Compositions and methods for purifying polyribonucleotides

Abstract

The present disclosure relates to compositions and methods for isolating and / or purifying polyribonucleotides, which may be separated from a mixture of polyribonucleotides and oligonucleotides that hybridize to target regions of the polyribonucleotides, and available for use as therapeutic agents.

Description

[Background technology]

[0001] Polyribonucleotides are useful in a variety of therapeutic and modifying applications. Thus, new compositions and methods for isolating and purifying polyribonucleotides would be useful. Summary of the Invention [Means for solving the problem]

[0002] In one aspect, the invention features a method of separating a polyribonucleotide having a target region from a plurality of polyribonucleotides. In the method, a sample is provided that includes a plurality of polyribonucleotides. A subset of the plurality of polyribonucleotides in the sample has the target region. The method further includes contacting the sample with an oligonucleotide that hybridizes to the target region, and separating the polyribonucleotide having the target region that is hybridized to the oligonucleotide from the plurality of polyribonucleotides in the sample.

[0003] In another aspect, the invention features a method of separating a linear polyribonucleotide having a target region from a plurality of polyribonucleotides, including a mixture of linear polyribonucleotides and cyclic polyribonucleotides. In the method, a sample is provided that includes a plurality of polyribonucleotides. A subset of the plurality of linear polyribonucleotides in the sample has the target region. The method further includes contacting the sample with an oligonucleotide that hybridizes to the target region, and separating the linear polyribonucleotide having the target region hybridized to the oligonucleotide from the plurality of polyribonucleotides in the sample. In some embodiments, the polyribonucleotide having the target region lacks a polyA sequence (i.e., a polyA tail). In some embodiments, the target region is not located at the 3' or 5' end of the polyribonucleotide having the target region. The cyclic polyribonucleotide or the subset thereof can lack the target region.

[0004] In another aspect, the invention features a method of separating a polyribonucleotide having a target region from a plurality of polyribonucleotides. The method includes providing a sample including a plurality of polyribonucleotides, where a subset of the plurality of polyribonucleotides has the target region. The target region is not located at the 3' or 5' end of the polyribonucleotide having the target region (i.e., the target region is located internally on the polyribonucleotide). The method further includes contacting the sample with an oligonucleotide that hybridizes to the target region, and separating the polyribonucleotide having the target region that is hybridized to the oligonucleotide from the plurality of polyribonucleotides in the sample.

[0005] In another aspect, the invention features a method for separating a linear polyribonucleotide having a target region from a plurality of circular polyribonucleotides having a target region. The method includes providing a sample including a plurality of polyribonucleotides. The method further includes contacting the sample with an oligonucleotide. The oligonucleotide hybridizes to the target region of the linear polyribonucleotide with a first binding affinity, and the oligonucleotide hybridizes to the target region of the circular polynucleotide with a second binding affinity different from the first binding affinity, and the linear polyribonucleotide having the target region hybridized to the oligonucleotide is separated from the plurality of circular polyribonucleotides in the sample. In some embodiments, the first binding affinity is lower than the second binding affinity, and the oligonucleotide preferentially binds to the linear polyribonucleotide.

[0006] In another aspect, the invention features a method for separating polyribonucleotides in a first arrangement having a target region from a plurality of polyribonucleotides in a second arrangement having a target region. The method includes providing a sample including a plurality of polyribonucleotides. The method further includes contacting the sample with an oligonucleotide. The oligonucleotide hybridizes to the target region of the polynucleotides in the first arrangement with a first binding affinity, and the oligonucleotide hybridizes to the target region of the polynucleotides in the second arrangement with a second binding affinity different from the first binding affinity, and the polyribonucleotides in the first arrangement having the target region hybridized to the oligonucleotide are separated from the plurality of polyribonucleotides in the second arrangement in the sample. In some embodiments, the first binding affinity is lower than the second binding affinity, and the oligonucleotide preferentially binds to the polyribonucleotides in the first arrangement.

[0007] In some embodiments of any of the aspects described herein, the separating comprises immobilizing the oligonucleotides.

[0008] In some embodiments of any of the methods described herein, the target region is not located at the 5' or 3' end of the polyribonucleotide that has the target region. For example, the target region may be located internally on the polyribonucleotide. In some embodiments, the target region is not located at the 3' end of the polyribonucleotide.

[0009] In some embodiments of any of the methods described herein, the target region is located at the 5' or 3' end of the polyribonucleotide and the target region does not contain a polyA sequence. In some embodiments, the target region is located at the 3' end of the polyribonucleotide and does not contain a polyA sequence.

[0010] In some embodiments of any of the methods described herein, the target region does not contain a polyA sequence.

[0011] In some embodiments of any of the above aspects, the oligonucleotide is conjugated to a particle. The particle may be, for example, a magnetic particle or a bead. The bead may be, for example, a cross-linked agarose, such as a SEPHAROSE® bead.

[0012] In some embodiments of any of the above aspects, the oligonucleotide is conjugated to a first capture agent.

[0013] In some embodiments, the oligonucleotide is conjugated to the first capture agent or particle via a chemical linker. The chemical linker may include, for example, triethylene glycol. In some embodiments, the chemical linker is conjugated to the 3'-end or 5'-end of the oligonucleotide.

[0014] In some embodiments, the first capture agent comprises an antigen (eg, biotin).

[0015] In some embodiments, the method further includes contacting the sample with a second capture agent that is bound to the first capture agent. The second capture agent may include, for example, an antibody or an antigen-binding fragment thereof (e.g., streptavidin).

[0016] In some embodiments, the second capture agent is conjugated to a particle. The particle may be, for example, a magnetic particle or a bead. The bead may be, for example, a cross-linked agarose, for example, a SEPHAROSE® bead. In some embodiments, the second capture agent is conjugated to a particle using a chemical linker. The chemical linker may include, for example, triethylene glycol.

[0017] In some embodiments, the polyribonucleotide having a target region comprises an intron or a portion thereof (e.g., a half-intron). The target region may comprise an intron or a portion thereof. In some embodiments, the intron or a portion thereof is a catalytic intron (e.g., a group I catalytic intron or a group II catalytic intron) or a portion thereof. In some embodiments, the intron or a portion thereof has a length of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some embodiments, the intron or a portion thereof has a length of 20-500, 20-400, 20-300, 20-200, 20-100, 50-500, 50-400, 50-300, 50-200, 100-500, 100-400, 100-300, or 100-200 nucleotides.

[0018] In some embodiments, the target region comprises a half intron (e.g., the 5' or 3' portion of a catalytic intron). In some embodiments, the target region comprises a 5' half-intron (e.g., a 5' half-intron corresponding to the 5' portion of a catalytic intron). In some embodiments, the target region comprises a 3' half-intron (e.g., a 3' half-intron corresponding to the 3' portion of a catalytic intron). In some embodiments, the target region comprises the 5' half of a group I catalytic intron. In some embodiments, the 5' half of the group I catalytic intron is derived from the 5' portion of the cyanobacterial Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, T4 phage td gene, or variants thereof. In some embodiments, the target region comprises the 3' half of a group I catalytic intron. In some embodiments, the 3' half of the Group I catalytic intron is selected from the 3' portion from the cyanobacterium Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, T4 phage td gene, or a mutant thereof.

[0019] The target region may be located 5' or 3' to an intron or portion thereof, or may include a portion of a region located 5' or 3' to an intron or portion thereof.

[0020] In some embodiments, the method includes separating the spliced ​​polyribonucleotide from the unspliced ​​or partially spliced ​​polyribonucleotide. In some embodiments, the spliced ​​polyribonucleotide is a circular polyribonucleotide. In some embodiments, the spliced ​​polyribonucleotide is a linear polyribonucleotide. In some embodiments, the spliced ​​polyribonucleotide lacks an intron or a portion thereof (e.g., the spliced ​​polyribonucleotide lacks a catalytic intron, e.g., a group I catalytic intron, or a portion thereof).

[0021] In some embodiments, the method enriches the amount of spliced ​​polyribonucleotide by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more) compared to the sample.

[0022] In some embodiments, the polyribonucleotide comprising an intron or a portion thereof is a linear polyribonucleotide.

[0023] In some embodiments, the oligonucleotide has a length of at least 5 nucleotides (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides). In some embodiments, the oligonucleotide is, for example, 5-100, 5-95, 10-90, 10-80, 12-60, 15-50, 15-40, 15-30, 18-30, 20-25, or 20-22 nucleotides in length. In some embodiments, the oligonucleotide is 23 nucleotides in length.

[0024] In some embodiments, the oligonucleotide may have a melting temperature (Tm) of, for example, about 45° C. to about 75° C., e.g., about 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., or 75° C. In some embodiments, the oligonucleotide has a Tm of about 45° C. to about 65° C.

[0025] In some embodiments, the method further comprises washing the polyribonucleotide having the captured target region one or more times (e.g., after the contacting and / or separating steps), hi some embodiments, the method further comprises washing the first and / or second capture agent one or more times (e.g., two, three, four, five or more times).

[0026] In some embodiments, the method further comprises performing a first elution step to release the polyribonucleotides having the captured target regions. The first elution step may include, for example, adding a first buffer and / or heating the sample, for example, to at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., or higher.

[0027] In some embodiments, the method further comprises performing a second elution step. The second elution step may comprise adding a second buffer and / or heating the sample, for example, to at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., or higher. In some embodiments, the second buffer comprises a denaturing agent, for example, formamide or urea. The second buffer may comprise, for example, about 40% to about 60% formamide (e.g., about 40%, 45%, 50%, 55%, or 60% formamide).

[0028] In some embodiments, the method includes incubating the sample with an oligonucleotide (e.g., conjugated to a first capture agent) for at least 10 minutes (e.g., at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, or more).

[0029] In some embodiments, the method includes recovering a portion of the sample that is not bound by the oligonucleotide. In some embodiments, the method includes recovering a portion of the sample that is not bound by the capture agent (e.g., the first and / or second capture agent).

[0030] In some embodiments, the method includes providing a plurality of oligonucleotides, each of which hybridizes to a different target region, hi some embodiments, each of the oligonucleotides is conjugated to a first capture agent.

[0031] In some embodiments, the method comprises providing an oligonucleotide that hybridizes to multiple target regions, hi some embodiments, the method comprises providing a single oligonucleotide that hybridizes to the 3' half intron and the 5' half intron.

[0032] In some embodiments, the methods include providing a first oligonucleotide that hybridizes to the 3' half-intron and a second oligonucleotide that hybridizes to the 5' half-intron, hi some embodiments, the methods include providing a plurality of oligonucleotides, each of which hybridizes to a different region on the 3' half-intron and / or the 5' half-intron.

[0033] In some embodiments, the oligonucleotides have at least 80% (eg, at least 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to an equal length portion of the target region.

[0034] In some embodiments, the method includes providing the oligonucleotide at a molar ratio of 10:1 to 1:10 (e.g., 10:1, 5:1, 2:1, 1:2, 1:5, or 1:10) to the polyribonucleotide.

[0035] In another aspect, the invention features a population of polyribonucleotides produced by the method of any of the embodiments described herein. The population can include, for example, cyclic polyribonucleotides that lack a target region, where the cyclic polyribonucleotides comprise at least 1% (e.g., at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) (mol / mol) of the total polyribonucleotides in the composition.

[0036] In some embodiments, the population has less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) (mol / mol) linear polyribonucleotides.

[0037] In another aspect, the invention features a composition comprising a mixture of polyribonucleotides, a first subset of the mixture comprising cyclic polyribonucleotides lacking a target region, and a second subset of the polyribonucleotides comprising linear polyribonucleotides having a target region, the first subset comprising at least 1% (e.g., at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) (mol / mol) of the total polyribonucleotides in the composition.

[0038] In another aspect, the invention features a composition that includes a polyribonucleotide having a target region and an oligonucleotide designed to hybridize to the target region, where the oligonucleotide is conjugated to a first capture agent (e.g., an antigen, e.g., biotin). The composition may further include, for example, a polyribonucleotide lacking a target region. The polyribonucleotide lacking a target region may be, for example, a cyclic polyribonucleotide. The composition may further include a second capture agent designed to bind to the first capture agent (e.g., an antibody or an antigen-binding fragment thereof, e.g., streptavidin). The second capture agent may be conjugated to a particle.

[0039] In some embodiments, the compositions are produced by the methods described herein.

[0040] In some embodiments of any of the compositions described herein, the linear polyribonucleotide comprises an intron or a portion thereof. The target region may comprise an intron or a portion thereof. The target region may be located 5' or 3' to the intron or a portion thereof.

[0041] In some embodiments of any of the above aspects, the polyribonucleotides and / or oligonucleotides may be modified.

[0042] In another aspect, the invention features a pharmaceutical composition including a composition described herein, e.g., produced by a method, and a diluent, carrier, or excipient described above.

[0043] definition To facilitate understanding of the present disclosure, several terms are defined below. Terms defined herein have the meanings as commonly understood by those skilled in the art in the areas relevant to the present disclosure. Terms such as "a", "an" and "the" are not intended to refer to a singular entity only, but also include general classes, specific examples of which may be used for illustration. The term "or" is used to mean "and / or" unless expressly specified to refer to alternatives only or the alternatives are not mutually exclusive, but the present disclosure supports a definition that refers to alternatives only and "and / or". Although the terms in this specification are used to describe specific embodiments, their use should not be construed as limiting, except as outlined in the claims.

[0044] As used herein, any value provided in a range of values ​​includes both the upper and lower limits, as well as any value subsumed within the upper and lower limits.

[0045] As used herein, the term "about" refers to a value within ±10% of the recited value.

[0046] 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) to a cell, via a partially or fully encapsulated agent, or a combination thereof, by covalent modification of the cyclic polyribonucleotide. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles (e.g., nanoparticles encapsulated or covalently attached to cyclic polyribonucleotides), liposomes, fusosomes, exosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently attached to polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents).

[0047] As used herein, the terms "cyclic polyribonucleotide", "circular RNA", and "circRNA" are used interchangeably and refer to polyribonucleotide molecules having a structure with no free ends (i.e., free 3' and / or 5' ends), e.g., polyribonucleotide molecules that form a circular or endless structure through covalent or non-covalent bonds. A circular polyribonucleotide may be, for example, a covalently closed polyribonucleotide.

[0048] As used herein, the terms "disease," "disorder," and "condition" each refer to a state of less than optimal health, e.g., a condition that is or would be normally diagnosed or treated by a medical professional.

[0049] "Heterologous" means occurring under a situation other than the naturally occurring (natural) situation. A "heterologous" polynucleotide sequence indicates that the polynucleotide sequence is used in a manner other than that found in the sequence's native genome. For example, a "heterologous promoter" is used to drive the transcription of a sequence that is not naturally transcribed by that promoter; therefore, a "heterologous promoter" sequence is often included in an expression construct using recombinant nucleic acid technology. The term "heterologous" is also used to refer to a given sequence that is placed in a non-naturally occurring relationship with another sequence; for example, a heterologous coding or non-coding nucleotide sequence is commonly inserted into a genome by genome transformation techniques, resulting in a genetically modified or recombinant genome.

