).

[0110] A modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified 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 (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) or in a given predetermined sequence region thereof. In some embodiments, a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) includes a pseudouridine. In some embodiments, a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) includes an inosine, which may facilitate the immune system to characterize the cyclic polyribonucleotide as endogenous to viral RNA. Incorporation of inosine may also mediate improved RNA stability / reduced degradation. See, e.g., Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as "self". Cell Res. 25, 1283-1284, incorporated by reference in its entirety.

[0111] In some embodiments, all nucleotides in the hybrid modified cyclic polyribonucleotide in a given sequence region (e.g., not the first portion or the unmodified portion) are modified. In some embodiments, the modifications include m6A, which can increase expression and / or attenuate immune response; inosine, which can attenuate immune response; pseudouridine (stagger element), which can increase RNA stability or translational read-through, m5C, which can increase stability and / or attenuate immune response; and 2,2,7-trimethylguanosine, which promotes intracellular translocation (e.g., nuclear localization).

[0112] Various sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in the cyclic polyribonucleotide. Those skilled in the art will understand that nucleotide analogs or other modifications can be located at any position of the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) so that the function of the modified cyclic polyribonucleotide is not substantially reduced. Modifications can also be modifications of non-coding regions. Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can have from about 1% to about 100% modified nucleotides (either with respect to the total nucleotide content or with respect 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%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10 ... %, 10%-70%, 10%-80%, 10%-90%, 10%-95%, 10%-100%, 20%-25%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%).

[0113] In some embodiments, the cyclic polyribonucleotide is a fully modified cyclic polyribonucleotide or a fully modified cyclic polyribonucleotide, and includes all or substantially all modified adenosine residues, all or substantially all modified uridine residues, all or substantially all modified guanine residues, all or substantially all modified cytidine residues, or any combination thereof. In some embodiments, the cyclic RNA may include at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% modified nucleotides. In some embodiments, the fully modified cyclic RNA includes substantially all (e.g., 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or more than about 100%) modified nucleotides. In some embodiments, the modified cyclic polyribonucleotide provided herein is a hybrid modified cyclic polyribonucleotide. The hybrid modified cyclic polyribonucleotide may have at least one modified nucleotide and may have a portion (e.g., a first portion / unmodified portion) of consecutive unmodified nucleotides. The unmodified portion of the hybrid modified cyclic polyribonucleotide can have at least about 5, 10, 15, or 20 contiguous unmodified nucleotides, or any number therebetween. In some embodiments, the unmodified portion of the hybrid modified cyclic polyribonucleotide has at least about 30, 40, 40, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 180, 200, 220, 250, 280, 300, 320, 350, 380, 400, 420, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 1000 contiguous unmodified nucleotides, or any number therebetween. In some embodiments, a hybrid modified cyclic polyribonucleotide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified portions. In some embodiments, a hybrid modified cyclic polyribonucleotide has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 70, 80, 100, 120, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or more modified nucleotides.In some embodiments, the hybrid modified cyclic polyribonucleotide has at least 1%, 2%, 5%, 7%, 8%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 99%, but less than 100%, modified nucleotides. In some embodiments, the unmodified portion comprises a binding site. In some embodiments, the unmodified portion comprises a binding site configured to bind to a peptide, protein, biomolecule, DNA, RNA, or cellular target. In some embodiments, the unmodified portion comprises an IRES.

[0114] In some cases, the hybrid modified cyclic polyribonucleotides described herein have similar immunogenicity compared to the corresponding cyclic polyribonucleotides that are otherwise completely modified.For example, a hybrid modified cyclic polyribonucleotide with 5' methylcytidine and pseudouridine throughout, except for its IRES element, may have similar or less immunogenicity compared to the corresponding cyclic polyribonucleotide with 5' methylcytidine and pseudouridine throughout, and without unmodified cytidine and uridine.In some embodiments, a hybrid modified cyclic polyribonucleotide with 5' methylcytidine and pseudouridine throughout, except for its IRES element, has a translation efficiency similar to or higher than the translation efficiency of the corresponding cyclic polyribonucleotide with 5' methylcytidine and pseudouridine throughout, and without unmodified cytidine and uridine.

[0115] In some embodiments, the hybrid modified cyclic polyribonucleotide has a binding site that is unmodified, e.g., has no modified nucleotides. In some embodiments, the hybrid modified cyclic polyribonucleotide has a binding site that is unmodified, e.g., has no modified nucleotides, and is configured to bind to a protein, DNA, RNA, or cellular target. In some embodiments, the hybrid modified cyclic polyribonucleotide has an internal ribosome entry site (IRES) that is unmodified, e.g., has no modified nucleotides. In some embodiments, the hybrid modified cyclic polyribonucleotide has 5% or less nucleotides in the internal ribosome entry site (IRES) that are modified nucleotides. In some embodiments, the nucleotides in the IRES are unmodified. In some embodiments, 0%, 1%, 2%, 3%, 4%, or 5% or less nucleotides in the IRES are modified. In some embodiments, the hybrid modified cyclic polyribonucleotide has modified nucleotides throughout, except for the binding site. In some embodiments, the hybrid modified cyclic polyribonucleotide has modified nucleotides throughout, except for the binding site that is configured to bind to a peptide, protein, biomolecule, DNA, RNA, or cellular target. In some embodiments, the hybrid modified circular polyribonucleotide has modified nucleotides throughout, except for the IRES element. In other embodiments, the hybrid modified circular polyribonucleotide has modified nucleotides throughout, except for the IRES element and one or more other portions. Without wishing to be bound by any particular theory, the unmodified IRES element confers translational competence on the hybrid modified circular polyribonucleotide, e.g., having a translation efficiency for one or more expressed sequences that is similar to or higher than a corresponding circular polyribonucleotide that does not have modified nucleotides.

[0116] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) has a half-life at least that of its linear equivalent, e.g., a linear expression sequence, or a linear cyclic polyribonucleotide. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) has an increased half-life over that of its linear equivalent. 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 modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) has a half-life or persistence in 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 modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) has a half-life or persistence in a 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 certain embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) has a half-life or persistence in a cell while the cell is dividing.In certain embodiments, the modified cyclic polyribonucleotide (eg, a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) has a half-life or persistence within the cell after cleavage. In certain embodiments, the modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) have 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.

[0117] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more expression sequences and is configured for sustained expression in a cell of a subject in vivo. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) is configured such that the expression of the one or more expression sequences in the cell at a later time point is equal to or greater than that at a previous time point. In such embodiments, the expression of the one or more expression sequences can be maintained at a relatively stable level or can increase over time. The expression of the expression sequences can be relatively stable over an extended period of time. For example, in some cases, the expression of the one or more expression sequences in the cell over 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 cells 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.

[0118] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) is non-immunogenic in a mammal, such as a human. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) is capable of or replicates in cells from aquaculture animals (fish, crabs, shrimp, oysters, etc.), mammalian cells, such as cells from pet or zoo animals (cats, dogs, lizards, birds, lions, tigers, and bears), cells from livestock or working animals (horses, cows, pigs, chickens, etc.), human cells, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, embryonic cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, the invention includes cells comprising modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) as described herein, where the cells are cells from aquaculture animals (fish, crabs, shrimp, oysters, etc.), mammalian cells, such as cells from pet or zoo animals (cats, dogs, lizards, birds, lions, tigers, bears, etc.), cells from livestock or working animals (horses, cows, pigs, chickens, etc.), human cells, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, the cells are engineered to comprise modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides).

[0119] In certain embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) modulates a cellular function, for example, transiently or long term. In certain embodiments, the cellular function, such as 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, a cellular function, such as regulation, is temporarily altered, for example, lasting for about 30 minutes or less to about 7 days, or for 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.

[0120] In certain embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified 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 modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) can be of a size sufficient to accommodate a binding site for a ribosome. One of skill in the art can understand that the maximum size of a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) can be any size within the technical constraints of generating and / or using the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide).Without being bound by theory, it is possible that multiple segments of RNA can be generated from DNA and their 5' and 3' free ends annealed to generate a "string" of RNA, which can eventually be circularized when only one 5' and one 3' free end remain. In certain embodiments, the maximum size of a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) can be limited by the ability to package and deliver the RNA to a target. In certain embodiments, the size of the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) is of sufficient length to encode a useful polypeptide, and thus lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides can be useful.

[0121] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more elements described elsewhere herein. In some embodiments, the elements can be separated from one another by a spacer sequence or linker. In some embodiments, the elements can 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, for example, lacking a spacer element. In some embodiments, one or more elements in the modified 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 modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a secondary or tertiary structure that corresponds to one or more desired functions or properties described herein, such as a binding site for ribosomes, such as translation, e.g., rolling circle translation.

[0122] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a specific sequence characteristic. For example, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may comprise a specific nucleotide composition. In some such embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may comprise one or more purine-rich regions (adenine or guanosine). In some such embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may comprise one or more purine-rich regions (adenine or guanosine). In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may comprise one or more AU-rich regions or elements (AREs). In certain embodiments, a modified cyclic polyribonucleotide (eg, a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) may contain one or more adenine-rich regions.

[0123] In certain embodiments, a modified cyclic polyribonucleotide (eg, a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) may contain one or more repeat elements as described elsewhere herein.

[0124] In certain embodiments, the modified cyclic polyribonucleotide (eg, a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) contains one or more modifications described elsewhere herein.

[0125] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) may contain one or more substitutions, insertions and / or additions, deletions, and covalent modifications relative to a reference sequence, particularly a parent polyribonucleotide, and are included within the scope of the present invention.

[0126] Expression sequence Peptides or Polypeptides In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified 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 grams per mole, less than about 2,000 grams per mole, less than about 1,000 grams per mole, less than about 500 grams per mole, as well as salts, esters and other pharma- ceutically 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.

[0127] The polypeptide may be linear or branched. The polypeptide 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, 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.

[0128] 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 natural bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degradative or self-destructive peptides, and degradative or self-destructive 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 (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7): 1076-113). Such antigen-binding peptides may bind to cytoplasmic antigens, nuclear antigens, and intraorganelle antigens.