[0050] As used herein, the term "half intron" refers to a portion of an intron, where a first half intron and a second half intron together form an intron, such as a catalytic intron. A half intron may be a 5' portion of an intron (e.g., the 5' portion of a catalytic intron) or a 3' portion of an intron (e.g., the 3' portion of a catalytic intron), whereby the 5' half-intron and the 3' half-intron together form a functional intron, e.g., a functional intron capable of catalytic self-splicing. The term half intron is intended to refer to an intron split into two portions. The term half intron is not intended to state, mean, or imply that the two portions or halves are equal in length. The term half intron is used synonymously with the term split intron and may be used in place of the term "intron fragment."

[0051] As used herein, the term "impurity" refers to an undesirable substance present in a composition, e.g., a pharmaceutical composition described herein. In some embodiments, an impurity is a process-related impurity. In some embodiments, an impurity refers to anything other than the desired product in the final composition, e.g., an active pharmaceutical ingredient, as described herein. other than, product-related substances such as circular polyribonucleotides. As used herein, the term "process-related impurities" are substances used, present, or produced in the making of a composition, preparation, or product that are undesirable in the final composition, preparation, or product, other than the linear polyribonucleotides described herein. In some embodiments, a process-related impurity is an enzyme used in the synthesis or circularization of polyribonucleotides. As used herein, the term "product-related substances" are substances or by-products produced during the synthesis of a composition, preparation, or product, or any intermediates thereof. In some embodiments, a product-related substance is a deoxyribonucleotide fragment. In some embodiments, a product-related substance is a deoxyribonucleotide monomer. In some embodiments, product-related materials are derivatives or fragments of polyribonucleotides described herein, e.g., one or more fragments of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acids, monoribonucleic acids, diribonucleic acids, or triribonucleic acids.

[0052] As used herein, "increasing fitness" or "promoting fitness" of a subject refers to any favorable alteration in the physiology or any activity carried out by a subject organism as a result of administration of a peptide or polypeptide described herein, including, but not limited to, any one or more of the following desired effects: (1) increased resistance to biotic or abiotic stress; (2) increased yield or biomass; (3) altered flowering time; (4) increased resistance to pests or pathogens, (5) increased resistance to herbicides; (6) increased population of a subject organism (e.g., an agriculturally important insect); (7) increased reproductive rate of a subject organism (e.g., an insect, e.g., a honeybee or silkworm); (8) increased motility of a subject organism (e.g., an insect, e.g., a honeybee or silkworm); (9) increased activity of a subject organism (e.g., an insect, e.g., a honeybee or silkworm); (10) increased activity of a subject organism (e.g., an insect, e.g., a honeybee or silkworm); (9) an increase in the body weight of a target organism (e.g., an insect, e.g., a honeybee or a silkworm); (10) an increase in pollination (e.g., the number of plants pollinated in a given period) by a target organism (e.g., an insect, e.g., a honeybee or a silkworm); (11) an increase in the production of by-products (e.g., honey from a honeybee or silkworm) by a target organism (e.g., an insect, e.g., a honeybee or a silkworm); (12) an increase in the nutrient content (e.g., protein, fatty acid, or amino acid) of a target organism (e.g., an insect); or (13) an increase in the resistance of a target organism to a pesticide (e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphate insecticide (e.g., a phosphorothioate, e.g., fenitrothion)), (14) an increase in the health or reduction of disease of a target organism, such as a human or non-human animal. The increase in host fitness can be determined in comparison to a target organism to which the modulator is not administered.Conversely, a "reduced fitness" of a subject refers to any undesirable alteration in the physiology or of any activity carried out by the subject organism as a result of administration of a peptide or polypeptide described herein, including, but not limited to, any one or more of the following intended effects: (1) reduced resistance to biotic or abiotic stress; (2) reduced yield or biomass; (3) altered flowering time; (4) reduced resistance to pests or pathogens; (5) reduced resistance to herbicides; (6) reduced population of the subject organism (e.g., an agriculturally important insect); (7) reduced reproductive rate of the subject organism (e.g., an insect, e.g., a honeybee or silkworm); (8) reduced motility of the subject organism (e.g., an insect, e.g., a honeybee or silkworm); (9) reduced body weight of the subject organism (e.g., an insect, e.g., a honeybee or silkworm). (9) a decrease in metabolic rate or activity of a target organism (e.g., an insect, such as a honeybee or silkworm); (10) a decrease in pollination (e.g., the number of plants pollinated in a given period) by a target organism (e.g., an insect, such as a honeybee or silkworm); (11) a decrease in the production of by-products (e.g., honey from honeybees or silkworms) by a target organism (e.g., an insect, such as a honeybee or silkworm); (12) a decrease in the nutrient content (e.g., protein, fatty acid, or amino acid) of a target organism (e.g., an insect); or (13) a decrease in the resistance of a target organism to a pesticide (e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphate insecticide (e.g., a phosphorothioate, such as fenitrothion)), (14) a decrease in the health or a decrease in disease of a target organism, such as a human or non-human animal. The decrease in host fitness can be determined in comparison to a target organism to which the modulator is not administered. It will be apparent to one of skill in the art that certain changes in a subject's physiology, phenotype, or activity, such as altering the flowering time in a plant, can be considered to increase the fitness of the subject or decrease the fitness of the subject, depending on the situation (e.g., to adapt to changes in climate or other environmental conditions).For example, a delay in flowering (e.g., about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% reduction in plants in a population that flower on a given calendar date) may be a beneficial adaptation to later or cooler spring seasons and therefore may be considered to increase the fitness of the plant; conversely, a similar delay in flowering in the context of earlier or warmer spring seasons may be considered to decrease the fitness of the plant.

[0053] As used herein, the terms "linear polyribonucleotide", "linear RNA", and "linear polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having a 5' and a 3' end. One or both of the 5' and 3' ends may be free ends or may be linked to another moiety. A linear polyribonucleotide may be a polyribonucleotide that has not been circularized (e.g., pre-circularized) and can be used as a starting material for circularization, for example, via split ligation or chemical, enzymatic, ribozyme or splicing catalyst circularization methods.

[0054] As used herein, the term "modified oligonucleotide" means an oligonucleotide containing nucleotides having at least one modification to the sugar, nucleobase, or internucleotide linkage.

[0055] As used herein, the term "modified ribonucleotide" means a ribonucleotide containing a nucleoside having at least one modification to the sugar, nucleobase, or internucleoside linkage.

[0056] As used herein, the term "naked delivery" refers to a formulation for delivery to cells without the aid of a carrier or covalent modification of moieties that aid in delivery to cells. Naked delivery formulations do not include transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation of cyclic polyribonucleotide is a formulation that includes cyclic polyribonucleotide without covalent modification and does not include a carrier.

[0057] As used herein, the terms "nicked RNA" or "nicked linear polyribonucleotide" or "nicked linear polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having 5' and 3' ends resulting from the nicking or degradation of a circular RNA. "Nicked circular RNA" refers to a circular RNA that has been nicked.

[0058] The term "optionally substituted X" as used herein is intended to be equivalent to "X, optionally substituted X" (e.g., "alkyl, optionally substituted alkyl"). It is not intended to imply that the feature "X" (e.g., alkyl) is optional in nature. The term "optionally substituted" as used herein refers to having 0, 1, or more substituents (e.g., 0-25, 0-20, 0-10, or 0-5 substituents). For example, a C1 alkyl group, i.e., methyl, can be substituted with oxo to form a formyl group, and further substituted with -OH or -NH2 to form a carboxyl group or an amide group.

[0059] The term "pharmaceutical composition" is also intended to disclose that the cyclic or linear polyribonucleotide contained within the pharmaceutical composition can be used for the treatment of the human or animal body by therapy.

[0060] As used herein, the term "polynucleotide" refers to a molecule that includes one or more nucleic acid subunits or nucleotides, and can be used interchangeably with "nucleic acid" or "oligonucleotide." A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. A nucleotide can include a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. A polyribonucleotide, ribonucleic acid, or RNA can refer to a polymer that includes multiple ribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0061] As used herein, the term "polyribonucleotide cargo" herein includes any sequence comprising at least one polyribonucleotide. In embodiments, the polyribonucleotide cargo comprises one or more expressed (or coding) sequences, each expressed (or coding) sequence encoding a polypeptide. In embodiments, the polyribonucleotide cargo comprises one or more non-coding sequences, such as polyribonucleotides having regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo comprises a combination of expressed and non-coding sequences. In embodiments, the polyribonucleotide cargo comprises one or more polyribonucleotide sequences as described herein, for example, one or more regulatory elements, internal ribosome entry site (IRES) elements, or spacer sequences.

[0062] As used interchangeably herein, the terms "polyA" and "polyA sequence" refer to an untranslated flanking region of a nucleic acid molecule that is at least 5 nucleotides in length and consists of adenosine residues. In some embodiments, the polyA sequence is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. In some embodiments, the polyA sequence is located 3' to (e.g., downstream of) an open reading frame (e.g., an open reading frame encoding a polypeptide), and the polyA sequence is present 3' to a termination sequence (e.g., a stop codon) such that the polyA is not translated. In some embodiments, the polyA sequence is located 3' to the termination sequence and the 3' untranslated region.

[0063] As used herein, nucleic acid elements are "operably connected" or "operably linked" when they are placed in a vector such that they can be transcribed to form a linear polyribonucleotide and then circularized to a circular polyribonucleotide using the methods provided herein.

[0064] Polydeoxyribonucleotide, deoxyribonucleic acid, and DNA refer to a polymer comprising multiple deoxyribonucleotides polymerized via phosphodiester bonds. Nucleotides can be nucleoside monophosphates or nucleoside polyphosphates. Nucleotides refer to deoxyribonucleoside polyphosphates, such as deoxyribonucleoside triphosphates (dNTPs), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTPs, including detectable tags (e.g., fluorophores), such as luminescent tags and markers. Nucleotides can include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T, or U, or any other subunits that are specific to one or more complementary A, C, G, T, or U, or that are complementary to purines (i.e., A or G, or variants thereof) or pyrimidines (i.e., C, T, or U, or variants thereof). In some instances, the polynucleotide is a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a derivative or variant thereof. In some instances, the polynucleotide is a small interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a small hairpin RNA (shRNA), a small nuclear RNA (snRNA), a messenger RNA (mRNA), a pre-mRNA (pre-mRNA), an antisense RNA (asRNA), to name a few, and includes both nucleotide sequences and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In some instances, the polynucleotide molecule is circular. The polynucleotide may have a variety of lengths. The nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. Polynucleotides can be isolated from cells or tissues.Polynucleotide sequence embodiments can include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0065] Polynucleotide embodiments, such as polyribonucleotides or polydeoxyribonucleotides, include polynucleotides that contain one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs, or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylketone, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, Examples of nucleotides include 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), butoxocine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. In some cases, the nucleotides include modifications of their phosphate moieties, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications of the thiol moiety (e.g., α-thiotriphosphate and β-thiotriphosphate).In embodiments, the nucleic acid molecules are also modified at the base moiety, sugar moiety, or phosphate backbone (e.g., at one or more atoms typically available to form hydrogen bonds with complementary nucleotides, or at one or more atoms typically not available to form hydrogen bonds with complementary nucleotides). In embodiments, the nucleic acid molecules include amine-modified groups such as aminoallyl-dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine-reactive moieties such as N-hydroxysuccinimide ester (NHS). Alternatives to standard DNA or RNA base pairs in the oligonucleotides of the present disclosure may provide high bit density per cubic mm, higher safety (resistance to accidental or deliberate synthesis of natural toxins), easier identification in photoprogrammed polymerases, or subsecondary structures. Such alternative base pairs compatible with natural and mutant polymerases for de novo or amplicon synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul, 8:612-4, incorporated herein by reference for all purposes.

[0066] As used herein, "polypeptide" refers to a polymer of amino acid residues (natural or non-natural) linked together, most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs thereof. Polypeptides can be single molecules or multi-molecular complexes such as dimers, trimers, or tetramers. Polypeptides can also include single chain or multi-chain polypeptides such as antibodies or insulin, which can be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0067] As used herein, the term "plant-modifying polypeptide" refers to a polypeptide that can modify a genetic property (e.g., increase gene expression, decrease gene expression, or otherwise modify the nucleotide sequence of DNA or RNA), an epigenetic property, or a biochemical or physiological property of a plant in a manner that results in a change in the physiology or phenotype of the plant, e.g., an increase or decrease in plant fitness.

[0068] As used herein, the terms "purify," "purifying," and "purification" refer to one or more steps or processes in which impurities (e.g., process-related impurities (e.g., enzymes), process-related substances (e.g., deoxyribonucleotide fragments, deoxyribonucleotide monomers)) or by-products (e.g., linear RNA) are removed from a sample containing a mixture of circular RNA and linear RNA, among other substances, to produce a composition containing an enriched population of circular RNA in which the levels of impurities (e.g., process-related impurities (e.g., enzymes), process-related substances (e.g., deoxyribonucleotide fragments, deoxyribonucleotide monomers)) or by-products (e.g., linear RNA) are reduced compared to the original mixture, or the linear RNA or substances are reduced by 40% or more (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% or more) on a mass basis compared to the starting mixture.

[0069] As used herein, the terms "pure" and "purity" refer to the degree to which an analyte (e.g., circular RNA) is isolated and free from other components. In the context of nucleic acids (e.g., polyribonucleotides), the purity of an isolated nucleic acid (e.g., circular RNA) can be expressed in terms of a population of nucleic acids that is free of any contaminants, impurities, or by-products (e.g., linear RNA and other substances). For example, the purity of a population of circular RNA indicates how much circular RNA is present in the population per total mass of isolated material, and can be determined, for example, using pure circular RNA as a reference. The purity levels found in the present disclosure can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, more than 95%, or more than 99% (w / w). In some embodiments, the level of contaminants or impurities or by-products is about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (w / w) or less. Purity can be determined by detecting the level of a particular analyte (e.g., circular RNA) or a particular impurity or by-product (e.g., linear RNA) using gel electrophoresis, spectrophotometry (e.g., NanoDrop by ThermoFisher Scientific), or other techniques suitable for measuring the purity of a population of nucleic acids, and calculating the percentage (w / w) of the analyte relative to the total nucleic acid content (e.g., as determined by assays known in the art).

[0070] As used herein, the phrase "substantially free of one or more impurities or by-products" refers to a characteristic of a sample that is free of one or more impurities or by-products (e.g., one or more impurities or by-products disclosed herein) or contains a minimal amount of one or more impurities or by-products, e.g., a sample that contains an enriched population of circular RNA. The minimal amount of one or more impurities or by-products may be 20% (w / w) or less (e.g., 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less, or less). In another example, a sample or enriched population of circular RNA is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount of less than 15% (w / w) (e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less, or less). In another example, a sample or enriched population of circular RNA is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount of less than 10% (w / w) (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less, or less). In another example, a sample or enriched population of circular RNA is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount of less than 5% (w / w) (e.g., 4%, 3%, 2%, 1% (w / w) or less or less). In yet another example, a sample or enriched population of circular RNA is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount of less than 1% (0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% (w / w) or less or less).

[0071] As used herein, a "regulatory element" is a portion of a nucleic acid sequence or the like that alters the expression of an expression sequence within a circular or linear polyribonucleotide.