[0129] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more RNA expression sequences, each of which may code for a polypeptide. The polypeptide may be produced in significant quantities. Thus, the polypeptide may be any protein molecule that may be produced. The polypeptide may be a polypeptide that may be secreted from a cell or localized to the cytoplasm, nuclear or membrane compartment of a cell. Some polypeptides include, but are not limited to, at least a portion of a viral envelope protein, metabolic regulatory enzymes (e.g., regulating lipid or steroid production), antigens, toleragens, cytokines, toxins, enzymes whose absence is associated with disease, and polypeptides that are not active in an animal (e.g., in the intestine of an animal) until cleaved, and hormones.

[0130] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises an expression sequence that encodes a protein, such as a therapeutic protein. In some embodiments, the therapeutic protein that can be expressed from the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) disclosed herein has antioxidant activity, binding, cargo receptor activity, catalytic activity, molecular carrier activity, molecular function regulator, molecular transducer activity, nutrient retention activity, protein tag, 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, proteins for replacement, protein vaccination, 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 the CRISPR system or components thereof.

[0131] In some embodiments, exemplary proteins that may be expressed from the modified cyclic polyribonucleotides disclosed herein (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) 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, such as therapeutic proteins for wound healing). In some embodiments, exemplary proteins that may be expressed from the modified cyclic polyribonucleotides disclosed herein (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) include EGF (epidermal growth factor). In some embodiments, exemplary proteins that may be expressed from the modified cyclic polyribonucleotides disclosed herein (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) include enzymes, such as oxidoreductase enzymes, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, and desaturases. In some embodiments, exemplary proteins that can be expressed from the modified cyclic polyribonucleotides disclosed herein (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) include intracellular or cytoplasmic proteins. In some embodiments, the modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) express NanoLuc® luciferase (nLuc). In some embodiments, exemplary proteins that can be expressed from the modified cyclic polyribonucleotides disclosed herein (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) include secreted proteins, e.g., secreted enzymes.In some cases, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) expresses a secreted protein that may have a therapeutically effective short half-life in blood, or may be a protein with a subcellular localization signal, or a protein with a secretory signal peptide. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) expresses Gaussia luciferase (gLuc). In some cases, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified 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 may be expressed from the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) includes GFP.In certain embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) can be a tagged protein, e.g., a fusion protein or a modified protein containing a protein tag, e.g., chitin-binding protein (CBP), maltose-binding protein (MBP), Fc tag, glutathione-S-transferase (GST), AviTag (GLNDIFEAQKIEWHE), calmodulin-tag (KRRWKKNFIAVS AANRFKKISSSGAL;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).

[0132] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) expresses an antibody, such as an antibody fragment, or a portion thereof. In some embodiments, the antibody expressed by the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) can be of any isotype, such as IgA, IgD, IgE, IgG, IgM, etc. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified 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, further portions thereof. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) expresses one or more portions of an antibody. For example, a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) can contain two or more expressed sequences, each of which expresses a portion of an antibody, the collection of which can constitute an antibody. In some cases, a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) contains one expressed sequence encoding the heavy chain of an antibody and another expressed sequence encoding the light chain of an antibody. In some cases, when a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) is expressed in a cell or in a cell-free environment, the light and heavy chains can be subjected to proper modification, folding, or other post-translational modification to form a functional antibody.

[0133] Adjustment element In certain embodiments, the modified cyclic polyribonucleotide (eg, a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) comprises a regulatory element, such as a sequence that regulates expression of an expression sequence within the modified cyclic polyribonucleotide.

[0134] Regulatory elements may include sequences located adjacent to the expression sequence that codes for the expression product. Regulatory elements may be operably linked to adjacent sequences. Regulatory elements may increase the amount of expressed product compared to the amount of expressed product in the absence of the regulatory element. Furthermore, one regulatory element may increase the amount of expressed product for multiple expression sequences that are linked side by side. 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.

[0135] 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 modified 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 modified circular polyribonucleotide or an expression product of a modified circular polyribonucleotide. Exemplary alternative degradation sequences may include riboswitch aptazymes and miRNA binding sites.

[0136] In some embodiments, the regulatory element is a translation modulator. The translation modulator may regulate the translation of the expressed sequence in the modified circular polyribonucleotide. The translation modulator may be a translation enhancer or suppressor. In some embodiments, the modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) comprises at least one translation modulator adjacent to at least one expressed sequence. In some embodiments, the modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) comprises a translation modulator adjacent to each expressed sequence. In some embodiments, the translation modulator is present on one or both sides of each expressed sequence, resulting in the separation of the expression products, such as peptides and / or polypeptides.

[0137] In some embodiments, the translation initiation sequence may function as a regulatory element. In some embodiments, the translation initiation sequence includes an AUG codon. In some embodiments, the translation initiation sequence includes any eukaryotic initiation codon, such as AUG, CUG, GUG, UUG, ACG, AUC, AUU, AAG, AUA, or AGG. In some embodiments, the translation initiation sequence includes a Kozak sequence. In some embodiments, translation is initiated from an alternative translation initiation sequence, such as a translation initiation sequence other than the AUG codon, under selective conditions, such as stress-induced conditions. As a non-limiting example, the translation of a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) is initiated from an alternative translation initiation sequence, such as ACG. As another non-limiting example, the translation of a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) may be initiated from an alternative translation initiation sequence, CTG / CUG. As yet another non-limiting example, modified circular polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) translation can initiate from alternative translation initiation sequences, GTG / GUG. As yet another non-limiting example, modified circular polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified 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.

[0138] Nucleotides adjacent to the codon that initiates translation, such as, but not limited to, the initiation codon or another initiation codon, are known to affect the translation efficiency, length and / or structure of the modified 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 position of translation initiation, translation efficiency, length and / or structure of the modified circular polyribonucleotide.

[0139] In one embodiment, a masking agent can be used near the start codon or an alternative start codon to mask or hide the codon to reduce the possibility 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 modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) to increase the possibility that translation will start from an alternative start codon.

[0140] 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 a modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) contains a GRSF-1 binding site, see, e.g., http: / / jvi.asm.org / content / 76 / 20 / 10417.full.

[0141] Translation initiation sequence In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may code for 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 modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified 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 modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified 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 modified cyclic polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single-stranded region of the modified cyclic polyribonucleotide.

[0142] A modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) may include two or more start codons, such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons. Translation may begin on the first start codon or may begin downstream of the first start codon.

[0143] In some embodiments, modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) can start from a codon that is not the first start codon, such as AUG. Translation of modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) can start from another translation start sequence, such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG (see Touriol et al. Biology of the Cell 95(2003)169-178 and Matsuda and Mauro PLoS ONE,2010 5:11, the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, translation starts from another translation start sequence under selective conditions, such as stress-inducing conditions. As a non-limiting example, the translation of the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may be initiated from an alternative translation initiation sequence, such as ACG. As another non-limiting example, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) translation may be initiated from an alternative translation initiation sequence, CTG / CUG. As yet another non-limiting example, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) translation may be initiated from an alternative translation initiation sequence, GTG / GUG. As yet another non-limiting example, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may be translated from a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA, e.g., CGG, GGGGCC, CAG, CTG.

[0144] In one embodiment, translation is initiated by eukaryotic initiation factor 4A (eIF4A) processing by Rocaglates (translation is inhibited by blocking 43S scanning, leading to early upstream translation initiation and reduced protein expression from transcripts with RocA-eIF4A target sequences, see e.g. www.nature.com / articles / nature17978).

[0145] IRES In some embodiments, the modified circular polyribonucleotides described herein (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) comprise an internal ribosome entry site (IRES) element. IRES elements suitable for inclusion in modified circular polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) comprise an RNA sequence capable of engaging with a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt. In one embodiment, the IRES element is derived from the DNA of an organism, including, but not limited to, a virus, a mammal, and Drosophila. Such viral DNA can be derived from picornavirus complementary DNA (cDNA), as well as, but not limited to, encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA from which the IRES element is derived includes, but is not limited to, the antennapedia gene from Drosophila melanogaster.

[0146] 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, It 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 certain embodiments, the IRES element comprises a synthetic IRES, such as (GAAA)16, (PPT19)4, KMI1, KMI1, KMI2, KMI2, KMIX, X1, or X2.

[0147] In some embodiments, the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified 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 at least one (e.g., 2, 3, 4, 5, or more) expressed sequence on both sides. In some embodiments, the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises one or more IRES sequences present on one or both sides of each expressed sequence, resulting in the separation of the resulting peptides and / or polypeptides.

[0148] End element In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more expression sequences, each of which may or may not have a termination element. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more expression sequences, each of which may or may not have a termination element. The expression sequences lack a termination element such that the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) is translated in a continuous manner. The elimination of the termination element may result in rolling circle translation or continuous expression of the expression product, such as a peptide or polypeptide, due to the absence of ribosome stalling or dropping. In such embodiments, the rolling circle translation expresses a continuous expression product through each expression sequence. In some other embodiments, the termination element of the expression sequence may be part of a stagger element. In some embodiments, one or more expression sequences in the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) contain a termination element. However, rolling circle translation or expression of the subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) occurs. In such cases, when the ribosome encounters a termination element, e.g., a stop codon, and terminates translation, the expression product may fall off the ribosome. In some embodiments, translation is terminated while the ribosome, e.g., at least one subunit of the ribosome, remains in contact with the modified cyclic polyribonucleotide.

[0149] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a termination element at the end of one or more expressed sequences. In some embodiments, the one or more expressed sequences comprise two or more termination elements in succession. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome completely disengages from the modified cyclic polyribonucleotide. In some such embodiments, the generation of a subsequent (e.g., second, third, fourth, fifth, etc.) expressed sequence in the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) may require the ribosome to re-engage with the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) before translation begins. In general, the termination element comprises an in-frame nucleotide triplet, e.g., UAA, UGA, UAG, which indicates the termination of translation. In some embodiments, one or more termination elements in the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular 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 include 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 cells.

[0150] Stagger Element In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises at least one stagger element adjacent to the expressed sequence. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a stagger element adjacent to each expressed sequence. In some embodiments, the stagger element is present on one or both sides of each expressed sequence, resulting in separation of the expression products, such as peptides and / or polypeptides. In some embodiments, the stagger element is part of one or more expressed sequences. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more expressed sequences, each of which is separated from the subsequent expressed sequence by a stagger element in the modified cyclic polyribonucleotide. In some embodiments, the stagger element prevents (a) the generation of a single polypeptide from two translations of a single expressed sequence, or (b) the generation of a single polypeptide from one or more translations of two or more expressed sequences. In certain embodiments, the stagger element is a sequence that is separate from one or more expressed sequences, hi certain embodiments, the stagger element comprises a portion of an expressed sequence of one or more expressed sequences.

[0151] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a stagger element. To avoid the generation of consecutive expression products, such as peptides or polypeptides, while maintaining rolling circle translation, the stagger element may be included to induce pausing of ribosomes during translation. In some embodiments, the stagger element is at the 3' end of at least one of the one or more expressed sequences. The stagger element may be configured to stall ribosomes during rolling circle translation of the modified cyclic polyribonucleotide. Stagger elements include, but are not limited to, 2A-like or CHYSEL (cis-acting hydrolase element) sequences. In some embodiments, the stagger element comprises, but is not limited to, X 1 X2 X 3 EX 5 NPGP (where X 1 is absent or is G or H, and X 2 is absent or is D or G, and X 3 is D, or V, or I, or S, or M, and X 5 In one embodiment, the sequence encodes a sequence with a C-terminal consensus sequence of -D(V / I)ExNPGP, where x=any amino acid. In one embodiment, the sequence contains a non-conserved sequence of amino acids with strong alpha helical propensity, followed by the consensus sequence -D(V / I)ExNPGP, where x=any amino acid. Some non-limiting examples of stagger elements include GDVESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, GDVEQNPGP, IESNPGP, GDIELNPGP, HDIETPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP.

[0152] In some embodiments, the stagger elements described herein cleave the expression product, such as between the G and P of the consensus sequences described herein. As one non-limiting example, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises at least one stagger element to cleave the expression product. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a stagger element adjacent to at least one expressed sequence. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a stagger element after each expressed sequence. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a stagger element, which 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.

[0153] In some embodiments, the stagger element comprises one or more modified or non-natural nucleotides that induce ribosome pausing during translation. Non-natural nucleotides may 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 may include any modification to the sugar, nucleobase, internucleoside bond (e.g., to the linking phosphate / phosphodiester bond / phosphodiester backbone) and any combination thereof that may induce ribosome pausing during translation. Some of the exemplary modifications provided herein are described elsewhere herein.

[0154] In some embodiments, the stagger element is present in the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) in other forms. For example, in some exemplary modified cyclic polyribonucleotides, the stagger element comprises a nucleotide spacer sequence that separates the termination element from the termination element of the first expression sequence in the modified cyclic polyribonucleotide and the first translation initiation sequence of the expression following the first expression sequence. In some examples, the first stagger element of the first expression sequence is upstream (5'-end side) of the first translation initiation sequence of the expression following the first expression sequence in the modified 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 modified 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 and 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 modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) that includes a first stagger element upstream of a first translation initiation sequence of a subsequent sequence in the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) is translated continuously, while a corresponding modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) that includes a stagger element of a second expression sequence upstream of a second translation initiation sequence of an expression sequence following the second expression sequence is not translated continuously. In some cases, there is only one expression sequence in the modified cyclic polyribonucleotide, and the first expression sequence and the subsequent expression sequence are the same expression sequence. In some exemplary modified cyclic polyribonucleotides, the stagger element includes a first termination element of the first expression sequence in the modified cyclic polyribonucleotide and a nucleotide spacer sequence that separates the termination element from the downstream translation initiation sequence.In some such instances, the first stagger element is upstream (5') of the first translation initiation sequence of the first expressed sequence in the modified circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence allows for continuous translation of the first expressed sequence and any subsequent expressed sequences. In some embodiments, the first stagger element separates the expression product of one round of the first expressed sequence from the expression product of the next round of the first expressed sequence, thereby forming separate expression products. In some cases, a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) that includes a first stagger element upstream of a first translation start sequence of a first expressed sequence in a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) is translated continuously, while a corresponding modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) that includes a stagger element upstream of a second translation start sequence of a second expressed sequence in a corresponding modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic 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, 3, 4, 5, 6, 7, 8, 9, or 10 times greater in the corresponding modified circular polyribonucleotide than the distance between the first stagger element and the first translation initiation in the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide). In some cases, the distance between the first stagger element and the first translation initiation is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt or 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 modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) comprises two or more expressed sequences.

[0155] regulatory nucleic acids In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) encodes a regulatory nucleic acid, for example, includes one or more expression sequences that regulate the expression of an endogenous gene and / or an exogenous gene. In some embodiments, the expression sequences of the modified cyclic polyribonucleotides provided herein (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) can include sequences that are antisense to a regulatory nucleic acid, such as, but not limited to, a non-coding RNA, such as tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA, and hnRNA.

[0156] In one embodiment, the regulatory nucleic acid targets a host gene. The regulatory nucleic acid can include any of the regulatory nucleic acids described in International Patent Publication No. WO2019118919A1, the entire contents of which are incorporated herein by reference, including

[0177] and

[0181] to

[0189] .

[0157] In some embodiments, the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a regulatory nucleic acid such as a guide RNA (gRNA). In some embodiments, the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises or encodes a guide RNA. The gRNA short synthetic RNA is composed of a "scaffold" sequence required to bind to an incomplete effector moiety and a user-defined, approximately 20 nucleotide targeting sequence for a genomic target. In practice, guide RNA sequences are generally designed to have a length of 17-24 nucleotides (e.g., 19, 20 or 21 nucleotides) and to be complementary to a targeting 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"), which are engineered (synthetic) single RNA molecules that mimic the natural crRNA-tracrRNA complex and contain both tracrRNA (to bind to the 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.

[0158] The gRNA may recognize a specific DNA sequence (e.g., a sequence adjacent to or within a gene's promoter, enhancer, silencer, or repressor).

[0159] In one embodiment, gRNA is used as part of the CRISPR system for gene editing.For gene editing, modified circular polyribonucleotide (for example, fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can be designed to include one or more guide RNA sequences corresponding to desired target DNA sequence, see for example 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 for DNA cleavage to occur; for Cpf1, at least about 16 nucleotides of gRNA sequence are required to achieve detectable DNA cleavage.

[0160] Modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can regulate the expression of RNA encoded by genes. Because multiple genes may share some degree of sequence homology with each other, in some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be designed to target a class of genes with sufficient sequence homology. In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can contain sequences that are shared between different gene targets or have complementarity to sequences unique to a particular gene target. In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be designed to target conserved regions of RNA sequences that share homology between several genes, thereby targeting several genes in a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In certain embodiments, modified cyclic polyribonucleotides (eg, fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be designed to target sequences unique to a particular RNA sequence of a single gene.

[0161] 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 in addition, be more than 10 bp (e.g., at least about 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, 5kb or more in length.

[0162] In certain 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.

[0163] Translation Efficiency In some embodiments, the translation efficiency of the modified cyclic polyribonucleotides provided herein (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) is higher than a reference, e.g., a linear equivalent, a linear expression sequence, a linear modified cyclic polyribonucleotide, or a fully modified cyclic polyribonucleotide equivalent. In certain embodiments, the modified cyclic polyribonucleotides provided herein (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) 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 a reference translation efficiency. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) has a translation efficiency 10% higher than that of its linear equivalent. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) has a translation efficiency 300% higher than that of its linear equivalent. In some embodiments, the hybrid modified cyclic polyribonucleotide has a translation efficiency 10% higher than that of its fully modified cyclic polyribonucleotide equivalent. In some embodiments, the hybrid modified cyclic polyribonucleotide has a translation efficiency 300% higher than that of its fully modified cyclic polyribonucleotide equivalent.In some embodiments, the hybrid modified cyclic polyribonucleotides 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 that of the corresponding cyclic polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotides have a translation efficiency that is at least about 10% higher than that of the corresponding cyclic polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotides have a translation efficiency at least about 20% higher than that of the corresponding cyclic polyribonucleotide. In some embodiments, the hybrid modified cyclic polyribonucleotides have a translation efficiency at least about 50% higher than that of the corresponding cyclic polyribonucleotide.

[0164] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) produces a stoichiometric ratio of expression products. Rolling circle translation produces expression products in succession in substantially equal ratios. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) has a stoichiometric translation efficiency such that expression products are produced in substantially equal ratios. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) has a stoichiometric translation efficiency of multiple expression products, e.g., products from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expressed sequences.

[0165] Rolling circle translation In some embodiments, once translation of a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) is initiated, a ribosome bound to the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) does not detach from the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) before completing at least one translation of the modified cyclic polyribonucleotide. In some embodiments, the modified cyclic polyribonucleotides described herein (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) are capable of rolling circle translation. In some embodiments, during rolling circle translation, once translation of a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) has been initiated, a ribosome bound to the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) is cleaved at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least one time, at least two times, at least one time ... 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 10000 times, at least 10 5 times, or at least 10 6 The modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) does not leave the modified circular polyribonucleotide before completing a translation round.

[0166] In certain embodiments, the rolling circle translation of a modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) results in the production of a polypeptide product (a "contiguous" expression product) that is translated from two or more translations of the modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide). In certain embodiments, the modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) comprises a stagger element, and the rolling circle translation of the modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) results in the production of a polypeptide product (a "distinct" expression product) that is generated from a single translation or less than one translation of the modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide). In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified 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 (moles / moles) produced during rolling circle translation of the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) are distinct polypeptides. In some embodiments, the amount ratio of distinct products across the total polypeptides is tested in an in vitro translation system. In some embodiments, the in vitro translation system used to test the amount ratio comprises rabbit reticulocyte lysate. In some embodiments, the amount ratio is tested in an in vivo translation system, such as eukaryotic or prokaryotic cells, cultured cells, or cells within an organism.

[0167] Untranslated Regions In some embodiments, the modified circular polyribonucleotide (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) comprises an untranslated region (UTR). The UTR of a genomic region that comprises 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.

[0168] In some embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) comprises a UTR having one or more stretches of adenosines and uridines embedded therein. These AU-rich signatures can increase the turnover rate of the expression product.

[0169] The introduction, removal, or modification of UTR AU-rich elements (AREs) can be useful for adjusting the stability or immunogenicity of modified cyclic polyribonucleotides. When modifying a particular modified cyclic polyribonucleotide, one or more copies of AREs can be introduced into the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide), and the copies of AREs can regulate the translation and / or production of expression products. Similarly, AREs can be identified and removed or modified in modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) to adjust the intracellular stability, thus affecting the translation and production of the resulting protein.