[0072] As used herein, "spacer" refers to any adjacent nucleotide sequence (eg, of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.

[0073] As used herein, the term "sequence identity" is determined by alignment of two peptide sequences or two nucleotide sequences using a global or local alignment algorithm. Sequences are called "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity when optimally aligned (aligned by a program such as GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, the default parameters of GAP are used: gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-19). Sequence alignment and sequence identity percentage scores are determined using computer programs such as, for example, GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the program "Needle"). Alternatively or additionally, percent identity is determined by searching in a database, for example, using algorithms such as FASTA, BLAST, etc. Sequence identity refers to sequence identity over the entire length of the sequence.

[0074] As used herein, "structured" with respect to RNA refers to an RNA sequence that is predicted by RNAFold software or similar prediction tools to form structures (e.g., hairpin loops) with itself or with other sequences in the same RNA molecule.

[0075] As used herein, the term "subject" refers to an organism, such as an animal, a plant, or a microorganism. In embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In embodiments, the subject is a human. In embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a bison, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In embodiments, the subject is an avian, such as a member of an avian taxonomic group such as Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate, such as an arthropod (e.g., insects, arachnids, crustaceans), nematodes, annelids, worms, or mollusks. In embodiments, the subject is an invertebrate that is an invertebrate agricultural pest or a parasite on an invertebrate or vertebrate host. In embodiments, the subject is a plant, such as an angiosperm (which may be dicotyledonous or monocotyledonous) or gymnosperm (e.g., conifers, cycads, Gnetophytes, ginkgo), fern, horsetail, club moss, or bryophyte. In embodiments, the subject is a eukaryotic alga (unicellular or multicellular). In embodiments, the subject is an agriculturally or horticulturally important plant, such as row crops, fruit-bearing plants and trees, vegetables, trees, and ornamentals, e.g., ornamental flowers, shrubs, trees, ground covers, and turf.

[0076] As used herein, the terms "treat" and "treating" refer to prophylactic or therapeutic treatment of a disease or disorder (e.g., an infection, cancer, toxicity, or allergic reaction) of interest. The effect of treatment may include reversing, alleviating, reducing the severity of, curing, inhibiting progression of, reducing the likelihood of recurrence of, the disease or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e., not worsening) the condition of the disease or disorder, or preventing the spread of the disease or disorder compared to the condition or symptoms of the disease or disorder in the absence of therapeutic treatment. Embodiments include treating plants to control a disease or deleterious symptoms caused by or associated with an invertebrate pest or a microbial (e.g., bacterial, fungal, oomycete, or viral) pathogen. Embodiments include treating plants to increase the plant's natural defense or immune ability to withstand pest or pathogen pressure.

[0077] As used herein, a "termination element" is a portion, such as a nucleic acid sequence, that terminates translation of an expressed sequence in a circular or linear polyribonucleotide.

[0078] As used herein, "translation efficiency" refers to the rate or amount of protein or peptide production from a ribonucleotide transcript. In an 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, a cell-free translation system such as rabbit reticulocyte lysate, for example, in a given period of time.

[0079] As used herein, a "translation initiation sequence" is a nucleic acid sequence that initiates the translation of an expression sequence in a circular or linear polyribonucleotide.

[0080] As used herein, a "therapeutic polypeptide" refers to a polypeptide that provides some therapeutic benefit when administered to or expressed in a subject. In embodiments, a therapeutic polypeptide is used to treat or prevent a disease, disorder, or condition in a subject by administration of the therapeutic polypeptide to the subject or by expression of the therapeutic polypeptide in the subject. In other embodiments, a therapeutic polypeptide is expressed in a cell, and the cell provides a therapeutic benefit when administered to a subject.

[0081] As used herein, "vector" refers to a piece of DNA that is synthesized (e.g., using PCR) or taken from a virus, plasmid, or cell of a higher organism into which a foreign DNA piece can or has been inserted for cloning or expression purposes. In some embodiments, the vector can be stably maintained in the organism. The vector can include, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, or a multiple cloning site (MCS). The term includes linear DNA pieces (e.g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like. In one embodiment, the vectors provided herein include a multiple cloning site (MCS). In another embodiment, the vectors provided herein do not include a MCS.

[0082] As used herein, the term "yield" refers to the relative amount of an analyte (e.g., a population of cyclic polyribonucleotides) obtained after a purification step or process compared to the amount of the analyte in the starting material (e.g., a mixed population of polyribonucleotides, e.g., cyclic polyribonucleotides and linear polyribonucleotides, etc.) (w / w). The yield can be expressed as a percentage. In the context of the present disclosure, the amount of the analyte (e.g., cyclic polyribonucleotides) in the starting material and the analyte obtained after a purification step can be measured using an assay (e.g., gel electrophoresis or spectrophotometry). The methods of the present disclosure can be used to provide a yield of an enriched population of cyclic polyribonucleotides of about 20% (w / w) or more compared to the amount present in the starting material, e.g., a mixed population of polyribonucleotides. For example, the methods can be used to result in yields of purified circular polyribonucleotide of about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more. [Brief description of the drawings]

[0083] [Figure 1] Schematic diagram of the method described herein. On the left is a mixture of linear polyribonucleotides and cyclic polyribonucleotides, some of which contain target regions. An oligonucleotide conjugated to a first capture agent is added to the mixture of polyribonucleotides and hybridizes to the polyribonucleotides that contain target regions. A second capture agent conjugated to a particle binds to the first capture agent, thereby separating the polyribonucleotides that contain target regions from the polyribonucleotides that lack target regions. [Diagram 2] 1 is a gel showing linear by-products in an in vitro transcription (IVT) mixture when circular RNA is generated by self-splicing. The gel shows the desired circular RNA product, unspliced ​​linear RNA, partially spliced ​​linear RNA, nicked circular RNA, and a spliced ​​intron. [Diagram 3]FIG. 1 is a schematic showing the design of a method for removing linear RNA by-products from circular RNA generated by self-splicing. [Figure 4] Gel showing enrichment of circular RNA by capturing linear by-products via oligo-streptavidin interactions. The construct was designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), polyribonucleotide cargo, exon fragment 1 (E1), and the 5' half of the catalytic intron. The open reading frame (ORF) is a model protein: Gaussia luciferase (Gluc). [Diagram 5] Gel showing enrichment of circular RNA by capturing linear by-products via oligo-streptavidin interactions. The construct was designed to contain, from 5' to 3': the 3' half of the catalytic intron, exon fragment 2 (E2), polyribonucleotide cargo, exon fragment 1 (E1), and the 5' half of the catalytic intron. The ORF is firefly luciferase (Fluc). [Figure 6] 13 is a gel showing enrichment of circular RNA by capturing linear by-products via oligo-streptavidin interactions. [Figure 7] 1 is a gel showing two different approaches to enrich for circular RNA by capturing linear by-products via oligo-streptavidin interactions: one method is to incubate the RNA-oligo mixture with streptavidin-SEPHAROSE® beads in a tube (batch method), and the other is to pre-pack the beads in a column and allow the RNA-oligomer mixture to pass through by gravity (column method). [Figure 8] 13 is a gel showing further enrichment of circular RNA by successive linear RNA pull-downs. [Figure 9]9A and 9B are gels showing the effect of salt concentration in the binding buffer on circular RNA enrichment by capturing linear by-products via oligo-streptavidin interactions. FIG. 9A shows a construct designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. The ORF is hEPO. FIG. 9B shows a similar construct to FIG. 9A, but the ORF is the SARS-CoV-2 spike protein (spike). [Figure 10] Figure 1 is a gel showing enhanced expression from circular RNA that has been purified by the linear RNA pull-down method to enrich for circular RNA by capturing linear by-products via oligo-streptavidin interactions. The construct was designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. The ORF is a model protein: Gaussia luciferase (Gluc). [Figure 11] 1 is a gel showing enhanced expression from circular RNA that has been purified by the linear RNA pull-down method to enrich for circular RNA by capturing linear by-products via oligo-streptavidin interactions. The construct was designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. The ORF is hEPO. [Figure 12] FIG. 1 is a schematic showing representative oligomer designs for the 3′ half-introns (#1-#4) and 5′ half-introns (#5 and #6) used in the linear pull-down method described herein. [Figure 13] 13 is a gel showing the effect of using a reduced number of oligomers to capture linear by-products via oligo-streptavidin interactions on circular RNA enrichment by linear RNA pull-down. [Figure 14]13 is a gel showing the effect of using a combination of two oligos (i.e., an oligomer against the 3' half-intron and an oligomer against the 5' half-intron) to capture the linear by-products via oligo-streptavidin interactions on circular RNA enrichment by linear RNA pull-down. [Figure 15] FIG. 1 is a bar graph showing expression from circular RNA that has been purified by a linear RNA pull-down method to enrich for circular RNA by capturing linear by-products via oligo-streptavidin interactions. [Figure 16] FIG. 1 is a schematic diagram showing a representative single oligomer targeting both the 3′ and 5′ half-introns. [Figure 17] FIG. 13 is a gel showing the effect of using a single oligomer targeting both the 3′ half-intron and the 5′ half-intron to capture linear by-products via oligo-streptavidin interactions on circular RNA enrichment by linear RNA pull-down. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] The present disclosure describes compositions and methods for processing, e.g., purifying, ribonucleotides. Polyribonucleotides, such as linear or cyclic polyribonucleotides, can be used for a variety of modification or therapeutic purposes. However, when polyribonucleotides are produced through certain biological reactions, various impurities, by-products, or incomplete products may be observed. The present invention features methods useful for reducing or removing these impurities, by-products, or incomplete products from a sample to produce a composition having a desired polyribonucleotide composition, amount, and / or purity, or a population containing a plurality of polyribonucleotides having a desired polyribonucleotide composition, amount, and / or purity.

[0085] In certain embodiments, the method is useful for purifying polyribonucleotides undergoing a splicing reaction. In such embodiments, the method can be used to separate spliced ​​polyribonucleotides from unspliced ​​polyribonucleotides, or unspliced ​​polyribonucleotides from spliced ​​polyribonucleotides. In some embodiments, the method can be used to separate circular polyribonucleotides (e.g., spliced) from linear polyribonucleotides, or linear polyribonucleotides from circular polyribonucleotides. Such purified compositions containing desired polyribonucleotides can be useful for various downstream applications, such as delivery of polynucleotide cargo (e.g., encoding genes or proteins) to target cells. The compositions and methods are described in more detail below.

[0086] method In some embodiments, the methods described herein include separating a polyribonucleotide having a target region from a plurality of polyribonucleotides. The method includes providing a sample comprising a plurality of polyribonucleotides. A subset of the plurality of polyribonucleotides has the target region. The method further includes contacting the sample with an oligonucleotide that hybridizes to the target region, and separating the polyribonucleotide having the target region that is hybridized to the oligonucleotide from the plurality of polyribonucleotides in the sample (FIG. 1).

[0087] In some embodiments, the methods described herein include separating a linear polyribonucleotide having a target region from a plurality of polyribonucleotides, the plurality of polyribonucleotides including a mixture of linear polyribonucleotides and cyclic polyribonucleotides. In the method, a sample is provided that includes a plurality of polyribonucleotides. A subset of the plurality of linear polyribonucleotides in the sample has the target region. The method further includes contacting the sample with an oligonucleotide that hybridizes to the target region, and separating the linear polyribonucleotide having the target region hybridized to the oligonucleotide from the plurality of polyribonucleotides in the sample. In some embodiments, the polyribonucleotide having the target region lacks a polyA sequence (e.g., a polyA tail). In some embodiments, the target region is not located at the 3' or 5' end of the polyribonucleotide having the target region. The cyclic polyribonucleotide or a subset thereof may lack the target region.

[0088] In some embodiments, the methods described herein include separating a polyribonucleotide having a target region from a plurality of polyribonucleotides. The method includes providing a sample including a plurality of polyribonucleotides, a subset of the plurality of polyribonucleotides having the target region. The target region is not located at the 3' or 5' end of the polyribonucleotide having the target region (e.g., the target region is located internally on the polyribonucleotide). The method further includes contacting the sample with an oligonucleotide that hybridizes to the target region, and separating the polyribonucleotide having the target region that is hybridized to the oligonucleotide from the plurality of polyribonucleotides in the sample.

[0089] In some embodiments, the methods described herein include separating polyribonucleotides by differential binding affinity to target regions. Differential binding can be achieved when the target binding regions are present within a differential secondary component that regulates access to the target site for binding. For example, an oligonucleotide can bind to a target region of a polyribonucleotide in a first structural configuration with a first affinity and to a target region of a polyribonucleotide in a second structural configuration with a second affinity, e.g., different from the first affinity. In some embodiments, differential binding or secondary components can occur when a target region is present within a linear polyribonucleotide or a cyclic polyribonucleotide, or in a polyribonucleotide that exists in two or more different structural configurations. For example, an oligonucleotide can bind to a target region of a linear polyribonucleotide with a first binding affinity and to a target region of a cyclic polyribonucleotide with a second binding affinity. The first binding affinity can be greater than the second binding affinity. Alternatively, the first binding affinity can be less than the second binding affinity. A lower binding affinity allows an oligonucleotide to bind to a target, for example, preferentially, rather than with greater binding affinity, to a target in a certain configuration.

[0090] In some embodiments, the method described herein comprises separating polyribonucleotides in a first arrangement having a target region from a plurality of polyribonucleotides in a second arrangement having a target region. The method comprises preparing a sample comprising a plurality of polyribonucleotides. The method further comprises contacting the sample with an oligonucleotide. The oligonucleotide hybridizes to the target region of the polynucleotides in the first arrangement with a first binding affinity, and the oligonucleotide hybridizes to the target region of the polynucleotides in the second arrangement with a second binding affinity different from the first binding affinity, and separating the polyribonucleotides in the first arrangement having the target region hybridized to the oligonucleotide from the plurality of polyribonucleotides in the second arrangement in the sample. In some embodiments, the first binding affinity is less than the second binding affinity, and the oligonucleotide preferentially binds to the polyribonucleotides in the first arrangement.

[0091] In some embodiments, the method includes contacting a polyribonucleotide with an oligonucleotide that is conjugated to a particle (eg, in the presence or absence of a capture agent).

[0092] In some embodiments, the method described herein comprises separating a linear polyribonucleotide having a target region from a plurality of circular polyribonucleotides having a target region. The method comprises preparing a sample comprising a plurality of polyribonucleotides. The method further comprises contacting the sample with an oligonucleotide. The oligonucleotide hybridizes to the target region of the linear polynucleotide with a first binding affinity, and the oligonucleotide hybridizes to the target region of the circular polynucleotide with a second binding affinity different from the first binding affinity, and separating the linear polyribonucleotide having a target region hybridized to the oligonucleotide from the plurality of circular polyribonucleotides in the sample. In some embodiments, the method comprises contacting the polyribonucleotide with an oligonucleotide conjugated to a particle (e.g., in the presence or absence of a capture agent).

[0093] In some embodiments of the methods described herein, the oligonucleotide is conjugated to a first capture agent. In some embodiments, the method further comprises preparing a second capture agent that binds to the first capture agent. The second capture agent can be conjugated to a particle. In some embodiments, the first capture agent is conjugated to a particle.