[0170] It should be understood that any UTR from any gene can be incorporated into each adjacent region of the modified circular polyribonucleotide.Exemplary UTRs that can be used in the modified circular polyribonucleotides (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) provided herein include those described in

[0200] to

[0201] of International Patent Publication No. WO2019118919A1, the entirety of which is incorporated herein by reference.

[0171] PolyA sequence In some embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) may include a poly-A sequence. In some embodiments, the length of the poly-A sequence is greater than 10 nucleotides in length. In one embodiment, the poly-A sequence is greater than 15 nucleotides in length (e.g., at least about or greater than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides in length). In one embodiment, the poly-A sequence is designed according to the description of poly-A sequences in paragraphs

[0202] to

[0204] of International Patent Publication No. WO2019118919A1, the entirety of which is incorporated herein by reference.

[0172] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises poly A, lacks poly A, or has a modified poly A to adjust one or more properties of the modified cyclic polyribonucleotide. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) lacking poly A or having a modified poly A improves one or more functional properties, such as immunogenicity, half-life, expression efficiency, etc.

[0173] RNA binding In some embodiments, the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular 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 modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular 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, U.S. Patent Application Publication No. 2005 / 0059005, and International Patent Publication No. WO2019118919A1, the entire contents of which are incorporated herein by reference, at

[0207] to

[0215] .

[0174] Protein binding In certain embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more protein binding sites that allow a protein, such as a ribosome, to bind to an internal site in the RNA sequence. By engineering a protein binding site, such as a ribosome binding site, into the modified cyclic polyribonucleotide, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) can evade or have reduced detection by the host's immune system by masking the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) from components of the host's immune system, and can have regulated degradation or regulated translation.

[0175] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified 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 some embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and serves to mask the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) as being exogenous. In some embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and serves to mask the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) as being exogenous or foreign.

[0176] The conventional mechanism of ribosome engagement into linear RNA involves ribosomes binding to the capped 5' end of the RNA, from which 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 modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) does not require a free or capped end. Rather, ribosomes bind to uncapped internal sites, whereby the ribosomes initiate polypeptide elongation at the start codon. In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) include one or more RNA sequences that include a ribosome binding site, e.g., a start codon.

[0177] 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 the 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.

[0178] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) encodes a protein binding sequence that binds to a protein. In some embodiments, the protein binding sequence targets or localizes the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) to a specific target. In some embodiments, the protein binding sequence specifically binds to an arginine-rich region of a protein.

[0179] In certain embodiments, the protein binding sites include, but are not limited to, ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, EL AVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC , HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, LIN28A, 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 RNA.

[0180] Encryptogen As described herein, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) contain an encryptogen to reduce, avoid or avoid the natural immune response of cells. In one aspect, provided herein are modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) that, when delivered to cells, result in a reduced immune response from the host compared to the response caused by a reference compound, such as the modified cyclic polyribonucleotide described (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides), the corresponding unmodified cyclic polyribonucleotides, the linear polynucleotides corresponding to the modified cyclic polyribonucleotides lacking the encryptogen. In some embodiments, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) have lower immunogenicity than their counterparts lacking the encryptogen.

[0181] In some embodiments, the encryptogen improves stability. There is increasing evidence of the regulatory role that UTR plays in the stability of nucleic acid molecules and translation. The regulatory features of UTR can be included in the encryptogen to improve the stability of modified circular polyribonucleotide.

[0182] In certain embodiments, the 5' or 3' UTR may constitute an encryptogen in a modified circular polyribonucleotide. For example, removal or modification of a UTR AU-rich element (ARE) may be useful to modulate the stability or immunogenicity of a modified circular polyribonucleotide.

[0183] In certain embodiments, removal of modifications of expressed sequences, such as AU-rich element (ARE) modifications in translatable regions, may be useful to modulate the stability or immunogenicity of modified circular polyribonucleotides.

[0184] 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 a modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) described herein can not only regulate expression in hematopoietic cells, but can also reduce or eliminate immune responses against proteins encoded in the modified cyclic polyribonucleotide.

[0185] In certain embodiments, the encryptogen comprises 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 modified cyclic polyribonucleotide, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) can evade or have reduced detection by the host's immune system by masking the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) from components of the host's immune system, and can have regulated degradation, or regulated translation. In certain embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises 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 serves to bind to an immune protein and mask the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) as being exogenous.

[0186] In some embodiments, the encryptogen comprises one or more modified nucleotides. Exemplary modifications may include any modification to the sugar, nucleobase, internucleoside linkage (e.g., to the linkage phosphate / phosphodiester linkage / phosphodiester backbone), and any combination thereof that may prevent or reduce immune response to modified cyclic polyribonucleotides. Some of the exemplary modifications provided herein are described in detail below.

[0187] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more modifications described elsewhere herein to reduce the immune response from the host compared to the response elicited by a reference compound, e.g., a modified cyclic polyribonucleotide lacking the modification (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide). In particular, the addition of one or more inosines has been shown to distinguish RNA as endogenous to the virus. See, e.g., Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as "self". Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.

[0188] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more expression sequences for shRNA or RNA sequences 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, resulting in the degradation of the exogenous cyclic RNA. Thus, a cyclic polynucleotide having a sequence for RIG-1 targeting shRNA, siRNA or any other regulatory nucleic acid can reduce immunity, such as host cell immunity, against the modified cyclic polyribonucleotide.

[0189] In some embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) lacks a sequence, element, or structure that assists the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) in reducing, avoiding, or evading a cellular innate immune response. In some such embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) can lack a polyA sequence, a 5' end, a 3' end, a phosphate group, a hydroxyl group, or any combination thereof.

[0190] Riboswitches In some embodiments, the modified cyclic polyribonucleotide (eg, a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) comprises one or more riboswitches.

[0191] Riboswitches are typically considered to be parts of modified circular polyribonucleotides (e.g., fully modified or hybrid modified circular polyribonucleotides) that can directly bind to small target molecules, and the binding of the target affects RNA translation, expression product stability and activity (Tucker BJ, Breaker RR (2005), Curr Opin Struct Biol 15(3):342-8). Thus, modified circular polyribonucleotides (e.g., fully modified or hybrid modified circular polyribonucleotides) that contain riboswitches are directly involved in regulating their own activity depending on the presence or absence of their target molecules. In some embodiments, riboswitches have regions of affinity like aptamers for distinct molecules. Thus, in the broader context of the present invention, any aptamer contained within a non-coding nucleic acid can be used for the sequestration of molecules from bulk volume. Downstream reporting of events by "(ribo)switch" activity can be particularly advantageous.

[0192] In some embodiments, riboswitches can affect gene expression, including but not limited to terminating transcription, inhibiting translation initiation, self-cleaving mRNA, and altering splicing pathways in eukaryotes. Riboswitches can function to control gene expression by binding or removing trigger molecules. Thus, modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) containing riboswitches are subjected to conditions that activate, inactivate, or block the riboswitch to alter expression. Expression can be altered, for example, as a result of terminating or blocking transcription of ribosomes that bind to the RNA. Binding of a trigger molecule or an analog thereof can reduce or prevent expression of the RNA molecule, or promote or increase expression of the RNA molecule, depending on the nature of the riboswitch. Some examples of riboswitches are described herein.

[0193] A cyclic di-GMP riboswitch, an FMN riboswitch (also known as an RFN element), a glmS riboswitch, a glutamine riboswitch, a glycine riboswitch, a lysine riboswitch (also known as an L-box), a PreQ1 riboswitch (e.g., PreQ1-l riboswitch and PreQ1-ll riboswitch), a purine riboswitch, a SAH riboswitch, a SAM riboswitch, a SAM-SAH riboswitch, a tetrahydrofolate riboswitch, a theophylline-binding riboswitch, a thymine pyrophosphate-binding riboswitch, a T. tengcongensis glmS catalytic riboswitch, a TPP riboswitch (also known as a THI-box), a Moco riboswitch, or an adenine-sensing add-A riboswitch, each of which is described in International Patent Publication No. WO 2019118919A1, the entire contents of which are incorporated herein by reference, at

[0235] to

[0252] .

[0194] Aptazymes In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises an aptazyme. An aptazyme is a switch for conditional expression in which an aptamer region is used as an allosteric control element and is linked 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 acid or viral protein.

[0195] Ribozymes (derived from ribonucleic acid enzymes, also called RNA enzymes or catalytic RNA) are RNA molecules that catalyze chemical reactions.

[0196] Some non-limiting examples of ribozymes include hammerhead ribozymes, VL ribozymes, leadzymes, hairpin ribozymes, and other ribozymes described in International Patent Publication No. WO 2019118919A1, the entire contents of which are incorporated herein by reference, at

[0254] to

[0259] .

[0197] In some embodiments, the modified circular RNA described herein can be used for RNA transcription and replication. For example, the circular RNA can be used to code non-coding RNA, lncRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA. In some embodiments, the circular RNA can include antisense miRNA and transcriptional elements. After transcription, such circular RNA can generate functional linear miRNA. Non-limiting examples of circular RNA expression and regulation applications are listed in Table 3.

[0198] [Table 3]

[0199] target binding In some embodiments, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) binds to one or more targets. In one embodiment, the circular RNA binds to both a DNA target and a protein target, e.g., mediates transcription. In another embodiment, the circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) brings together protein complexes, e.g., mediates signal transduction. In another embodiment, the circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) binds to two or more different targets, such as proteins, e.g., transports these proteins to the cytoplasm. In one embodiment, the pharmaceutical composition comprises a hybrid modified cyclic polyribonucleotide, wherein the hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding portion of a first target, e.g., an RNA, DNA, protein, or cellular target (wherein the first binding portion is a first cyclic polyribonucleotide (cyclic RNA) binding motif composed of unmodified nucleotides); and wherein the first target and the hybrid modified cyclic polyribonucleotide form a complex. In one embodiment, the pharmaceutical composition comprises a hybrid modified cyclic polyribonucleotide, wherein the hybrid modified cyclic polyribonucleotide comprises at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding portion of a first target, e.g., an RNA, DNA, protein, or cellular target, wherein the first binding portion is a first cyclic polyribonucleotide (cyclic RNA) binding motif consisting of unmodified nucleotides; and a second binding portion configured to bind to a second binding portion of a second target, wherein the second binding portion is a second cyclic RNA binding motif, wherein the first binding portion is different from the second binding portion, and wherein the first target, the second target, and the hybrid modified cyclic polyribonucleotide form a complex, and wherein the first target or the second target is not a microRNA.In one embodiment, a pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising at least one modified nucleotide; a first portion comprising a first binding site configured to bind to a first binding portion of a first target, where the first binding portion is a first cyclic polyribonucleotide (cyclic RNA) binding motif; and a second binding portion configured to bind to a second binding portion of a second target, where the second binding portion is a second cyclic RNA binding motif, the first binding portion being different from the second binding portion, and both the first target and the second target being microRNA. In one embodiment, the hybrid modified cyclic polyribonucleotide comprises a first portion comprising a binding site configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, where the binding portion is comprised of unmodified nucleotides. In one embodiment, the first portion described herein comprises a binding site configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, where the binding portion is comprised of unmodified nucleotides.

[0200] In some embodiments, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) binds to at least one of DNA, RNA, and protein, thereby regulating a cellular process (e.g., altering protein expression). In some embodiments, the synthetic modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for interaction with at least one portion, e.g., a binding moiety, of a selected DNA, RNA, or protein, thereby competing for binding with an endogenous counterpart.

[0201] In one embodiment, the synthetic modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) binds and / or sequesters miRNA. In another embodiment, the synthetic modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) binds and / or sequesters protein. In another embodiment, the synthetic modified circular RNA binds and / or sequesters mRNA. In another embodiment, the synthetic modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) binds and / or sequesters ribosomes. In another embodiment, the synthetic modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) binds and / or sequesters modified circular RNA. In another embodiment, the synthetic modified circular RNA binds and / or sequesters long non-coding RNA (lncRNA) or any other non-coding RNA, e.g., miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, long non-coding RNA, shRNA. In addition to the binding and / or sequestration sites, the modified circular RNA may contain a degradation element, which results in degradation of the bound and / or sequestered RNA and / or protein.

[0202] In one embodiment, the modified circular RNA (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) comprises the sequence of a lncRNA or a lncRNA, e.g., the modified circular RNA comprises the sequence of a naturally occurring, non-circular lncRNA or a fragment thereof. In one embodiment, the lncRNA or the sequence of a lncRNA is circularized with or without a spacer sequence to form a synthetic modified circular RNA (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide).

[0203] In one embodiment, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) has ribozyme activity. In one embodiment, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can act as a ribozyme and be used to cleave pathogenic or endogenous RNA, DNA, small molecules or proteins. In one embodiment, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) has enzymatic activity. In one embodiment, the synthetic modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can specifically recognize and cleave RNA (e.g., viral RNA). In another embodiment, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can specifically recognize and cleave proteins. In another embodiment, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can specifically recognize and degrade small molecules.

[0204] In one embodiment, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) is an immolating or self-cleaving or cleavable modified circular RNA. In one embodiment, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can be used to deliver RNA, such as miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, long non-coding RNA, shRNA. In one embodiment, the synthetic modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) is composed of microRNA separated by (1) a self-cleavable element (e.g., hammerhead, splicing element), (2) a cleavage recruitment site (e.g., ADAR), (3) a degradable linker (glycerol), (4) a chemical linker, and / or (5) a spacer sequence. In another embodiment, the synthetic modified circular RNA is composed of siRNAs separated by (1) a self-cleavable element (e.g., hammerhead, splicing element), (2) a cleavage recruitment site (e.g., ADAR), (3) a degradable linker (glycerol), (4) a chemical linker, and / or (5) a spacer sequence.

[0205] In one embodiment, the modified circular RNA is a transcription / replicative modified circular RNA. The modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can encode any type of RNA. In one embodiment, the synthetic modified circular RNA has an antisense miRNA and a transcription element. In one embodiment, after transcription, a linear functional miRNA is generated from the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide).

[0206] In one embodiment, the modified circular RNA (eg, a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) in combination with a translation element has one or more of the above attributes.

[0207] target The modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises at least one binding site for a binding moiety of a target. Targets include, but are not limited to, nucleic acids (e.g., RNA, DNA, RNA-DNA hybrids), small molecules (e.g., drugs), aptamers, polypeptides, proteins, lipids, carbohydrates, antibodies, viruses, virus particles, membranes, multicomponent complexes, cells, other cellular parts, fragments of any of them, and any combination thereof. (See, e.g., Fredriksson et al., (2002) Nat Biotech 20:473-77; Gullberg et al., (2004) PNAS, 101:8420-24). For example, the target can be single-stranded RNA, double-stranded RNA, single-stranded DNA, double-stranded DNA, DNA or RNA containing one or more double-stranded regions and one or more single-stranded regions, an RNA-DNA hybrid, a small molecule, an aptamer, a polypeptide, a protein, a lipid, a carbohydrate, an antibody, an antibody fragment, a mixture of antibodies, a virus particle, a membrane, a multi-component complex, a cell, a cell part, any fragment thereof, or any combination thereof.

[0208] In certain embodiments, the target is a polypeptide, a protein, or any fragment thereof. For example, the target can be a purified polypeptide, an isolated polypeptide, a fusion tagged polypeptide, a polypeptide bound to or spanning a membrane virus or virion of a cell, a cytoplasmic protein, an intracellular protein, an extracellular protein, a kinase, a phosphatase, an aromatase, a helicase, a protease, an oxidoreductase, a reductase, a transferase, a hydrolase, a lyase, an isomerase, a glycosylase, an extracellular matrix protein, a ligase, an ion transporter, a channel, a pore, an apoptotic protein, a cell adhesion protein, a pathogenicity protein, an aberrantly expressed protein, a transcription factor, a transcription regulator, a translation protein, a chaperone, a secreted protein, a ligand, a hormone, a cytokine, a chemokine, a nuclear protein, a receptor, a transmembrane receptor, a signal transducer, an antibody, a membrane protein, an integral membrane protein, a peripheral membrane protein, a cell wall protein, a globular protein, a fibrous protein, a glycoprotein, a lipoprotein, a chromosomal protein, any fragment thereof, or any combination thereof. In some embodiments, the target is a heterologous polypeptide. In some embodiments, the target is a protein that is overexpressed in a cell using molecular techniques such as transfection. In some embodiments, the target is a recombinant polypeptide. For example, the target is in a sample (e.g., a protein overexpressed by an organism) produced from a bacterial (e.g., E. coli), yeast, mammalian, or insect cell. In some embodiments, the target is a polypeptide that has a mutation, insertion, deletion, or polymorphism. In some embodiments, the target is an antigen, such as a polypeptide, that is used to immunize the organism or generate an immune response in the organism, such as antibody production.

[0209] In certain embodiments, the target is an antibody. An antibody may specifically bind to a particular spatial and polar structure of another molecule. The antibody may be monoclonal, polyclonal, or recombinant and may be prepared by techniques well known in the art, such as immunizing a host and collecting serum (polyclonal), or by preparing continuous hybrid cell lines and collecting secreted proteins (monoclonal), or by cloning and expressing nucleotide sequences encoding at least the amino acid sequence required for specific binding of a natural antibody, or a mutated form thereof. A natural antibody may be a protein comprising at least two heavy (H) chains and two light (L) chains interlinked by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H The heavy chain constant region can be composed of three domains: H1 , C H2 and C H3 Each light chain can be composed of a light chain variable region (V L ) and a light chain constant region. The light chain constant region can be composed of one domain, C L It may consist of V H and V L The regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs) interspersed with regions that are more conserved, called framework regions (FRs). H and V L The order is as follows: FR 1 , CDR 1 , F.R. 2 , CDR 2 , F.R. 3 , CDR 3 The constant region of an antibody may be composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus at FR1, FR2, and FR3. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class ... 1 , IgG 2 , IgG 3 , IgG4 , lgA 1 and lgA 2 ), subclass or modified forms thereof. Antibodies may include complete immunoglobulins or fragments thereof. Antibody fragments may refer to one or more fragments of an antibody that retain the ability to specifically bind to a binding moiety such as an antigen. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments are included so long as binding affinity for a particular molecule is maintained. Examples of antibody fragments include Fab fragments, V L , V H , C L and C H1 F(ab) is a monovalent fragment consisting of a domain. 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; V H and C H1 Fd fragment containing the domain; V of a single arm of an antibody L and V H Fv fragment consisting of domains; V H Single domain antibody (dAb) fragments consisting of domains (Ward et al., (1989) Nature 341:544-46); and isolated CDR and V L and V H Antibody fragments include single chain fragments (scFv) in which regions are paired to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-26; and Huston et al., (1988) PNAS 85:5879-83). Thus, antibody fragments include Fab, F(ab) and F(c) fragments. 2 , scFv, Fv, dAb, etc. Two domains V L and V HAlthough the antibodies are encoded by separate genes, they can be linked by an artificial peptide linker that allows them to be made as a single protein chain using recombinant methods. Such single chain antibodies contain one or more antigen-binding portions. These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same way as intact antibodies. The antibodies can be human, humanized, chimeric, isolated, canine, feline, donkey, ovine, any plant, animal, or mammalian.

[0210] In some embodiments, the target is a polymeric form of ribonucleotides and / or deoxyribonucleotides (adenine, guanine, thymine, or cytosine), such as DNA or RNA (e.g., mRNA). DNA includes double-stranded DNA found in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In some embodiments, the polynucleotide target is single-stranded, double-stranded, small interfering RNA (siRNA), messenger RNA (mRNA), transfer RNA (tRNA), chromosome, gene, non-coding genomic sequence, genomic DNA (e.g., fragmented genomic DNA), purified polynucleotide, isolated polynucleotide, hybridized polynucleotide, transcription factor binding site, mitochondrial DNA, ribosomal RNA, eukaryotic polynucleotide, prokaryotic polynucleotide, synthetic polynucleotide, ligated polynucleotide, recombinant polynucleotide, polynucleotide containing nucleic acid analogs, methylated polynucleotide, demethylated polynucleotide, any fragment thereof, or any combination thereof. In some embodiments, the target is a recombinant polynucleotide. In some embodiments, the target is a heterologous polynucleotide. For example, the target is a polynucleotide produced from a bacterial (e.g., E. coli), yeast, mammalian, or insect cell (e.g., a polynucleotide heterologous to the organism). In some embodiments, the target is a polynucleotide that has a mutation, insertion, deletion, or polymorphism.