[0094] In some embodiments, the oligonucleotide is conjugated to a particle.

[0095] In some embodiments, the method described herein includes separating a polyribonucleotide having a target region from a plurality of polyribonucleotides. The method includes preparing a sample including a plurality of polyribonucleotides and an oligonucleotide conjugated to a first capture agent. A subset of the plurality of polyribonucleotides has a target region, and the oligonucleotide hybridizes to the target region. The method further includes contacting the sample with a second capture agent that binds to the first capture agent, and separating the polyribonucleotide having the target region hybridized to the oligonucleotide from the plurality of polyribonucleotides in the sample.

[0096] In some embodiments, the method described herein includes separating a polyribonucleotide in a first confirmation having a target region from a plurality of polyribonucleotides having a target region in a second confirmation. The method includes preparing a sample including a plurality of polyribonucleotides and an oligonucleotide conjugated to a first capture agent. The oligonucleotide hybridizes to the target region of the polynucleotide in the first confirmation with a first binding affinity, and the oligonucleotide hybridizes to the target region of the polynucleotide in the second confirmation with a second binding affinity different from the first binding affinity. The method further includes contacting the sample with a second capture agent that binds to the first capture agent, and separating the polyribonucleotide in the first confirmation having a target region hybridized to the oligonucleotide from the plurality of polyribonucleotides in the second confirmation in the sample. In some embodiments, the first binding affinity is less than the second binding affinity, and the oligonucleotide preferentially binds to the polyribonucleotide in the first confirmation.

[0097] In some embodiments, the method described herein includes separating a linear polyribonucleotide having a target region from a plurality of circular polyribonucleotides that include the target region. The method includes preparing a sample including a plurality of polyribonucleotides and an oligonucleotide conjugated to a first capture agent. The oligonucleotide hybridizes to the target region of the linear polynucleotide with a first binding affinity, and the oligonucleotide hybridizes to the target region of the circular polynucleotide with a second binding affinity different from the first binding affinity. The method further includes contacting the sample with a second capture agent that binds to the first capture agent, and separating the linear polyribonucleotide having the target region hybridized to the oligonucleotide from the plurality of circular polyribonucleotides in the sample.

[0098] In some embodiments, the method described herein includes separating a polyribonucleotide having a target region from a plurality of polyribonucleotides, for example, using an oligonucleotide without a first or second capture agent. The oligonucleotide can be directly conjugated to a particle. The method includes preparing a sample including a plurality of polyribonucleotides and an oligonucleotide conjugated to a particle. A subset of the plurality of polyribonucleotides has a target region, and the oligonucleotide hybridizes to the target region.

[0099] In some embodiments of any of the methods described herein, the target region is not located at the 5' or 3' end of the polyribonucleotide that has the target region. For example, the target region may be located internally on the polyribonucleotide. In some embodiments, the target region is not located at the 3' end of the polyribonucleotide.

[0100] In some embodiments of any of the methods described herein, the target region is located at the 5' or 3' end of the polyribonucleotide and the target region does not contain a polyA sequence. In some embodiments, the target region is located at the 3' end of the polyribonucleotide and does not contain a polyA sequence.

[0101] In some embodiments of any of the methods described herein, the target region does not contain a polyA sequence (eg, a polyA tail).

[0102] In some embodiments of any of the methods described herein, the separating comprises immobilizing the oligonucleotide. The method may comprise, for example, immobilizing the oligonucleotide, the first capture agent, the second capture agent, the particle, or a combination thereof.

[0103] In some embodiments, the particles are magnetic particles. The method may include applying a force, such as a magnetic force, to the magnetic particles. The particles or beads may be, for example, cross-linked agarose, such as SEPHAROSE® beads. The method may include applying a force, such as a mechanical force, an optical force, a centrifugal force, or an acoustic force, to the beads or particles.

[0104] In some embodiments, the polyribonucleotide having a target region comprises an intron or a portion thereof (e.g., a half intron). The target region may comprise an intron or a portion thereof. In some embodiments, the intron or a portion thereof is a catalytic intron (e.g., a group I catalytic intron or a group II catalytic intron) or a portion thereof. In some embodiments, the intron or a portion thereof has a length of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some embodiments, the intron or a portion thereof has a length of 20-500, 20-400, 20-300, 20-200, 20-100, 50-500, 50-400, 50-300, 50-200, 100-500, 100-400, 100-300, or 100-200 nucleotides.

[0105] In some embodiments, the target region comprises a half intron (e.g., the 5' or 3' portion of a catalytic intron). In some embodiments, the target region comprises a 5' half-intron (e.g., a 5' half-intron corresponding to the 5' portion of a catalytic intron). In some embodiments, the target region comprises a 3' half-intron (e.g., a 3' half-intron corresponding to the 3' portion of a catalytic intron). In some embodiments, the target region comprises the 5' half of a group I catalytic intron. In some embodiments, the 5' half of the group I catalytic intron is derived from the 5' portion of the cyanobacterial Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, T4 phage td gene, or variants thereof. In some embodiments, the target region comprises the 3' half of a group I catalytic intron. In some embodiments, the 3' half of the Group I catalytic intron is selected from the 3' portion from the cyanobacterium Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, T4 phage td gene, or a mutant thereof.

[0106] As described herein, the method can be used to separate, e.g., splice, from unspliced ​​polyribonucleotides. In some embodiments, the method described herein includes separating spliced ​​polyribonucleotides from unspliced ​​or partially spliced ​​polyribonucleotides. In some embodiments, the spliced ​​polyribonucleotide is a circular polyribonucleotide. In some embodiments, the spliced ​​polyribonucleotide is a linear polyribonucleotide. In some embodiments, the spliced ​​polyribonucleotide lacks an intron or a portion thereof, e.g., after a splicing (e.g., self-splicing) event during production. In some embodiments, the polyribonucleotide having an intron or a portion thereof is a linear polyribonucleotide.

[0107] In some embodiments, the method further comprises washing the captured polyribonucleotide having the target region and / or the capture agent (e.g., the first and / or second capture agent) one or more times (e.g., two, three, four, five, or more). Washing may occur after the contacting step and / or after the separating step.

[0108] In some embodiments, the method further comprises performing a first elution step to release the captured polyribonucleotides and / or capture agents (e.g., the first and / or second capture agents) that comprise the target region from the polyribonucleotides having the target region. The first elution step may comprise adding a first buffer and / or heating the sample, for example, to at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., or higher.

[0109] In some embodiments, the method further comprises performing a second elution step. The second elution step may comprise adding a second buffer and / or heating the sample, for example, to at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., or higher. In some embodiments, the second buffer comprises a denaturing agent, for example, formamide or urea. The second buffer may comprise, for example, about 40% to about 60% formamide (e.g., about 40%, 45%, 50%, 55%, or 60% formamide).

[0110] In some embodiments, the method includes incubating the sample with the oligonucleotide and / or capture agent (e.g., the first and / or second capture agent) for at least 10 minutes (e.g., at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, or more).

[0111] In some embodiments, the method includes recovering a portion of the sample that is not bound by the capture agent (eg, the first and / or second capture agent) and / or the oligonucleotide.

[0112] In some embodiments, the method includes providing a plurality of oligonucleotides, each of which hybridizes to a different target region, each of which can be conjugated to a first capture agent or particle, such as a magnetic particle or bead, for example.

[0113] In some embodiments, the method includes providing oligonucleotides that hybridize to a plurality of target regions, hi some embodiments, the method includes providing oligonucleotides that hybridize to a 3' half intron and a 5' half intron.

[0114] In some embodiments, the methods include providing a first oligonucleotide that hybridizes to the 3' half-intron and a second oligonucleotide that hybridizes to the 5' half-intron, hi some embodiments, the methods include providing a plurality of oligonucleotides, each of which hybridizes to a different region on the 3' half-intron and / or the 5' half-intron.

[0115] In some embodiments, the method includes providing the oligonucleotide at a molar ratio of 10:1 to 1:10 (e.g., 10:1, 5:1, 2:1, 1:2, 1:5, or 1:10) to the polyribonucleotide.

[0116] In some embodiments, the method includes preparing a sample of particles, e.g., beads, e.g., magnetic beads. The particles can be in a container, e.g., a microcentrifuge tube, or can be packed in a column. The particles can be conjugated to an oligonucleotide. The method can include flowing a mixture of polyribonucleotides (e.g., with an oligonucleotide conjugated to a first capture agent) through a column containing the particles. As such, the polyribonucleotides bound by the oligonucleotides will bind to the column. In some embodiments, the particles are conjugated to a second capture agent, e.g., designed to bind to the first capture agent conjugated to the oligonucleotide. In other embodiments, the particles are conjugated directly to an oligonucleotide, e.g., designed to hybridize to a target region of the polyribonucleotide.

[0117] In some embodiments, for example when using magnetic particles, the method may include pelleting the magnetic particles in a container (eg, a microcentrifuge tube), for example by providing a permanent magnet.

[0118] In some embodiments, the methods described herein enrich the amount of a desired polyribonucleotide in a sample. For example, the methods may enrich the amount of a desired (e.g., spliced) polyribonucleotide by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more) compared to the sample prior to purification.

[0119] In some embodiments, the method of purification results in cyclic polyribonucleotides having less than 50% (mol / mol) (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% (mol / mol)) linear polyribonucleotides.

[0120] Oligonucleotides The oligonucleotides described herein are designed to hybridize to a target region of a polyribonucleotide. In some embodiments, the oligonucleotide has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) complementarity with an equal length portion of the target region. In some embodiments, the oligonucleotide has at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatch to the target region of the polyribonucleotide. In some embodiments, the oligonucleotide has no mismatch to the target region. In some embodiments, the oligonucleotide may be a modified oligonucleotide (e.g., with modified phosphates, sugars, or bases). In some embodiments, the oligonucleotide contains a portion designed to hybridize to the target region and a portion that does not hybridize to the target region (e.g., terminal regions).

[0121] The oligonucleotides may be, for example, at least 5 nucleotides (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides) in length. In some embodiments, the oligonucleotides are, for example, 5-100, 5-95, 10-90, 10-80, 12-60, 15-50, 15-40, 15-30, 18-30, 20-25, or 20-22 nucleotides in length.

[0122] The oligonucleotide may have a GC content of, for example, 30 to 70%, for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. The oligonucleotide may have a melting temperature (Tm) of, for example, about 45°C to about 75°C, for example, about 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or 75°C.

[0123] Scavenger As described herein, the oligonucleotide and / or particle can be conjugated to a capture agent. The capture agent is designed to bind to, e.g., capture, a target molecule. The capture agent or set of capture agents, e.g., the first and / or second capture agent, can include any affinity pair, such as an antibody or its antigen-binding fragment and an antigen. Similarly, the affinity pair can include a receptor or its fragment and its cognate ligand. The first capture agent and the second capture agent can be any two molecules designed to interact with sufficient binding energy to allow covalent or non-covalent (e.g., ionic or van der Waals) interactions.

[0124] The oligonucleotides used in the compositions or methods described herein can be conjugated to a first capture agent. In some embodiments, the first capture agent comprises an antigen. The antigen can be, for example, biotin.

[0125] As described herein, a capture agent or oligonucleotide (e.g., an oligonucleotide conjugated to a capture agent) can be bound by or designed to be bound by a second capture agent. In some embodiments, the second capture agent comprises, for example, an antibody or an antigen-binding fragment thereof designed to bind to an antigen. The second capture agent can be, for example, streptavidin. In some embodiments, the oligonucleotide is not conjugated to or does not comprise a first capture agent, and the second capture agent binds directly to the oligonucleotide.

[0126] In some embodiments, the first capture agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the second capture agent is an antigen.

[0127] particle The oligonucleotides and / or capture agents described herein can be conjugated to particles, such as magnetic particles or beads. In some embodiments, the oligonucleotides and / or capture agents are conjugated to multiple particles. In some embodiments, a particle is conjugated to multiple capture agents and / or oligonucleotides.

[0128] The magnetic particles include at least one component that is responsive to magnetic forces. The magnetic particles may be entirely magnetic or may contain components that are non-magnetic. The magnetic particles may be magnetic beads, e.g., substantially spherical magnetic beads. The magnetic particles may be entirely magnetic or may contain one or more magnetic cores surrounded by one or more additional materials, e.g., one or more functional groups and / or modifications for binding to one or more target molecules. In some examples, the magnetic particles may contain a magnetic component and a surface modified with one or more silanol groups. This type of magnetic particle may be used to bind to target nucleic acid molecules.

[0129] The particles, e.g., magnetic particles or beads, may be porous, non-porous, hollow, solid, semi-solid, semi-fluid, fluid, and / or combinations thereof. In some cases, the particles, e.g., beads, may be dissolvable or degradable. In some cases, the particles, e.g., beads, may not be degradable. In some embodiments, the beads are composed of cross-linked agarose, e.g., SEPHAROSE®.

[0130] The particles, e.g., magnetic particles or beads, may comprise natural and / or synthetic materials. For example, the particles, e.g., beads, may comprise natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include proteins and sugars, e.g., deoxyribonucleic acid, gums, cellulose, starches (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac, karaya gum, xanthan gum, corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginates, or natural polymers thereof. Examples of synthetic polymers include acrylic, nylon, silicone, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride), and / or combinations (e.g., copolymers) thereof. Beads may also be formed from materials other than polymers, including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and others.

[0131] The crosslinks may be permanent or reversible, depending on the particular crosslinker used. Reversible crosslinks may allow the polymer to linearize or dissociate under appropriate conditions. In some cases, reversible crosslinks may also allow for reversible binding of materials bound to the surface of the beads.

[0132] The particles, e.g., beads or magnetic particles, may be of uniform or non-uniform size. In some cases, the diameter of the particles, e.g., beads, may be at least about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or more. In some cases, the particles, e.g., beads, may have a diameter of less than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or less. In some cases, the particles, e.g., beads, may have a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm, 500 μm-1 mm, 1 mm-2 mm, 1-5 mm, or 1-10 mm.

[0133] The particles may be of any suitable shape. Examples of particle, e.g., magnetic particle or bead, shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, toroidal, cylindrical, and variations thereof.

[0134] Linker In some embodiments, a linker is used to conjugate two or more components used in the compositions or methods described herein. For example, a linker can be used to conjugate an oligonucleotide to a capture agent, a capture agent to a particle (e.g., a bead), an oligonucleotide to a particle (e.g., a bead), or any combination or variation thereof. In some embodiments, an oligonucleotide is conjugated to a first capture agent by a chemical linker. The chemical linker can be conjugated to the 3'-end or 5'-end of the oligonucleotide. Alternatively, the chemical linker can be conjugated to an internal region of the oligonucleotide.

[0135] In some embodiments, the second capture agent is conjugated to a particle. The particle may be, for example, a magnetic particle or a bead. The bead may be, for example, a cross-linked agarose, such as a SEPHAROSE® bead. In some embodiments, the oligonucleotide is directly conjugated to a particle (e.g., a bead, such as a magnetic bead or a cross-linked agarose, such as a SEPHAROSE® bead).