[0211] In one embodiment, the target is an aptamer. An aptamer is an isolated nucleic acid molecule that binds to a binding moiety, such as a protein, with high specificity and affinity. An aptamer is a three-dimensional structure held in a specific conformation that provides chemical contact for specific binding to its given target. Although aptamers are nucleic acid-based molecules, there is a fundamental difference between aptamers and other nucleic acid molecules, such as genes and mRNA. In the latter case, the nucleic acid structure codes information through its linear base sequence, and this sequence is therefore important for the function of information storage. In complete contrast, aptamer function, which is based on specific binding of a target molecule, does not entirely depend on a conserved linear base sequence (non-coding sequence), but on a specific secondary / tertiary / quaternary structure. Any coding potential that an aptamer may have is entirely incidental and does not play any role in the binding of the aptamer to its cognate target. Aptamers must also be distinguished from natural nucleic acid sequences that bind to specific proteins. These latter sequences are natural sequences embedded in the genome of organisms that bind to a special subgroup of proteins involved in the transcription, translation, and transport of natural nucleic acids (e.g., nucleic acid binding proteins). Aptamers, on the other hand, are short, isolated, non-natural nucleic acid molecules. Although aptamers that bind to nucleic acid binding proteins can be identified, in most cases, such aptamers have little or no sequence identity to the sequences recognized by the nucleic acid binding proteins in nature. More importantly, aptamers can bind to almost any partner of interest, including virtually all proteins (not just nucleic acid binding proteins) as well as small molecules, carbohydrates, peptides, and the like. For most partners, even proteins, there is no natural nucleic acid sequence to which it binds. For partners with such sequences, e.g., nucleic acid binding proteins, such sequences differ from aptamers as a result of the relatively low binding affinity used in nature compared to tightly binding aptamers. Aptamers can specifically bind to selected partners and modulate the activity or binding interaction of the partners, e.g., through binding, aptamers can block the ability of their partners to function.The functional property of specific binding to a partner is an inherent property of an aptamer. A typical aptamer is 6-35 kDa in size (20-100 nucleotides), binds to its partner with micromolar to subnanomolar affinity, and can distinguish closely related targets (e.g., an aptamer can selectively bind related proteins from the same gene family). Aptamers are capable of using commonly found intermolecular interactions such as hydrogen bonding, electrostatic complementarity, hydrophobic contacts, and steric exclusion to bind to a specific partner. Aptamers have several desirable characteristics for use as therapeutic and diagnostic agents, including high specificity and affinity, low immunogenicity, biological potency, and excellent pharmacokinetic properties. Aptamers can include molecular stem and loop structures (e.g., hairpin loop structures) formed from the hybridization of covalently linked complementary polynucleotides. The stem contains the hybridized polynucleotide, and the loop is the region that covalently binds the two complementary polynucleotides.

[0212] In some embodiments, the target is a small molecule. For example, the small molecule can be a macrocycle, an inhibitor, a drug, or a chemical compound. In some embodiments, the small molecule contains 5 or fewer hydrogen bond donors. In some embodiments, the small molecule contains 10 or fewer hydrogen bond acceptors. In some embodiments, the small molecule has a molecular weight of 500 Daltons or less. In some embodiments, the small molecule has a molecular weight of about 180-500 Daltons. In some embodiments, the small molecule has an octanol-water partition coefficient lop P of 5 or less. In some embodiments, the small molecule has a partition coefficient log P of -0.4 to 5.6. In some embodiments, the small molecule has a molar refractive index of 40 to 130. In some embodiments, the small molecule contains about 20 to about 70 atoms. In some embodiments, the small molecule has a molecular weight of 140 Angstroms. 2 It has the following polar surface area:

[0213] In some embodiments, the target is a cell. For example, the target is an intact cell, a cell treated with a compound (e.g., a drug), a fixed cell, a lysed cell, or any combination thereof. In some embodiments, the target is a single cell. In some embodiments, the target is a plurality of cells.

[0214] In some embodiments, a single target or multiple (e.g., two or more) targets have multiple binding moieties. In one embodiment, a single target can have 2, 3, 4, 5, 6, 7, 8, 9, 10, or more binding moieties. In one embodiment, the two or more targets, such as a mixture or library of targets, are in a sample, and the sample comprises two or more binding moieties. In some embodiments, a single target or multiple targets comprise multiple different binding moieties. For example, the multiple targets can include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, or 30,000 binding moieties.

[0215] A target can include multiple binding moieties, including at least two different binding moieties. For example, the binding moieties can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, The molecule may comprise a plurality of binding moieties, including 0, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, or 25,000 different binding moieties.

[0216] Binding Sites and Binding Moieties In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises one binding site. In some embodiments, the first portion comprises a binding site configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, which is composed of unmodified nucleotides. In some embodiments, the first portion comprises one or more binding sites configured to bind to a protein, peptide, biomolecule, DNA, RNA, or cellular target, which is composed of unmodified nucleotides, or a combination thereof. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises at least two binding sites. For example, the modified circular RNA may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more binding sites. In some embodiments, the modified circular RNA described herein (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) is a molecular scaffold that binds to one or more binding moieties of one or more targets. Each target can be, but is not limited to, a different or the same nucleic acid (e.g., RNA, DNA, RNA-DNA hybrid), small molecule (e.g., drug), aptamer, polypeptide, protein, lipid, carbohydrate, antibody, virus, virus particle, membrane, multi-component complex, cell, cell part, any fragment thereof, and any combination thereof. In some embodiments, one or more binding moieties bind to one or more binding moieties of the same target. In some embodiments, one or more binding moieties bind to one or more binding moieties of different targets. In some embodiments, the modified circular RNA serves as a scaffold for one or more binding moieties of one or more targets. In some embodiments, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) specifically binds to one or more binding moieties of one or more targets to regulate a cellular process.In some embodiments, the modified circular RNA described herein (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for one or more specific targets of interest. In some embodiments, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises multiple binding sites or a combination of binding sites for each binding moiety of interest. For example, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a polynucleotide target, such as DNA or RNA. For example, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for an mRNA target. For example, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for an rRNA target. For example, the modified circular RNA comprises a binding site for a tRNA target. For example, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a genomic DNA target.

[0217] In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in a single stranded DNA. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in a double stranded DNA. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in an antibody. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in a viral particle. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in a small molecule. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in a cell or in a cellular environment. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in an RNA-DNA hybrid. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in a methylated polynucleotide. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in an unmethylated polynucleotide. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in an aptamer. In some cases, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in a polypeptide.In some cases, the modified circular RNA (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) comprises a binding site for a binding moiety in a polypeptide, a protein, a protein fragment, a tagged protein, an antibody, an antibody fragment, a small molecule, a viral particle (e.g., a viral particle containing a transmembrane protein), or a cell.

[0218] In some cases, the binding moiety contains at least two amide bonds. In some cases, the binding moiety does not contain phosphodiester bonds. In some cases, the binding moiety is not DNA or RNA.

[0219] The modified circular RNA provided herein (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) may include one or more binding sites for binding moieties in the complex. The modified circular RNA provided herein (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) may include one or more binding sites for a target to form a complex. The modified circular RNA provided herein (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) may form a complex between the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) and a target. In some embodiments, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) forms a complex with a single target. In some embodiments, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) forms a complex with two or more target complexes. In some embodiments, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) forms a complex with three or more target complexes. In some embodiments, two or more modified circular RNAs (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) are complexed with a single target. In some embodiments, two or more modified circular RNAs (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) are complexed with two or more targets. In some embodiments, a first modified circular RNA (e.g., fully modified circular polyribonucleotides or hybrid modified circular polyribonucleotides) is complexed with a first binding moiety of a first target and a second different binding moiety of a second target.In one embodiment, a first modified circular RNA (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) is complexed with a first binding moiety of a first target, and a second modified circular RNA (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) is complexed with a second binding moiety of a second target.

[0220] In certain embodiments, the modified circular RNA (e.g., a fully modified circular polyribonucleotide or a hybrid modified circular polyribonucleotide) may comprise binding sites for one or more binding moieties in one or more antibody-polypeptide complexes, polypeptide-polypeptide complexes, polypeptide-DNA complexes, polypeptide-RNA complexes, polypeptide-aptamer complexes, virus particle-antibody complexes, virus particle-polypeptide complexes, virus particle-DNA complexes, virus particle-RNA complexes, virus particle-aptamer complexes, cell-antibody complexes, cell-polypeptide complexes, cell-DNA complexes, cell-RNA complexes, cell-aptamer complexes, small molecule-polypeptide complexes, small molecule-DNA complexes, small molecule-aptamer complexes, small molecule-cell complexes, small molecule-virus particle complexes, and combinations thereof.

[0221] In some cases, the binding moiety is on a polypeptide, protein, or fragment thereof. In some embodiments, the binding moiety comprises a domain, fragment, epitope, region, or portion of a polypeptide, protein, or fragment thereof. For example, the binding moiety comprises a domain, fragment, epitope, region, or portion of an isolated, cellular, purified, or recombinant polypeptide. For example, the binding moiety comprises a domain, fragment, epitope, region, or portion of an antibody or fragment thereof. For example, the binding moiety comprises a domain, fragment, epitope, region, or portion of a transcription factor. For example, the binding moiety comprises a domain, fragment, epitope, region, or portion of a receptor. For example, the binding moiety comprises a domain, fragment, epitope, region, or portion of a transmembrane receptor. The binding moiety may be on or comprise a domain, fragment, epitope, region, or portion of an isolated, purified, and / or recombinant polypeptide. The binding moiety comprises or comprises a domain, fragment, epitope, region, or portion of a mixture of analytes (e.g., lysates). For example, the binding moiety is on or includes a domain, fragment, epitope, region, or moiety derived from multiple cells or from the lysate of a single cell.

[0222] In some cases, the binding moiety is on or comprises a domain, fragment, epitope, region, or portion of a small molecule. For example, the binding moiety is on or comprises a domain, fragment, epitope, region, or portion of an agent. For example, the binding moiety is on or comprises a domain, fragment, epitope, region, or portion of a compound. For example, the binding moiety is on or comprises a domain, fragment, epitope, region, or portion of an organic compound. In some cases, the binding moiety is on or comprises a domain, fragment, epitope, region, or portion of a small molecule having a molecular weight of 900 Daltons or less. In some cases, the binding moiety is on or comprises a domain, fragment, epitope, region, or portion of a small molecule having a molecular weight of 500 Daltons or more. The binding moiety is obtained, for example, from a library of compounds generated by combinatorial means, i.e., a compound diversity combinatorial library, of natural or synthetic molecules. Combinatorial libraries and methods for their generation and screening are known in the art and include, for example, U.S. Patent Nos. 5,741,713; 5,734,018; 5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696; 5,684,711; 5,641,862; 5,639,603; Nos. 5,593,853; 5,574,656; 5,571,698; 5,565,324; 5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564; 5,440,016; 5,438,119; and 5,223,409, the disclosures of which are incorporated herein by reference.

[0223] A binding moiety may be on or comprise a domain, fragment, epitope, region, or portion of a member of a specific binding pair (e.g., a ligand). A binding moiety may be on or comprise a domain, fragment, epitope, region, or portion of a monovalent (monoepitopic) or multivalent (polyepitopic) portion. A binding moiety may be antigenic or haptenic. A binding moiety may be on or comprise a domain, fragment, epitope, region, or portion of a single molecule or multiple molecules that share at least one common epitope or determinant. A binding moiety may be on or comprise a domain, fragment, epitope, region, or portion of a cell (e.g., a bacterial cell, a plant cell, or an animal cell). A binding moiety may be either in the natural environment (e.g., a tissue), in cultured cells, or in a microorganism (e.g., a bacterium, a fungus, a protozoan, or a virus), or in a lysed cell. The binding moiety may be modified (e.g., chemically) to provide one or more additional binding sites, including, but not limited to, a dye (e.g., a fluorescent dye), a polypeptide modifying moiety such as a phosphate group, a carbohydrate group, or a polynucleotide modifying moiety such as a methyl group.

[0224] In some cases, the binding moiety is on or comprises a domain, fragment, epitope, region, or part of a molecule found in a sample from a host. Samples from a host include bodily fluids (e.g., urine, blood, plasma, serum, saliva, semen, stool, saliva, cerebrospinal fluid, tears, mucus, etc.). Samples can be examined directly or may be pretreated to make the binding moiety more easily detectable. A sample comprises a quantity of material derived from an organism or former organism. A sample can be natural, recombinant, synthetic, or non-natural. A binding moiety can be any of the above expressed naturally or recombinantly from a cell, in a cell lysate or cell culture medium, in an in vitro translation sample, or immunoprecipitation from a sample (e.g., a cell lysate).

[0225] In some cases, the target binding moiety is expressed in a cell-free system or in vitro. For example, the target binding moiety is in a cell extract. In some cases, the target binding moiety is in a cell extract together with a DNA template and reagents for transcription and translation. Exemplary sources of cell extracts that can be used include wheat germ, Escherichia coli, rabbit reticulocytes, hyperthermophile, hybridoma, Xenopus oocytes, insect cells, and mammalian cells (e.g., human cells). Exemplary cell-free methods that can be used to express target polypeptides (e.g., generate target polypeptides on an array) include protein in situ arrays (PISA), multiple spotting technology (MIST), self-assembly mRNA translation, nucleic acid programmable protein arrays (NAPPA), nanowell NAPPA, DNA array to protein arrays (DAPA), membrane-free DAPA, nanowell copy and μIP-microintaglio printing, and pMAC-protein microarray copy (see Kilb et al., Eng. Life Sci. 2014, 14, 352-364).

[0226] In some cases, a target binding moiety is synthesized in situ from a DNA template (e.g., on an array of solid substrates). In some cases, multiple binding moieties are synthesized in situ from multiple corresponding DNA templates, either in parallel or in a single reaction. Exemplary methods for in situ target polypeptide expression include those described in Stevens, Structure 8(9):R177-R185 (2000); Katzen et al., Trends Biotechnol. 23(3):150-6. (2005); He et al., Curr. Opin. Biotechnol. 19(1):4-9. (2008); Ramachandran et al., Science 305(5680):86-90. (2004); He et al., Nucleic Acids Res. 29(15):E73-3 (2001); Angenendt et al., Mol. Cell Proteomics 5(9):1658-66 (2006); Tao et al., Nat Biotechnol 24(10):1253-4 (2006); Angenendt et al. al., Anal. Chem. 76(7):1844-9(2004); Kinpara et al., J. Biochem. 136(2):149-54(2004); Takulapalli et al., J. Proteome Res. 11(8):4382-91(2012); He et al., Nat. Methods 5(2):175-7(2008); Chatterjee and J. LaBaer, ​​Curr Opin Biotech 17(4):334-336(2006); He and Wang, Biomol Eng 24(4):375-80(2007); and He and Taussig, J. Immunol. Methods 274(1-2):265-70(2003).

[0227] In some cases, the binding portion of a nucleic acid target comprises a range of at least 6 nucleotides, e.g., at least 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, or 100 nucleotides. In some cases, the binding portion of a protein target comprises a contiguous stretch of nucleotides. In some cases, the binding portion of a protein target comprises a discontinuous stretch of nucleotides. In some cases, the binding portion of a nucleic acid target comprises a site of mutation or functional mutation, including deletion, addition, swap, or truncation of nucleotides in the nucleic acid sequence.

[0228] In some cases, the binding portion of a protein target comprises a range of at least 6 amino acids, e.g., at least 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, or 100 amino acids. In some cases, the binding portion of a protein target comprises a contiguous stretch of amino acids. In some cases, the binding portion of a protein target comprises a discontinuous stretch of amino acids. In some cases, the binding portion of a protein target comprises a site of mutation or functional mutation, including deletion, addition, exchange, or truncation of amino acids in the polypeptide sequence.

[0229] In certain embodiments, the binding moiety is on or comprises a domain, fragment, epitope, region, or portion of a membrane-bound protein. Exemplary membrane-bound proteins include, but are not limited to, GPCRs (e.g., adrenergic receptors, angiotensin receptors, cholecystokinin receptors, muscarinic acetylcholine receptors, neurotensin receptors, galanin receptors, dopamine receptors, opioid receptors, serotonin receptors, somatostatin receptors, etc.), ion channels (e.g., nicotinic acetylcholine receptors, sodium channels, potassium channels, etc.), receptor tyrosine kinases, receptor serine / threonine kinases, receptor guanylate cyclases, growth factor and hormone receptors (e.g., epidermal growth factor (EGF) receptor), and others. The binding moiety may also be on or comprise a domain, fragment, epitope, region, or portion of a mutant or modified variant of a membrane-bound protein. For example, certain point mutations in GPCRs retain function and are involved in disease (see, e.g., Stadel et al., (1997) Trends in Pharmacological Review 18:430-37).

[0230] In some embodiments, the modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) may contain other binding motifs for binding to other intracellular molecules. Non-limiting examples of the application of modified circular RNA (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) are listed in Table 4.

[0231] [Table 4]

[0232] RNA binding site In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more RNA binding sites. In some embodiments, the first portion comprises one or more RNA binding sites consisting of unmodified nucleotides. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises an RNA binding site that regulates the expression of an endogenous gene and / or an exogenous gene. In some embodiments, the RNA binding site regulates the expression of a host gene. The RNA binding site may comprise a sequence that hybridizes to an endogenous gene (e.g., a sequence for miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA described herein), a sequence that hybridizes to an exogenous nucleic acid such as viral DNA or RNA, a sequence that hybridizes to RNA, a sequence that interferes with gene transcription, a sequence that interferes with RNA translation, a sequence that destabilizes RNA, such as by stabilizing RNA or targeting it for degradation, or a sequence that regulates a DNA- or RNA-binding factor.

[0233] In some embodiments, the RNA binding site may be one of a tRNA, lncRNA, lincRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA, and hnRNA binding site. RNA binding sites are well known to those of skill in the art.

[0234] Specific RNA binding sites can inhibit gene expression through the biological process of RNA interference (RNAi). In certain embodiments, the modified circular polyribonucleotide comprises an RNA or RNA-like structure RNAi molecule having a nucleic acid base sequence that is identical (complementary) or nearly identical (substantially complementary) to a coding sequence in a target gene expressed in a cell, typically having 15-50 base pairs (such as about 18-25 base pairs). 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.

[0235] In some embodiments, the RNA binding site comprises an siRNA or shRNA. siRNA and shRNA resemble intermediates in the processing pathway of endogenous miRNA genes. In some embodiments, siRNA can function as miRNA and vice versa. MicroRNAs, like siRNA, use RISC to downregulate target genes, but unlike siRNA, most animal miRNAs do not cleave mRNAs. Instead, miRNAs reduce protein output by translational repression or polyA removal and mRNA degradation. Known miRNA binding sites are 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. This region is known as the seed region. Because siRNA and miRNA are replaceable, exogenous siRNA can downregulate mRNAs with seed complementarity to the siRNA. Multiple target sites within the 3'-UTR can confer stronger downregulation.

[0236] MicroRNA (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. Modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can contain one or more miRNA target sequences, miRNA sequences, or miRNA seeds. Such sequences can correspond to any miRNA.

[0237] The miRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature miRNA, which has Watson-Crick complementarity to the miRNA target sequence. The miRNA seed may comprise positions 2-8 or 2-7 of the mature miRNA. In certain embodiments, the miRNA seed may comprise seven nucleotides (e.g., nucleotides 2-8 of the mature miRNA), where the seed-complementary site in the corresponding miRNA target is adjacent to an adenine (A) opposite miRNA position 1. In certain embodiments, the miRNA seed may comprise six nucleotides (e.g., nucleotides 2-7 of the mature miRNA), where the seed-complementary site in the corresponding miRNA target is adjacent to an adenine (A) opposite miRNA position 1.

[0238] The base of miRNA seed can be substantially complementary to the target sequence.By engineering miRNA target sequence into modified cyclic polyribonucleotide, modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) can evade or be detected by the host's immune system, and can have regulated degradation or regulated translation.This process reduces the risk of off-target effect during delivery of modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide).

[0239] The modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) can contain an miRNA sequence identical to about 5 to about 25 contiguous nucleotides of a 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 have a high percentage of identity to 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.

[0240] Conversely, miRNA binding sites can be engineered out (i.e., removed) from modified cyclic polyribonucleotides (e.g., fully modified or hybrid modified cyclic polyribonucleotides) to regulate protein expression in specific tissues. Regulation of expression in multiple tissues can be achieved through the introduction or removal or through one or several miRNA binding sites.

[0241] Examples of tissues in which miRNAs 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). MiRNAs may also regulate complex biological processes such as angiogenesis (miR-132). In the modified cyclic polyribonucleotides described herein, binding sites for miRNAs involved in such processes can be removed or introduced to regulate expression from the modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) for biologically relevant cell types or in the context of relevant biological processes. In some embodiments, the miRNA binding site includes, for example, miR-7.