[0136] A chemical linker provides space, rigidity, and / or flexibility, for example, between an oligonucleotide and a capture agent (e.g., a first capture agent) and / or a capture agent (e.g., a second capture agent) and a particle. In some embodiments, the linker can be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a CO bond, a CN bond, an NN bond, a CS bond, or any type of bond resulting from a chemical reaction, e.g., chemical conjugation. In some embodiments, the linker is 250 or less atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1- 190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atoms; 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom).In some embodiments, the linker has 250 or fewer non-hydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-19 0, 1 to 200, 1 to 210, 1 to 220, 1 to 230, 1 to 240, or 1 to 250 non-hydrogen atoms; 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-hydrogen atom). In some embodiments, the backbone of the linker is 250 or less atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom). The "backbone" of a linker refers to the atoms in the linker that together form the shortest path from one portion of the conjugate to the other portion of the conjugate. The atoms in the backbone of the linker are directly involved in connecting one part of the conjugate to another part of the conjugate.For example, a hydrogen atom attached to a carbon in the backbone of a linker is not considered to be directly involved in linking one portion of the conjugate to another portion of the conjugate.

[0137] In some embodiments, the linker may include a synthetic group, for example, derived from a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). A chemical linker may include, for example, triethylene glycol (TEG). In some embodiments, the linker may include one or more amino acid residues. In some embodiments, the linker may be an amino acid sequence (e.g., 1-25 amino acids, 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, 1-2 amino acids, or 1 amino acid sequence). In some embodiments, the linker may include one or more optionally substituted C1-C 20 Alkylene, optionally substituted C1-C 20 Heteroalkylene (e.g., PEG units), optionally substituted C-C 20 Alkenylene (e.g., C alkenylene), optionally substituted C-C 20 Heteroalkenylene, optionally substituted C-C 20 Alkynylene, optionally substituted C-C 20 Heteroalkynylene, optionally substituted C-C 20 Cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C-C 20 Heterocycloalkylene, optionally substituted C4-C 20 Cycloalkenylene, optionally substituted C4-C 20 Heterocycloalkenylene, optionally substituted C-C 20 Cycloalkynylene, optionally substituted C-C 20 Heterocycloalkynylene, optionally substituted C-C 15 Arylene (e.g., C6 arylene), optionally substituted C3-C 15Heteroarylene (e.g., imidazole, pyridine), O, S, NRi (where Ri is H, optionally substituted C1-C 20 Alkyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C-C 20 Alkenyl, optionally substituted C-C 20 Heteroalkenyl, optionally substituted C-C 20 Alkynyl, optionally substituted C-C 20 Heteroalkynyl, optionally substituted C-C 20 Cycloalkyl, optionally substituted C-C 20 Heterocycloalkyl, optionally substituted C4-C 20 Cycloalkenyl, optionally substituted C4-C 20 Heterocycloalkenyl, optionally substituted C-C 20 Cycloalkynyl, optionally substituted C-C 20 Heterocycloalkynyl, optionally substituted C5-C 15 Aryl or optionally substituted C-C 15 Heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.

[0138] Covalent conjugation of two or more components in a conjugate using a linker can be achieved using well-known organic chemical synthesis techniques and methods. Complementary functional groups on two components can react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine. Site-specific conjugation to a polypeptide can be achieved using techniques known in the art.

[0139] composition As described herein, the invention features compositions comprising a population of polyribonucleotides produced by the methods described herein. The population may, for example, comprise cyclic polyribonucleotides lacking a target region, where the cyclic polyribonucleotides comprise at least 1% (e.g., at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) (mol / mol) of the total polyribonucleotides in the composition. In some embodiments, the population has less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) (mol / mol) of linear polyribonucleotides in the composition.

[0140] In other embodiments, the population may comprise, for example, polyribonucleotides in a first configuration having a target region and polyribonucleotides in a second configuration having a target region, where the polyribonucleotides in the first configuration comprise at least 1%, e.g., at least 5%, e.g., at least 10%, at least 20%, at least 30%, or at least 40% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) (mol / mol) of the total polyribonucleotides in the composition.

[0141] In some embodiments described herein, the invention features a composition comprising a mixture of polyribonucleotides. A first subset of the mixture comprises cyclic polyribonucleotides lacking a target region, and a second subset of the polyribonucleotides comprises linear polyribonucleotides having a target region. The first subset comprises at least 1%, e.g., at least 5%, e.g., at least 10%, at least 20%, at least 30%, or at least 40% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) (mol / mol) of the total polyribonucleotides in the composition.

[0142] In some embodiments described herein, the invention features a composition comprising a polyribonucleotide having a target region and an oligonucleotide designed to hybridize to the target region, where the oligonucleotide is conjugated to a first capture agent (e.g., an antigen, e.g., biotin). The composition may further comprise, for example, a polyribonucleotide lacking a target region. The polyribonucleotide lacking a target region may be, for example, a cyclic polyribonucleotide. The composition may further comprise a second capture agent (e.g., an antibody or an antigen-binding fragment thereof, e.g., streptavidin) designed to bind to the first capture agent. The second capture agent may be conjugated to the particle, for example, via a linker.

[0143] In some embodiments of any of the compositions described herein, the linear polyribonucleotide comprises an intron or a portion thereof. The target region may comprise an intron or a portion thereof. The target region may be located 5' or 3' to the intron or a portion thereof.

[0144] In some embodiments, the polyribonucleotide may be a modified polyribonucleotide.

[0145] In certain embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) is at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), or 100% (w / w) pure by mass. Purity can be measured by any one of several analytical techniques known to those skilled in the art, including, for example and without limitation, the use of separation techniques such as chromatography (column-based, paper-based, gel-based, HPLC-based, UHPLC-based, etc., or by IC, SEC, reversed-phase, anion-exchange, mixed-mode, etc.) or electrophoresis (urea-PAGE, chip-based, polyacrylamide gel, RNA, capillary, c-IEF, etc.), with or without the use of detection techniques based on mass spectrometry, UV-visible light, fluorescence, light scattering, refractive index, or pre- or post-separation derivatization methods using silver or dye stains or radioactive decay for detection. Alternatively, purity can be measured without the use of separation techniques by mass spectrometry, by microscopy, by circular dichroism (CD) spectroscopy, by UV or UV-vis spectrophotometry, by fluorescence measurements (e.g., Qubit), by RNase H analysis, by surface plasmon resonance (SPR), or by methods using silver or dye stains or radioactive decay for detection.

[0146] In some embodiments, purity can be measured by biological testing methods (e.g., cell-based or receptor-based tests). In some embodiments, at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), or 100% (w / w) of the total ribonucleotide population in the preparations described herein is contained in circular polyribonucleotide molecules. The percentage can be measured by any one of several analytical techniques known to those skilled in the art, including, for example and without limitation, using separation techniques such as chromatography (column-based, paper-based, gel-based, HPLC-based, UHPLC-based, etc., or by IC, SEC, reversed-phase, anion-exchange, mixed-mode, etc.) or electrophoresis (urea-PAGE, chip-based, polyacrylamide gel, RNA, capillary, c-IEF, etc.), with or without the use of detection techniques based on mass spectrometry, UV-visible light, fluorescence, light scattering, refractive index, or pre- or post-separation derivatization methods using silver or dye stains or radioactive decay for detection. Alternatively, the purity can be measured without the use of separation techniques by mass spectrometry, by microscopy, by circular dichroism (CD) spectroscopy, by UV or UV-vis spectrophotometry, by fluorescence measurements (e.g., Qubit), by RNase H analysis, by surface plasmon resonance (SPR), or by methods using silver or dye stains or radioactive decay for detection.

[0147] In certain embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) has a concentration of at least 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL , 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL, 100,000 μg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / mL, 650 mg / mL, 700 mg / mL, or 750 mg / mL of cyclic polyribonucleotide. In some embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) is substantially free of mononucleotides or has a mononucleotide content of less than or equal to 1 pg / mL, 10 pg / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL, or 100,000 μg / mL.In some embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) has a mononucleotide content from a limit of detection of at most 1 pg / mL, 10 pg / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL, or 100,000 μg / mL.

[0148] In certain embodiments, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) comprises, by mass, 0.1% (w / w), 0.2% (w / w), 0.3% (w / w), 0.4% (w / w), 0.5% (w / w), 0.6% (w / w), 0.7% (w / w), 0.8% (w / w), 0.9% (w / w), 1% (w / w) of total nucleotides. ), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), or any percentage therebetween, where the total nucleotide content is the total mass of deoxyribonucleotide and ribonucleotide molecules.

[0149] In certain embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) is administered at any concentration of 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 25 μg / mL, 30 ...20 μg / mL, In some embodiments, the linear RNA content is less than or equal to 1 mg / mL, 50 μg / mL, 100 μg / mL, 200 g / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / mL, 650 mg / mL, 700 mg / mL, or 750 mg / mL of linear RNA, e.g., linear RNA counterparts or RNA fragments.In some embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) has a concentration of at most 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 1 μg / mL, 10 μg / mL, or more. In some embodiments, the linear RNA content, e.g., linear RNA counterparts or RNA fragments, from a detection limit of 100 μg / mL, 50 μg / mL, 100 μg / mL, 200 g / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / mL, 650 mg / mL, 700 mg / ml, or 750 mg / ml.

[0150] In certain embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) has a nicked RNA content of less than or equal to 10% (w / w), 9.9% (w / w), 9.8% (w / w), 9.7% (w / w), 9.6% (w / w), 9.5% (w / w), 9.4% (w / w), 9.3% (w / w), 9.2% (w / w), 9.1% (w / w), 9% (w / w), 8% (w / w), 7% (w / w), 6% (w / w), 5% (w / w), 4% (w / w), 3% (w / w), 2% (w / w), 1% (w / w), 0.5% (w / w), or 0.1% (w / w), or any percentage therebetween. In certain embodiments, a circular polyribonucleotide preparation (e.g., a circular polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a circular polyribonucleotide preparation) has a nicked RNA content as low as zero or is substantially free of nicked RNA.

[0151] In certain embodiments, a circular polyribonucleotide preparation (e.g., a circular polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a circular polyribonucleotide preparation) has a combined linear and nicked RNA content of less than or equal to 30% (w / w), 25% (w / w), 20% (w / w), 15% (w / w), 10% (w / w), 9% (w / w), 8% (w / w), 7% (w / w), 6% (w / w), 5% (w / w), 4% (w / w), 3% (w / w), 2% (w / w), 1% (w / w), 0.5% (w / w), or 0.1% (w / w), or any percentage therebetween. In certain embodiments, a circular polyribonucleotide preparation (e.g., a circular polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a circular polyribonucleotide preparation) has a combined content of nicked RNA and linear RNA as low as zero, or is substantially free of nicked and linear RNA.

[0152] In some embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) has linear RNA content, e.g., linear RNA counterparts or RNA fragments, below the detection limit of an analytical method such as a method using mass spectrometry, UV spectroscopy or fluorescence detectors, light scattering techniques, surface plasmon resonance (SPR), with or without the use of HPLC, including HPLC, chip or gel-based electrophoretic separation methods, derivatization methods either before or after separation, detection methods using silver or dye staining or radioactive decay, or with or without the use of microscopy, visual methods or spectrophotometers.

[0153] In certain embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) has no more than 0.1% (w / w), 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w) or less linear RNA, e.g., as measured by the method in Example 2.

[0154] In some embodiments, the linear polyribonucleotide molecules of the circular polyribonucleotide preparation comprise a linear counterpart of a circular polyribonucleotide molecule or a fragment thereof. In some embodiments, the linear polyribonucleotide molecules of the circular polyribonucleotide preparation comprise a linear counterpart (e.g., a pre-circularized version). In some embodiments, the linear polyribonucleotide molecules of the circular polyribonucleotide preparation comprise a non-counterpart to a circular polyribonucleotide or a fragment thereof. In some embodiments, the linear polyribonucleotide molecules of the circular polyribonucleotide preparation comprise a non-counterpart to a circular polyribonucleotide. In some embodiments, the linear polyribonucleotide molecules comprise a combination of a counterpart of a circular polyribonucleotide and a non-counterpart of a circular polyribonucleotide or a fragment thereof. In some embodiments, the linear polyribonucleotide molecules comprise a combination of a counterpart of a circular polyribonucleotide and a non-counterpart of a circular polyribonucleotide. In some embodiments, linear polyribonucleotide molecule fragments are fragments that are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, or more nucleotides in length, or any number of nucleotides in between.

[0155] In some embodiments, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) has an A260 / A280 absorbance ratio of about 1.6 to about 2.3, e.g., as measured by a spectrophotometer. In some embodiments, the A260 / A280 absorbance ratio is about 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any number therebetween. In some embodiments, the cyclic polyribonucleotide (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide) has an A260 / A280 absorbance ratio of greater than about 1.8, e.g., as measured by a spectrophotometer. In some embodiments, the A260 / A280 absorbance ratio is about 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or greater.

[0156] In some embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) is substantially free of impurities or by-products. In various embodiments, the level of at least one impurity or by-product in a composition comprising a cyclic polyribonucleotide is reduced by at least 30% (w / w), at least 40% (w / w), at least 50% (w / w), at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), at least 90% (w / w), or at least 95% (w / w) compared to the composition prior to purification or treatment to remove the impurity or by-product. In some embodiments, the level of at least one process-related impurity or by-product is reduced by at least 30% (w / w), at least 40% (w / w), at least 50% (w / w), at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), at least 90% (w / w), or at least 95% (w / w) when compared to the composition prior to purification or treatment to remove the impurity or by-product. In some embodiments, the level of at least one product-related substance is reduced by at least 30% (w / w), at least 40% (w / w), at least 50% (w / w), at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), at least 90% (w / w), or at least 95% (w / w) when compared to the composition prior to purification or treatment to remove the impurity or by-product. In some embodiments, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of the cyclic polyribonucleotide preparation) is further substantially free of process-related impurities or by-products.In some embodiments, the process-related impurities or by-products include proteins (e.g., cellular proteins such as host cell proteins), deoxyribonucleic acids (e.g., cellular deoxyribonucleic acids such as host cell deoxyribonucleic acids), monodeoxyribonucleotide or dideoxyribonucleotide molecules, enzymes (e.g., nucleases, e.g., endonucleases or exonucleases, or ligases), reagent components, gel components, or chromatographic materials. In some embodiments, the impurities or by-products are selected from buffer reagents, ligases, nucleases, ribonuclease inhibitors, ribonuclease R, deoxyribonucleotide molecules, acrylamide gel strips, and monodeoxyribonucleotide molecules. In some embodiments, the pharmaceutical preparation contains less than 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or 500 ng of protein contaminants, impurities, or by-products per milligram (mg) of circular polyribonucleotide molecule.

[0157] In some embodiments, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediates in the production of the cyclic polyribonucleotide preparation) is substantially free of DNA content, e.g., the template DNA or cellular DNA (e.g., host cell DNA) has a DNA content as low as 0, or as low as 1 pg / mL, 10 pg / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, , 15ng / mL, 20ng / mL, 25ng / mL, 30ng / mL, 35ng / mL, 40ng / mL, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 200ng / mL, 300ng / mL, 400ng / mL, 500ng / mL, 1000μg / mL, 5000μg / mL, 10,000μg / mL, or 100,000μg / mL.

[0158] In certain embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) is substantially free of DNA content, has a DNA content as low as 0, or has a DNA content of 0.001% (w / w), 0.01% (w / w), 0.1% (w / w), 1% (w / w), 2% (w / w), 3% (w / w), or less of total nucleotides by mass. and having a DNA content of 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w) or less, where total nucleotide molecules is the total mass of deoxyribonucleotide molecules and ribonucleotide molecules. In some embodiments, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) is substantially free of DNA content, as low as zero, as measured by quantitative liquid chromatography-mass spectrometry (LC-MS) following total DNA digestion with a nucleoside digesting enzyme (the DNA content is back-calculated from a calibration curve for each base (i.e., A, C, G, T) as measured by LC-MS). or has a DNA content of less than 0.001% (w / w), 0.01% (w / w), 0.1% (w / w), 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w) of total nucleotides on a mass basis.

[0159] In some embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) has less than 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / ml, or 500 ng / ml of protein (e.g., cellular proteins (CPs), e.g., enzymes, product-associated proteins, e.g., carrier proteins), contaminants, impurities, or by-products. In some embodiments, the cyclic polyribonucleotide (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide) has protein (e.g., cellular protein (CP), product-associated protein such as an enzyme), contaminant, impurity, or by-product from a detection limit of at most 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / ml, or 500 ng / ml.

[0160] In certain embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) has less than 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or 500 ng of protein (e.g., cellular protein (CP), product-associated protein such as an enzyme), contaminant, impurity, or by-product per milligram (mg) of cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide) has a detection level of at most 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or 500 ng of protein (e.g., cellular protein (CP), product-associated protein such as an enzyme), contaminant, impurity, or by-product per milligram (mg) of cyclic polyribonucleotide.

[0161] In certain embodiments, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) has low levels of endotoxins or is substantially free of endotoxins, for example, as measured by the Limulus Amebocyte Lysate (LAL) test. In some embodiments, the pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide contains less than 20 EU / kg (by weight), 10 EU / kg, 5 EU / kg, 1 EU / kg, or is devoid of endotoxins, as measured by the Limulus Amebocyte Lysate Test. In certain embodiments, the cyclic polyribonucleotide composition has low levels or is free of nucleases or ligases.

[0162] In some embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or an intermediate in the production of a cyclic polyribonucleotide preparation) is about 50% (w / w), 45% (w / w), 40% (w / w), 35% (w / w), 30% (w / w), 25% (w / w), 20% (w / w), 19% (w / w), 18% (w / w), 17% (w / w), 20% (w / w), 25 ... ), 16% (w / w), 15% (w / w), 14% (w / w), 13% (w / w), 12% (w / w), 11% (w / w), 10% (w / w), 9% (w / w), 8% (w / w), 7% (w / w), 6% (w / w), 5% (w / w), 4% (w / w), 3% (w / w), 2% (w / w), 1% (w / w) or less of at least one enzyme, e.g., a polymerase, e.g., an RNA polymerase.

[0163] In certain embodiments, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) is sterile or substantially free of microorganisms, e.g., the composition or preparation supports the growth of less than 100 viable microorganisms when tested under sterile conditions, the composition or preparation meets the USP <71> and / or the composition or preparation meets the criteria of U.S.P. <85> In some embodiments, the pharmaceutical preparation comprises a bioburden of less than 100 CFU / 100 ml, 50 CFU / 100 ml, 40 CFU / 100 ml, 30 CFU / 100 ml, 200 CFU / 100 ml, 10 CFU / 100 ml, or 10 CFU / 100 ml prior to sterilization.

[0164] In some embodiments, the circular polyribonucleotide preparation can be further purified to remove impurities or by-products using techniques known in the art, such as column chromatography or pH, vial inactivation, etc.

[0165] Polynucleotides The present invention features polyribonucleotides used in methods of separation and / or purification and present in compositions described herein. The polyribonucleotides described herein may be linear polyribonucleotides, cyclic polyribonucleotides, or combinations thereof. In some embodiments, cyclic polyribonucleotides are generated from linear polyribonucleotides (e.g., by splicing compatible ends of a linear polyribonucleotide). In some embodiments, linear polyribonucleotides are transcribed from a deoxyribonucleotide template (e.g., a vector, a linearized vector, or a cDNA). Thus, the present invention features linear deoxyribonucleotides, cyclic deoxyribonucleotides, linear polyribonucleotides, and cyclic polyribonucleotides and compositions thereof that are useful in the generation of polyribonucleotides.

[0166] Linear Polyribonucleotides The invention features linear polyribonucleotides that can include one or more of the following: a 3' half-intron; a 3' splice site; a 3' exon; a polyribonucleotide cargo; a 5' exon; a 5' splice site; and a 5' half-intron. In some embodiments, the 3' half-intron corresponds to the 3' portion of a catalytic group I intron, such as from the cyanobacteria Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, T4 phage td gene, or a mutant thereof. In some embodiments, the 5' half-intron corresponds to the 5' portion of a catalytic group I intron, such as from the cyanobacteria Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, T4 phage td gene, or a mutant thereof.

[0167] A linear polyribonucleotide may include additional elements, for example, outside or between any of the above elements. For example, any of the above elements may be separated by a spacer sequence, as described herein. A target region as described herein may be present within any region of a linear polyribonucleotide as described herein. In some embodiments, a target region is present within an intron or a portion thereof.

[0168] In certain embodiments, provided herein are methods of generating linear polyribonucleotides by performing transcription in a cell-free system (e.g., in vitro transcription) using a deoxyribonucleotide provided herein (e.g., a vector, a linearized vector, or a cDNA) as a template (e.g., a vector, a linearized vector, or a cDNA provided herein in which an RNA polymerase promoter is positioned upstream of a region encoding the linear polyribonucleotide).

[0169] A deoxyribonucleotide template can be transcribed to produce a linear polyribonucleotide containing the components described herein. Upon expression, the linear polyribonucleotide can produce a splicing-compatible polyribonucleotide that can be spliced ​​to produce a circular polyribonucleotide, for example, for later use.

[0170] In some embodiments, the linear polyribonucleotide is 50 to 20,000, e.g., 300 to 20,000 (e.g., 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1, A linear polyribonucleotide can be, for example, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, or at least 5,000 ribonucleotides in length.

[0171] Circular Polyribonucleotides In some embodiments, the invention features a circular polyribonucleotide. The circular polyribonucleotide may include a splice junction connecting a 5' exon and a 3' exon. The target region described herein may be present within any region of the circular polyribonucleotide described herein. The circular polyribonucleotide may lack an intron, for example, after splicing.

[0172] The circular polynucleotide may further comprise a polyribonucleotide cargo. The polyribonucleotide cargo may comprise an expressed sequence, a non-coding sequence, or a combination of expressed and non-coding sequences. The polyribonucleotide cargo may comprise an expressed sequence encoding a polypeptide. The polyribonucleotide may comprise an IRES operably linked to the expressed sequence encoding the polypeptide. In some embodiments, the circular polyribonucleotide further comprises a spacer region between the IRES and the 5' exon fragment or the 3' exon fragment. The spacer region may be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. The spacer region may be, for example, 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length. In some embodiments, the spacer region comprises a polyA sequence. In some embodiments, the spacer region comprises poly AC, poly AG, poly AU, or other heterogeneous or random sequences.

[0173] In some embodiments, a 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.

[0174] In some embodiments, the size of the circular polyribonucleotide can be sufficient to accommodate the binding site for ribosome.In some embodiments, the size of the circular polyribonucleotide can be sufficient to code useful polypeptides, and thus can generate lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, 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 1400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides.

[0175] In some embodiments, the circular polyribonucleotide comprises one or more elements as described herein. In some embodiments, the elements can be separated from each other by a spacer sequence. In some embodiments, the elements can be separated from each other 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 adjacent to each other, e.g., lacking a spacer element.

[0176] In some embodiments, the cyclic polyribonucleotide may include one or more repeat elements. In some embodiments, the cyclic polyribonucleotide includes one or more modifications described herein. In one embodiment, the cyclic polyribonucleotide includes at least one nucleoside modification. In one embodiment, up to 100% of the nucleosides of the cyclic polyribonucleotide are modified. In one embodiment, the at least one nucleoside modification is a uridine modification or an adenosine modification.

[0177] As a result of its circularization, a cyclic polyribonucleotide may contain certain features that distinguish it from linear polyribonucleotides. For example, a cyclic polyribonucleotide may contain more or less accessible target regions than a linear polyribonucleotide. In some embodiments, a cyclic polyribonucleotide may be less susceptible to degradation by exonucleases when compared to linear polyribonucleotides. As such, a cyclic polyribonucleotide may be more stable than a linear polyribonucleotide, especially when incubated in the presence of an exonuclease. The increased stability of a cyclic polyribonucleotide compared to a linear polyribonucleotide makes it more useful as a cell transformation reagent for producing polypeptides, and it can be stored more easily and for longer than a linear polyribonucleotide. The stability of an exonuclease-treated cyclic polyribonucleotide can be tested using methods standard in the art that determine whether RNA degradation has occurred (e.g., by gel electrophoresis). Furthermore, unlike linear polyribonucleotides, circular polyribonucleotides may be less susceptible to dephosphorylation when the circular polyribonucleotides are incubated with a phosphatase, such as calf intestinal phosphatase.

[0178] Polyribonucleotide Cargo The polyribonucleotide cargo described herein includes any sequence comprising at least one polyribonucleotide. In some embodiments, the polyribonucleotide cargo comprises an expressed sequence, a non-coding sequence, or an expressed sequence and a non-coding sequence. In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide. In some embodiments, the polyribonucleotide cargo comprises an IRES operably linked to an expressed sequence encoding a polypeptide. In some embodiments, the polyribonucleotide cargo comprises an expressed sequence encoding a polypeptide having a biological effect on a subject.

[0179] The polyribonucleotide cargo may comprise, for example, 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 polyribonucleotide cargo comprises from 1 to 20,000 nucleotides, from 1 to 10,000 nucleotides, from 1 to 5,000 nucleotides, from 100 to 20,000 nucleotides, from 100 to 10,000 nucleotides, from 100 to 5,000 nucleotides, from 500 to 20,000 nucleotides, from 500 to 10,000 nucleotides, from 500 to 5,000 nucleotides, from 1,000 to 20,000 nucleotides, from 1,000 to 10,000 nucleotides, or from 1,000 to 5,000 nucleotides.

[0180] In embodiments, the polyribonucleotide cargo comprises one or more expressed (or coding) sequences, where each expressed (or coding) sequence encodes a polypeptide. In embodiments, the polyribonucleotide cargo comprises one or more non-coding sequences. In embodiments, the polyribonucleotide cargo consists entirely of non-coding sequences. In embodiments, the polyribonucleotide cargo comprises a combination of expressed (or coding) sequences and non-coding sequences.

[0181] In some embodiments, the polyribonucleotides produced as described herein are used as effectors in therapy or agriculture.For example, the cyclic polyribonucleotides produced by the methods described herein (e.g., the cell-free methods described herein) can be administered to a subject (e.g., in pharmaceutical, veterinary, or agricultural compositions).In another example, the cyclic polyribonucleotides produced by the methods described herein (e.g., the cell-free methods described herein) can be delivered to a cell.

[0182] In some embodiments, the polyribonucleotide comprises any feature, or any combination of features, as disclosed in PCT Publication No. WO 2019 / 118919, the entirety of which is incorporated herein by reference.

[0183] In some embodiments, the polyribonucleotide cargo comprises an open reading frame. In some embodiments, the open reading frame is operably linked to an IRES. The open reading frame may encode an RNA or a polypeptide. In some embodiments, the open reading frame encodes a polypeptide and the polyribonucleotide (e.g., a cyclic polyribonucleotide) provides increased expression of the polypeptide (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more), e.g., when compared to a linear polyribonucleotide encoding the polypeptide. In some embodiments, increasing the purity of polyribonucleotides, e.g., circular polyribonucleotides, results in increased expression of polypeptides (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more), e.g., when compared to a population of circular and linear polyribonucleotides.

[0184] Polypeptide Expression Sequences In some embodiments, a polyribonucleotide described herein (e.g., the polyribonucleotide cargo of a circular polyribonucleotide) comprises one or more expressed (or coding) sequences, where each expressed sequence encodes a polypeptide. In some embodiments, a circular polyribonucleotide comprises two, three, four, five, six, seven, eight, nine, ten or more expressed (or coding) sequences.

[0185] Each encoded polypeptide may be linear or branched. In an embodiment, the polypeptide has 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 some embodiments, polypeptides have 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 that may be useful.

[0186] The polypeptides included herein may include naturally occurring or non-naturally occurring polypeptides. In some embodiments, the polypeptide is or includes a functional fragment or variant of a reference polypeptide (e.g., an enzymatically active fragment or variant of an enzyme). For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of a polypeptide described herein or a naturally occurring polypeptide, e.g., over a specific region or over the entire sequence. In some cases, a polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identity to a protein of interest.

[0187] Some examples of polypeptides include, but are not limited to, a fluorescent tag or marker, an antigen, a therapeutic polypeptide, or a polypeptide for agricultural use.

[0188] The therapeutic polypeptide may be a hormone, a neurotransmitter, a growth factor, an enzyme (e.g., oxidoreductases, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, lysosomal enzymes, desaturases), a cytokine, an antigen-binding polypeptide (e.g., an antigen-binding antibody or antibody-like fragment, such as a single chain antibody, nanobody or other polypeptide comprising an Ig heavy or light chain), an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an interferon, an interleukin, and a thrombolytic agent.

[0189] Polypeptides for agricultural use may be bacteriocins, lysins, antibacterial polypeptides, antifungal polypeptides, nodule C-rich peptides, bacteriocyte regulatory peptides, peptide toxins, pesticidal polypeptides (e.g., insecticidal or nematicidal polypeptides), antigen-binding polypeptides (e.g., antigen-binding antibodies or antibody-like fragments, such as single chain antibodies, nanobodies or other Ig heavy or light chain-containing polypeptides), enzymes (e.g., nucleases, amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones, and transcription factors.

[0190] In some cases, the polyribonucleotide expresses a non-human protein.

[0191] In some embodiments, the polyribonucleotide expresses an antibody, such as an antibody fragment, or a portion thereof. In some embodiments, the antibody expressed by the cyclic polyribonucleotide can be any isotype, such as IgA, IgD, IgE, IgG, IgM, etc. In some embodiments, the cyclic polyribonucleotide expresses a portion of an antibody, such as a light chain, a heavy chain, an Fc fragment, a CDR (complementarity determining region), an Fv fragment, or a Fab fragment, or further portions thereof. In some embodiments, the cyclic polyribonucleotide expresses one or more portions of an antibody. For example, the cyclic polyribonucleotide can include two or more expression sequences, each of which expresses a portion of an antibody, the sum of which can constitute an antibody. In some cases, the cyclic polyribonucleotide includes one expression sequence that encodes a heavy chain of the antibody, and another expression sequence that encodes a light chain of the antibody. In some cases, when the cyclic polyribonucleotide is expressed in a cell or in a cell-free environment, the light and heavy chains can undergo appropriate modification, folding, or other post-translational modifications to form a functional antibody.