[0242] By understanding the expression patterns of miRNAs in various cell types, the modified cyclic polyribonucleotides described herein (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be engineered for more targeted expression only in specific cell types or under specific biological conditions. By introducing tissue-specific miRNA binding sites, the modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) can be designed for optimal protein expression in a tissue or in relation to a biological condition.

[0243] Furthermore, miRNA seed sites can be incorporated into modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) to regulate expression in certain cells, resulting in biological improvements. An example of this is the incorporation of miR-142 sites. Incorporation of miR-142 sites into modified cyclic polyribonucleotides (e.g., fully modified cyclic polyribonucleotides or hybrid modified cyclic polyribonucleotides) described herein can not only regulate expression in hematopoietic cells, but can also reduce or eliminate immune responses against proteins encoded in the modified cyclic polyribonucleotides.

[0244] In some embodiments, the modified cyclic polyribonucleotide comprises at least one miRNA, e.g., 2, 3, 4, 5, 6, or more. In some embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) comprises a miRNA having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% nucleotide sequence identity with any one of the nucleotide sequences or sequences complementary to the target sequence.

[0245] Lists of known miRNA sequences can be found in databases maintained by research organizations such as the Wellcome Trust Sanger Institute, the Penn Center for Bioinformatics, the Memorial Sloan Kettering Cancer Center, and the European Molecule Biology Laboratory. RNAi molecules can be easily designed and produced by techniques known in the art. Furthermore, computerized means can be used to determine effective and specific sequence motifs.

[0246] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a long non-coding RNA. Long non-coding RNA (lncRNA) comprises non-protein-coding transcripts longer than 100 nucleotides. The longer length distinguishes lncRNA from small regulatory RNAs such as miRNAs, siRNAs, and other short RNAs. In general, the majority (about 78%) of lncRNAs are characterized as tissue-specific. Branched lncRNAs that are transcribed in the opposite direction to nearby protein-coding genes (which account for a significant proportion of about 20% of all lncRNAs in mammalian genomes) can regulate the transcription of nearby genes.

[0247] The length of the RNA binding site can be 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 RNA binding site with the intended target can be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0248] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises one or more large intergenic non-coding RNA (lincRNA) binding sites. LincRNAs constitute the majority of long-chain non-coding RNA. LincRNAs are non-coding transcripts and, in some embodiments, are greater than about 200 nucleotides in length. In some embodiments, lincRNAs have an exon-intron-exon structure similar to protein-coding genes, but do not contain open reading frames and do not code for proteins. LincRNA expression can be highly tissue-specific compared to coding genes. LincRNAs are typically co-expressed with their neighboring genes, although not to the same extent as their neighboring protein-coding gene pairs. In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a circularized lincRNA.

[0249] In some embodiments, the modified circular polyribonucleotides disclosed herein include one or more lincRNAs, such as FIRRE, LINC00969, PVT1, LINC01608, JPX, LINC01572, LINC00355, C1orf132, C3orf35, RP11-734, LINC01608, CC-499B15.5, CASC15, LINC00937, and RP11-191.

[0250] Lists of known lincRNA and lncRNA sequences can be found in databases maintained by research organizations such as the Institute of Genomics and Integrative Biology, the Diamantina Institute at the University of Queensland, Ghent University, and Sun Yat-sen University. LincRNA and lncRNA molecules can be readily designed and generated by techniques known in the art. Additionally, computational means can be used to determine effective and specific sequence motifs.

[0251] An RNA binding site may comprise a sequence that is substantially complementary or completely complementary to all or a fragment of an endogenous gene or gene product (e.g., mRNA). A complementary sequence may complement a sequence at a boundary between an intron and an exon to prevent maturation of a newly generated nuclear RNA transcript of a particular gene into an mRNA for transcription. A complementary sequence may be specific to a gene by hybridizing with the mRNA for that gene and preventing its translation. An RNA binding site may comprise a sequence that is antisense or substantially antisense to all or a fragment of an endogenous gene or gene product, such as DNA, RNA, or derivatives or hybrids thereof.

[0252] In certain embodiments, the modified cyclic polyribonucleotide (e.g., a fully modified cyclic polyribonucleotide or a hybrid modified cyclic polyribonucleotide) typically has an RNA or RNA-like structure of about 5-5000 base pairs (depending on the particular RNA structure, e.g., miRNA 5-30 bp, lncRNA 200-500 bp) and includes an RNA binding site having a nucleobase sequence identical (complementary) or nearly identical (substantially complementary) to a coding sequence in a target gene expressed in a cell.

[0253] DNA binding site In some embodiments, the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a DNA binding site, such as a sequence for a guide RNA (gRNA). In some embodiments, the first portion comprises one or more DNA binding sites consisting of unmodified nucleotides. In some embodiments, the modified circular polyribonucleotide (e.g., fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) comprises a complement to a guide RNA or gRNA sequence. The gRNA short synthetic RNA is composed of a "scaffold" sequence required to bind to the incomplete effector moiety and a user-defined approximately 20 nucleotide targeting sequence for the genomic target. The guide RNA sequence can have a length of 17 to 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to the targeting nucleic acid sequence. Custom gRNA generators and algorithms can be used to design effective guide RNAs. Gene editing can be performed using chimeric "single guide RNAs" ("sgRNAs"), which are 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 can be effective for genome editing.

[0254] The gRNA may recognize a specific DNA sequence, such as a sequence adjacent to or within a gene's promoter, enhancer, silencer, or repressor.

[0255] In some embodiments, gRNA is part of the CRISPR system for gene editing.For gene editing, modified circular polyribonucleotide (for example, fully modified circular polyribonucleotide or hybrid modified circular polyribonucleotide) can be designed to include one or more guide RNA sequences corresponding to desired target DNA sequence.The gRNA sequence can include at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides for interaction with Cas9 or other exonucleases to cleave DNA, for example, Cpf1 interacts with at least about 16 nucleotides of the gRNA sequence for detectable DNA cleavage.

[0256] In some embodiments, the modified cyclic polyribonucleotide (e.g., fully modified cyclic polyribonucleotide or hybrid modified cyclic polyribonucleotide) comprises a sequence that binds to the major groove in double-stranded DNA. In one such case, the specificity and stability of the triplet structure formed by the modified cyclic polyr...

Claims

1. A pharmaceutical composition for use in a method of reducing the immunogenicity of a cyclic polyribonucleotide in a subject, the method comprising the steps of: (a) administering to a subject a cyclic polyribonucleotide comprising: a first portion of contiguous nucleotides comprising an IRES consisting of unmodified nucleotides; and a second portion of contiguous nucleotides consisting of cytidine, adenine, guanine and modified uridine; The method comprises: administering the pharmaceutical composition to the subject; obtaining a reduced immunogenicity for said hybrid modified cyclic polyribonucleotide compared to a corresponding unmodified cyclic polyribonucleotide in a cell or tissue of said subject. The modified uridine is 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-methyl-uridine 2. The pharmaceutical composition of claim 1, comprising 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, or 4-methoxy-2-thio-pseudouridine.

3. The pharmaceutical composition of claim 1, wherein the modified uridine is pseudouridine.

4. The pharmaceutical composition of claim 3, wherein the modified uridine is N1-methyl-pseudouridine.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the hybrid modified cyclic polyribonucleotide is translatable.

6. The pharmaceutical composition of claim 1, wherein the second portion of consecutive nucleotides comprises one or more expressed sequences.

7. The one or more expression sequences of the hybrid modified circular polyribonucleotides are (a) higher translation efficiency than the fully modified circular polyribonucleotide equivalent; (b) 0.7- to 3-fold higher translation efficiency than the equivalent fully modified circular polyribonucleotide; (c) have a higher translation efficiency than the corresponding unmodified circular polyribonucleotide; (d) a translation efficiency that is 0.7- to 3-fold higher than the corresponding unmodified circular polyribonucleotide; (e) has 0.7- to 3-fold higher expression than the corresponding unmodified circular polyribonucleotide; (f) has a half-life that is 1.1 to 10.0 times longer than the corresponding unmodified circular polyribonucleotide; (g) has a longer half-life than the corresponding unmodified circular polyribonucleotide; or (h) is 1.1-fold to 10.0-fold less immunogenic than the corresponding unmodified cyclic polyribonucleotide as assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, and IFN-β; The pharmaceutical composition of claim 6, having the formula:

8. A pharmaceutical composition for use in a method of expressing one or more expression sequences in a subject, comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion of contiguous nucleotides comprising an IRES consisting of unmodified nucleotides and a second portion of contiguous nucleotides consisting of cytidine, adenine, guanine and modified uridine; The method comprises: administering the pharmaceutical composition to the subject; and obtaining increased expression of said one or more expressed sequences compared to the expression of the corresponding one or more expressed sequences in a fully modified circular polyribonucleotide equivalent in said subject's cells or tissues.

9. A pharmaceutical composition for use in a method of increasing stability of a cyclic polyribonucleotide in a subject, comprising a hybrid modified cyclic polyribonucleotide, the hybrid modified cyclic polyribonucleotide comprising a first portion of contiguous nucleotides comprising an IRES consisting of unmodified nucleotides and a second portion of contiguous nucleotides consisting of cytidine, adenine, guanine and modified uridine; The method comprises: administering the pharmaceutical composition to the subject; obtaining increased stability for said hybrid modified cyclic polyribonucleotides as compared to corresponding unmodified cyclic polyribonucleotides in said cells or tissues of said subject; 13. A pharmaceutical composition comprising:

10. A pharmaceutical composition comprising a hybrid modified cyclic polyribonucleotide and a pharma- ceutically acceptable carrier or excipient, wherein the hybrid modified cyclic polyribonucleotide comprises a first portion of contiguous nucleotides comprising an IRES consisting of unmodified nucleotides, and a second portion of contiguous nucleotides consisting of cytidine, adenine, guanine and modified uridine. The modified uridine is selected from the group consisting of 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, and 5-methyl-uridine.

11. The pharmaceutical composition of claim 10, comprising 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, or 4-methoxy-2-thio-pseudouridine.

12. The pharmaceutical composition described in claim 11, wherein the modified uridine is pseudouridine.

13. The pharmaceutical composition of claim 12, wherein the modified uridine is N1-methyl-pseudouridine.

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