[0192] In embodiments, the polypeptide comprises multiple polypeptides, e.g., multiple copies of a single polypeptide sequence, or multiple different polypeptide sequences, In embodiments, the multiple polypeptides are connected by linker or spacer amino acids.

[0193] In an embodiment, the polynucleotide cargo comprises a sequence encoding a signal peptide. Many signal peptide sequences have been described, for example, the Tat (twin arginine translocation) signal sequence is typically an N-terminal peptide sequence containing a consensus SRRxFLK "twin arginine" motif that serves to translocate folded proteins containing such Tat signal peptides through lipid bilayers. Also see, for example, the publicly available signal peptide database at www.signalpeptide.de. Signal peptides are also useful for directing proteins to specific organelles; see, for example, the experimentally measured and computationally predicted signal peptides disclosed in the publicly available Spdb signal peptide database at proline.bic.nus.edu.sg / spdb.

[0194] In embodiments, the polynucleotide cargo comprises a sequence encoding a cell penetrating peptide (CPP). Hundreds of CPP sequences have been described; see, for example, CPPsite, a database of publicly available cell penetrating peptides at crdd[dot]osdd[dot]net / raghava / cppsite / . An example of a commonly used CPP sequence is a poly-arginine sequence, such as octoarginine or nonoarginine, which can be fused to the C-terminus of a CGI peptide.

[0195] In embodiments, the polynucleotide cargo comprises a sequence encoding a self-assembling peptide; see, e.g., Miki et al. (2021) Nature Communications, 21:3412, DOI:10.1038 / s41467-021-23794-6.

[0196] In some embodiments, the expressed sequence includes a polyA sequence (e.g., at the 3' end of the expressed sequence). In some embodiments, the length of the polyA sequence is more than 10 nucleotides in length. In one embodiment, the polyA sequence is more than 15 nucleotides in length (e.g., is at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides in length). In some embodiments, the polyA sequence is designed according to the description of polyA sequences in paragraphs

[0202] to

[0204] of WO 2019 / 118919 A1, the entirety of which is incorporated herein by reference. In some embodiments, the expression sequence lacks a polyA sequence (e.g., at the 3' end of the expression sequence).

[0197] In some embodiments, the cyclic polyribonucleotide comprises a polyA, lacks a polyA, or has a modified polyA to modulate one or more characteristics of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide lacks a polyA or has a modified polyA to improve one or more functional properties, such as immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response), half-life, and / or expression efficiency.

[0198] Therapeutic Polypeptides In some embodiments, the polyribonucleotides described herein (e.g., the polyribonucleotide cargo of a cyclic polyribonucleotide) comprise at least one expression sequence that encodes a therapeutic polypeptide. A therapeutic polypeptide is a polypeptide that provides some therapeutic benefit when administered to or expressed in a subject. Administration of a therapeutic polypeptide to a subject or expression in a subject can be used to treat or prevent a disease, disorder, or condition or a symptom thereof. In some embodiments, a cyclic polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten, or more therapeutic polypeptides.

[0199] In some embodiments, the polyribonucleotide comprises an expression sequence that encodes a therapeutic protein. The protein can treat a disease in a subject in need thereof. In some embodiments, the therapeutic protein can compensate for a mutated, under-expressed, or absent protein in a subject in need thereof. In some embodiments, the therapeutic protein can target, interact with, or bind to a cell, tissue, or virus in a subject in need thereof.

[0200] A therapeutic polypeptide can be a polypeptide that can be secreted from the cell or that can be localized in the cytoplasm, nucleus, or membrane compartment of the cell.

[0201] Therapeutic polypeptides can be any of a variety of therapeutic polypeptides, including hormones, neurotransmitters, growth factors, enzymes (e.g., oxidoreductases, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, lysosomal enzymes, desaturases), cytokines, transcription factors, antigen-binding polypeptides (e.g., antigen-binding antibodies or antibody-like fragments, such as single chain antibodies, nanobodies, or other polypeptides comprising an Ig heavy or light chain), Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, interferons, interleukins, thrombolytic agents, antigens (e.g., tumor, viral, or bacterial antigens), nucleases (e.g., Cas proteins, endonucleases such as Cas9), membrane proteins (e.g., chimeric antigen receptors (CARs), transmembrane receptors, G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), antigen receptors, ion channels, or membrane transporters), secreted proteins, gene editing proteins (e.g., CRISPR-Cas, TALENs, or Zn fingers), or gene writing proteins. The present invention may also be directed to a method for the preparation of a medicament for the treatment of a cancer, such as the administration of a medicament for the treatment of a cancer.

[0202] In some embodiments, the therapeutic polypeptide is an antibody, e.g., a full-length antibody, an antibody fragment, or a portion thereof. In some embodiments, the antibody expressed by the polyribonucleotide (e.g., a cyclic polyribonucleotide) can be of any isotype, such as IgA, IgD, IgE, IgG, IgM, etc. In some embodiments, the polyribonucleotide expresses a portion of the expressed antibody, e.g., a light chain, a heavy chain, an Fc fragment, a CDR (complementarity determining region), an Fv fragment, or a Fab fragment, or further portions thereof. In some embodiments, the polyribonucleotide expresses one or more portions of an antibody. For example, the polyribonucleotide can include two or more expression sequences, each of which expresses a portion of an antibody, the sum of which can constitute an antibody. In some cases, the polyribonucleotide includes one expression sequence that encodes a heavy chain of the antibody, and another expression sequence that encodes a light chain of the antibody. When the polyribonucleotide is expressed subcellularly, the light and heavy chains can undergo appropriate modification, folding, or other post-translational modifications to form a functional antibody.

[0203] In some embodiments, polyribonucleotides (e.g., cyclic polyribonucleotides) made as described herein are used as effectors in therapy or agriculture. For example, polyribonucleotides made by the methods described herein can be administered to a subject (e.g., in a pharmaceutical, veterinary, or agricultural composition). In embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In embodiments, the subject is a human. In embodiments, the subject of the method is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In embodiments, the subject is an avian, such as a member of an avian taxonomic group such as Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate, such as an arthropod (e.g., insects, arachnids, crustaceans), nematodes, annelids, worms, or mollusks. In embodiments, the subject is an invertebrate that is an invertebrate agricultural pest or a parasite on an invertebrate or vertebrate host. In embodiments, the subject is a plant, such as an angiosperm (which may be dicotyledonous or monocotyledonous) or gymnosperm (e.g., conifers, cycads, Gnetophytes, ginkgo), fern, horsetail, club moss, or bryophyte. In embodiments, the subject is a eukaryotic alga (unicellular or multicellular). In embodiments, the subject is an agriculturally or horticulturally important plant, such as row crops, fruit-bearing plants and trees, vegetables, trees, and ornamentals, e.g., ornamental flowers, shrubs, trees, ground covers, and turf.

[0204] Plant Modified Polypeptides In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide cargo of a polyribonucleotide) described herein comprises at least one expressible sequence that encodes a plant modified polypeptide. A plant modified polypeptide refers to a polypeptide that can modify the genetic characteristics of a plant (e.g., increase gene expression, decrease gene expression, or otherwise modify the nucleotide sequence of DNA or RNA), modify the epigenetic characteristics, or modify the physiological or biochemical characteristics in a manner that results in a change in the plant's physiology or phenotype, such as an increase or decrease in the fitness of the plant. In some embodiments, a polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten, or more different plant modified polypeptides, or multiple copies of one or more plant modified polypeptides. A plant modified polypeptide can be one that can modify the physiology or phenotype of various plants, or increase or decrease the fitness of various plants, or that can effect such changes in one or more particular plants (e.g., a particular species or genus of plants).

[0205] Examples of polypeptides that can be used herein include enzymes (e.g., metabolic recombinases, helicases, integrases, ribonucleases, deoxyribonucleases, or ubiquitinating proteins), pore-forming proteins, signaling ligands, cell-penetrating peptides, transcription factors, receptors, antibodies, nanobodies, gene editing proteins (e.g., CRISPR-Cas endonucleases, TALENs, or Zn fingers), riboproteins, protein aptamers, or chaperones.

[0206] Agricultural polypeptide In some embodiments, the polyribonucleotides described herein (e.g., the polyribonucleotide cargo of the polyribonucleotide) comprise at least one expression sequence encoding an agricultural polypeptide. An agricultural polypeptide is a polypeptide suitable for agricultural use. In embodiments, the agricultural polypeptide is applied to a plant or seed (e.g., by foliar spray, dusting, injection, or seed coating) or to the plant's environment (e.g., by soil drench or granular soil application) resulting in an alteration of the plant's physiology, phenotype, or fitness. Embodiments of agricultural polypeptides include polypeptides that alter the level, activity, or metabolism of one or more microorganisms resident in or on a plant or non-human animal host, which alteration results in an increase in the fitness of the host. In some embodiments, the agricultural polypeptide is a plant polypeptide. In some embodiments, the agricultural polypeptide is an insect polypeptide. In some embodiments, the agricultural polypeptide has a biological effect when contacted with a non-human vertebrate, invertebrate, microorganism, or plant cell.

[0207] In some embodiments, the polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten or more agricultural polypeptides, or multiple copies of one or more agricultural polypeptides.

[0208] Embodiments of polypeptides useful for agricultural applications include, for example, bacteriocins, lysins, antimicrobial peptides, rhizocine C-rich peptides, and fungal cell regulatory peptides. Such polypeptides can be used to modify the levels, activity, or metabolism of target microorganisms to increase the fitness of insects such as honeybees and silkworms. Embodiments of agriculturally useful polypeptides include peptide toxins, such as those naturally produced by insect pathogens (e.g., Bacillus thuringiensis, Photorhabdus luminescens, Serratia entomophila, or Xenorhabdus nematophila), as known in the art. Embodiments of agriculturally useful polypeptides include polypeptides (including small peptides such as cyclodipeptides or diketopiperazines) for controlling agriculturally important pests or pathogens, e.g., antibacterial or antifungal polypeptides for controlling disease in plants, or pesticidal polypeptides (e.g., insecticidal or nematocidal polypeptides) for controlling invertebrate pests such as insects or nematodes. Embodiments of agriculturally useful polypeptides include antibodies, nanobodies, and fragments thereof, e.g., antibodies or nanobody fragments that retain at least a portion (e.g., at least 10%) of the specific binding activity of an intact antibody or nanobody. Embodiments of agriculturally useful polypeptides include transcription factors, e.g., plant transcription factors; see, e.g., the "AtTFDB" database, which lists transcription factor families identified in the model plant Arabidopsis thaliana, publicly available at agris-knowledgebase[dot]org / AtTFDB / . Embodiments of agriculturally useful polypeptides include nucleases, e.g., exonucleases or endonucleases (e.g., Cas nucleases, such as Cas9 or Cas12a).Embodiments of agriculturally useful polypeptides further include cell penetrating peptides, enzymes (e.g., amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones (e.g., yeast mating pheromones, invertebrate reproductive and larval signaling pheromones, see, e.g., Altstein (2004) Peptides, 25:1373-76).

[0209] Internal ribosome entry site (IRES) In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide cargo of a polyribonucleotide) described herein comprises one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences). In embodiments, the IRES is located between the heterologous promoter and the 5' end of the coding sequence.

[0210] IRES elements suitable for inclusion within a polyribonucleotide comprise an RNA sequence capable of associating with a eukaryotic ribosome, hi 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.

[0211] In some embodiments, the IRES element is derived from DNA of organisms including, but not limited to, viruses, mammals, and Drosophila. Such viral DNA can be derived from, but is not limited to, encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA, as well as picornavirus complementary DNA (cDNA). In one embodiment, the Drosophila DNA from which the IRES element is derived includes, but is not limited to, the antennapedia gene from Drosophila melanogaster.

[0212] In some embodiments, if present, the IRES sequence is selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invictavirus 1, Rhopalopsis aphid virus, Reticuloendotheliosis virus, Fuman poliovirus 1, German winged stink bug enteric virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Tea budworm picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila melanogaster C virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black queen brood virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus virus), classical swine fever virus, human FGF2, human SFTPAl, human AMLl / RUNXl, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, dog Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Salivirus, cosavirus, parechovirus, Drosophila hairless, yeast (S. cerevisiae) TFIID, yeast (S.cerevisiae YAP1, human c-src, human FGF-1, picomavirus, Turnip crinkle virus, an aptamer for eIF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 / 2) IRES sequence. In yet another embodiment, the IRES is a Coxsackievirus B3 (CVB3) IRES sequence. In a further embodiment, the IRES is an encephalomyocarditis virus IRES sequence.

[0213] In some embodiments, the polyribonucleotide comprises at least one IRES flanking at least one (e.g., two, three, four, five or more) expressed sequence. In some embodiments, an IRES flanks both sides of at least one (e.g., two, three, four, five or more) expressed sequence. In some embodiments, the polyribonucleotide comprises one or more IRES sequences on either or both sides of each expressed sequence, providing for isolation of the resulting peptides and / or polypeptides.

[0214] In some embodiments, the polyribonucleotide cargo comprises an IRES. For example, the polyribonucleotide cargo may comprise a circular RNA IRES, for example, as described in Chen et al. Mol. Cell 81:1-19, 2021, the entirety of which is incorporated herein by reference.

[0215] Adjustment element In some embodiments, a polyribonucleotide described herein (e.g., a polyribonucleotide cargo of a polyribonucleotide) comprises one or more regulatory elements. In some embodiments, a polyribonucleotide comprises a regulatory element, e.g., a sequence that regulates expression of an expression sequence within the polyribonucleotide.

[0216] A regulatory element may comprise a sequence located adjacent to an expression sequence that codes for an expression product. A regulatory element may be operably linked to an adjacent sequence. A regulatory element may increase the amount of an expressed product compared to the amount or number of products expressed in the absence of the regulatory element. Furthermore, a single regulatory element may increase the amount of an expressed product for multiple expression sequences linked side by side. Thus, a single regulatory element can promote the expression of one or more expression sequences. Multiple regulatory elements are well known to those skilled in the art.

[0217] In some embodiments, the regulatory element is a translation modulator. The translation modulator may regulate the translation of an expressed sequence in the polyribonucleotide. The translation modulator may be a translation enhancer or suppressor. In some embodiments, the polyribonucleotide comprises at least one translation modulator adjacent to at least one expressed sequence. In some embodiments, the polyribonucleotide comprises a translation modulator adjacent to each expressed sequence. In some embodiments, the translation modulator is on one or both sides of each expressed sequence, resulting in the separation of the expression products, e.g., peptides and / or polypeptides.

[0218] In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.

[0219] Further examples of regulatory elements are described, for example, in paragraphs

[0154] to

[0161] of International Publication No. 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0220] Translation initiation sequence In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide cargo of a polyribonucleotide) described herein comprises at least one translation initiation sequence. In some embodiments, a polyribonucleotide comprises a translation initiation sequence that is operably linked to an expression sequence.

[0221] In some embodiments, the polyribonucleotide may encode a polypeptide and include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence, e.g., a Kozak sequence, flanking the 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 to provide separation of the expression products. In some embodiments, the polyribonucleotide includes at least one translation initiation sequence flanking the expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single-stranded region of the circular polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs

[0163] to

[0165] of WO 2019 / 118919, which is incorporated herein by reference in its entirety.

[0222] A polyribonucleotide may comprise 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 at the first start codon or may begin downstream of the first start codon.

[0223] In some embodiments, the polyribonucleotide may initiate at the first start codon, e.g., a codon that is not AUG. Translation of the polyribonucleotide may initiate at an alternative translation start sequence, e.g., but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG. In some embodiments, translation initiates at an alternative translation start sequence under selective conditions, e.g., stress-inducing conditions. As a non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, e.g., ACG. As another non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, CTG / CUG. As another non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, GTG / GUG. As another non-limiting example, a polyribonucleotide may initiate translation at alternative translation initiation sequences, including repeat-associated non-AUG (RAN) sequences, e.g., short stretches of repetitive RNA, e.g., CGG, GGGGCC (SEQ ID NO: 1), CAG, CTG.

[0224] Termination sequence In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide cargo of a polyribonucleotide) described herein comprises at least one termination sequence. In some embodiments, the polyribonucleotide comprises a termination sequence that is operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination sequence.

[0225] In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence. In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence, such that the polyribonucleotide is continuously translated. Exclusion of the termination sequence may result in rolling circle translation or continuous expression of the expression product.

[0226] In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, each of which may lack a termination sequence, such that the circular polyribonucleotide is translated continuously. The exclusion of the termination sequence may result in rolling circle translation or continuous expression of expression products, such as peptides or polypeptides, due to lack of ribosome stalling or shedding. In such embodiments, the rolling circle translation expresses continuous expression products through each expression sequence. In some other embodiments, the termination sequence of the expression sequence may be part of a stagger element. In some embodiments, one or more expression sequences in the circular polyribonucleotide include a termination sequence. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the circular polyribonucleotide is performed. In such cases, the expression product may be shedding from the ribosome when the ribosome encounters a termination sequence, such as a stop codon, and terminates translation. In some embodiments, translation is terminated while the ribosome, eg, at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.

[0227] In some embodiments, the circular polyribonucleotide comprises a termination sequence at the end of one or more expressed sequences. In some embodiments, the one or more expressed sequences comprise two or more subsequent termination sequences. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome is completely separated from the circular polyribonucleotide. In some such embodiments, the generation of the subsequent (e.g., second, third, fourth, fifth, etc.) expressed sequences in the circular polyribonucleotide may require the ribosome to reassociate with the circular polyribonucleotide before the start of translation. Generally, the termination sequence comprises an in-frame nucleotide triplet, e.g., UAA, UGA, UAG, that signals the termination of translation. In some embodiments, one or more termination sequences in the circular polyribonucleotide are frame-shifted termination sequences, such as, but not limited to, off-frame or -1 and +1 shifted reading frames (e.g., cryptic stops), that may terminate translation. Frameshifted termination sequences include the triple nucleotides TAA, TAG, and TGA that occur in the second and third reading frames of the expressed sequence.Frameshifted termination sequences can be important to prevent misreading of mRNA, which is often harmful to cells.In some embodiments, the termination sequence is a stop codon.

[0228] Further examples of termination sequences are described in paragraphs

[0169] to

[0170] of International Publication No. 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0229] Untranslated Regions In some embodiments, the 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 the expression sequence described herein. In some embodiments, the UTR may be included downstream of the 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.

[0230] Exemplary untranslated regions are described in paragraphs

[0197] to

[0201] of International Publication No. 2019 / 118919, the entirety of which is incorporated herein by reference.

[0231] In some embodiments, the cyclic polyribonucleotide comprises a polyA sequence. Exemplary polyA sequences are described in paragraphs

[0202] to

[0205] of WO 2019 / 118919, the entirety of which is incorporated herein by reference. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence.

[0232] In some embodiments, the cyclic polyribonucleotide comprises a UTR that contains one or more stretches of adenosines and uridines. These AU-rich signatures may increase the turnover rate of the expression product.

[0233] The introduction, removal, or modification of AU-rich elements (AREs) in the UTRs can be useful for modulating the stability or immunogenicity (e.g., the level of one or more markers of immune or inflammatory response) of a cyclic polyribonucleotide. When modifying a particular cyclic polyribonucleotide, one or more copies of an ARE may be introduced into the cyclic polyribonucleotide, and the copies of the ARE may modulate the translation and / or production of the expression product. Similarly, AREs can be identified and removed or modified into a cyclic polyribonucleotide to modulate the intracellular stability, which in turn affects the translation and production of the resulting protein.

[0234] It should be understood that any UTR from any gene may be incorporated into each flanking region of the circular polyribonucleotide.

[0235] In some embodiments, the circular polyribonucleotide lacks a 5'UTR and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 3'UTR and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a polyA sequence and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a termination sequence and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks an internal ribosome entry site and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a cap and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 5'UTR, a 3'UTR, and an IRES and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide comprises one or more of the following sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding an exogenous gene, a sequence encoding a therapeutic, a regulatory element (e.g., a translation modulator, e.g., a translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids (e.g., siRNA, lncRNA, shRNA) targeting an endogenous gene, and a sequence encoding a therapeutic mRNA or protein.

[0236] In some embodiments, the cyclic polyribonucleotide lacks a 5' UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3' UTR. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence. In some embodiments, the cyclic polyribonucleotide lacks a termination sequence. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks susceptibility to degradation by exonucleases. In some embodiments, the fact that the cyclic polyribonucleotide lacks susceptibility to degradation can mean that the cyclic polyribonucleotide is not degraded by exonucleases or is degraded to a limited extent in the presence of exonucleases, e.g., is equivalent or similar in the absence of exonucleases. In some embodiments, the cyclic polyribonucleotide is not degraded by exonucleases. In some embodiments, the cyclic polyribonucleotide has reduced degradation when exposed to exonucleases. In some embodiments, the cyclic polyribonucleotide lacks binding to cap-binding proteins. In some embodiments, the circular polyribonucleotide lacks a 5' cap.

[0237] Stagger Element In some embodiments, the cyclic polyribonucleotide comprises at least one stagger element adjacent to the expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element adjacent to each expressed sequence. In some embodiments, the stagger element is on one or both sides of each expressed sequence, resulting in separation of the expression products, e.g., peptides and / or polypeptides. In some embodiments, the stagger element is a portion of one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide comprises one or more expressed sequences, each of which is separated from the subsequent expressed sequence by a stagger element in the cyclic polyribonucleotide. In some embodiments, the stagger element prevents the generation 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.

[0238] In some embodiments, the circular polyribonucleotide comprises a stagger element. To maintain rolling circle translation while avoiding the production of continuous expression products, such as peptides or polypeptides, the inclusion of a stagger element may induce ribosome stalling during translation. In some embodiments, the stagger element is at the 3' end of at least one of the one or more expressed sequences. The stagger element may be designed to stall the ribosome during rolling circle translation of the circular polyribonucleotide. The stagger element may include, but is not limited to, a 2A-like or CHYSEL (SEQ ID NO: 72) (cis-acting hydrolase element) sequence. In some embodiments, the stagger element encodes a sequence with a C-terminal consensus sequence that is X1X2X3EX5NPGP (wherein X1 is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid) (SEQ ID NO: 83). In some embodiments, the sequence contains a non-conserved sequence of amino acids with strong alpha-helical character followed by the consensus sequence -D(V / I)EXNPGP, where x=any amino acid (SEQ ID NO:4). Some non-limiting examples of stagger elements include GDVESNPGP (SEQ ID NO:55), GDIEENPGP (SEQ ID NO:56), VEPNPGP (SEQ ID NO:57), IETNPGP (SEQ ID NO:58), GDIESNPGP (SEQ ID NO:59), GDVELNPGP (SEQ ID NO:10), GDIETNPGP (SEQ ID NO:11), GDVENPGP (SEQ ID NO:12), GDVEENPGP (SEQ ID NO:13), GDVEQNPGP (SEQ ID NO:14), IESNPGP (SEQ ID NO:15), GDIELNPGP (SEQ ID NO:16), HDIETPGP (SEQ ID NO:17), HDVETNPGP (SEQ ID NO:18), HDVEMNPGP (SEQ ID NO:19), GDMESNPGP (SEQ ID NO:20), GDVETNPGP (SEQ ID NO:21) GDIEQNPGP (SEQ ID NO:22), and DSEFNPGP (SEQ ID NO:23).

[0239] 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 cyclic polyribonucleotide comprises at least one stagger element for cleaving the expression product. In some embodiments, the cyclic polyribonucleotide comprises a stagger element adjacent to at least one expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element after each expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element present on one or both sides of each expressed sequence to cause translation of individual peptides and / or polypeptides from each expressed sequence.

[0240] In some embodiments, the stagger element comprises one or more modified or non-natural nucleotides that induce ribosome stalling 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 naturally occurring DNA or RNA by changes to the backbone of the molecule. Modifications may include any modification to the sugar, nucleobase, internucleoside bond (e.g., linking phosphate / phosphodiester bond / phosphodiester backbone), and any combination thereof, that may induce ribosome stalling during translation. Some of the exemplary modifications provided herein are described elsewhere herein.

[0241] In some embodiments, the stagger element is present in other forms in the circular polyribonucleotide. For example, in some exemplary circular polyribonucleotides, the stagger element comprises a termination sequence of the first expressed sequence in the circular polyribonucleotide and a nucleotide spacer sequence that separates the termination sequence from the first translation initiation sequence of the expression contiguous to the first expressed sequence. In some examples, the first stagger element of the first expressed sequence is present upstream (5' to) the first translation initiation sequence of the expression contiguous to the first expressed sequence in the circular polyribonucleotide. In some cases, the first expressed sequence and the expression sequence contiguous to the first expressed sequence are two separate expressed sequences in the circular polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence may allow for the continuous translation of the first expressed sequence and its contiguous expressed sequence.

[0242] In some embodiments, the first stagger element comprises a termination sequence and separates the expression product of the first expressed sequence from the expression product of the contiguous expressed sequence, thereby creating a separate expression product. In some cases, a circular polyribonucleotide comprising a first stagger element upstream of a first translation initiation sequence of a contiguous sequence in the circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide comprising a stagger element of a second expressed sequence present upstream of a second translation initiation sequence of a contiguous expressed sequence in the second expressed sequence is not continuously translated. In some cases, there is only one expressed sequence in the circular polyribonucleotide, and the first expressed sequence and the contiguous expressed sequence are the same expressed sequence. In some exemplary circular polyribonucleotides, the stagger element comprises a first termination sequence of the first expressed sequence in the circular polyribonucleotide and a nucleotide spacer sequence that separates the termination sequence from the downstream translation initiation sequence. In some such examples, the first stagger element is upstream (5' to) of the first translation initiation sequence of the first expressed sequence in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence allows for continuous translation of the first expressed sequence and any consecutive expressed sequences.

[0243] In some embodiments, the first stagger element separates the product of one expression of the first expression sequence from the product of the next expression of the first expression sequence, thereby creating separate expression products. In some cases, a circular polyribonucleotide that includes a first stagger element upstream of a first translation initiation sequence of a first expression sequence in a circular polyribonucleotide is continuously translated, while a corresponding circular polyribonucleotide that includes a stagger element upstream of a second translation initiation sequence of a second expression sequence in a corresponding circular polyribonucleotide is not continuously translated. In some cases, the distance between the second stagger element and the second translation initiation sequence is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater in the corresponding circular polyribonucleotide than the distance between the first stagger element and the first translation initiation sequence in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence 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 stagger element and the second translation initiation sequence 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 first stagger element and the first translation initiation sequence. In some embodiments, the circular polyribonucleotide comprises two or more expressed sequences.

[0244] Examples of stagger elements are described in paragraphs

[0172] to

[0175] of International Publication No. WO 2019 / 118919, the entirety of which is incorporated herein by reference.

[0245] Non-coding sequences In some embodiments, a polyribonucleotide described herein (e.g., the polyribonucleotide cargo of a polyribonucleotide) comprises one or more non-coding sequences, e.g., sequences that do not code for expression of a polypeptide. In some embodiments, a polyribonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 non-coding sequences. In some embodiments, a polyribonucleotide does not code for a polypeptide expression sequence.

[0246] The non-coding sequence may be a natural or synthetic sequence. In some embodiments, the non-coding sequence may modify cell behavior, such as lymphocyte behavior. In some embodiments, the non-coding sequence is antisense to a cellular RNA sequence.

[0247] In some embodiments, the polyribonucleotide comprises a regulatory nucleic acid that is typically an RNA or RNA-like structure of about 5-500 base pairs (bp) (depending on the particular RNA structure (e.g., miRNA of 5-30 bp, lncRNA of 200-500 bp) and may have a nucleobase sequence that is identical (complementary) or nearly identical (substantially complementary) to a coding sequence in an expressed target gene in a cell. In embodiments, the circular polyribonucleotide comprises a regulatory nucleic acid that encodes an RNA precursor, e.g., a miRNA precursor, that is processable into a smaller RNA, which may be about 50 to about 1000 bp, that is processable into a smaller miRNA intermediate or mature miRNA.

[0248] Long non-coding RNA (IncRNA) is defined as a non-protein-coding transcript longer than 100 nucleotides. Many IncRNAs are characterized as tissue-specific. Diverse IncRNAs that are transcribed in the opposite direction to nearby protein-coding genes comprise a significant proportion (e.g., about 20% of all IncRNAs in mammalian genomes) and likely regulate the transcription of nearby genes. In one embodiment, the polyribonucleotide provided herein comprises the sense strand of an IncRNA. In one embodiment, the polyribonucleotide provided herein comprises the antisense strand of an IncRNA.

[0249] In embodiments, the polyribonucleotide encodes a regulatory nucleic acid that is substantially complementary or completely complementary to all or at least a fragment of an endogenous gene or gene product (e.g., mRNA). In embodiments, the regulatory nucleic acid complements sequences at intron-exon boundaries, between exons, or adjacent to exons to prevent the maturation of newly generated nuclear RNA transcripts of a particular gene into mRNA during transcription. A regulatory nucleic acid that is complementary to a particular gene may hybridize with the mRNA for that gene and block its translation. An antisense regulatory nucleic acid may be DNA, RNA, or derivatives or hybrids thereof. In some embodiments, the regulatory nucleic acid comprises a protein binding site that may bind to a protein involved in regulating the expression of an endogenous or exogenous gene.

[0250] In embodiments, the length of the polyribonucleotide encodes a regulatory nucleic acid that hybridizes to a transcript of interest, where the regulatory RNA has a length of 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 nucleotides or more than 30 nucleotides. In embodiments, the degree of sequence identity of the regulatory RNA to the targeted transcript is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0251] In embodiments, the polyribonucleotide encodes a microRNA (miRNA) molecule identical to about 5 to about 25 contiguous nucleotides of the target gene, or encodes a precursor of that miRNA. In some embodiments, the miRNA has a sequence that enables the mRNA to recognize and bind to a specific target mRNA. In embodiments, the miRNA sequence starts with the dinucleotide AA, contains about 30-70% (about 30-60%, about 40-60%, or about 45%-55%) GC content, and does not share a high percentage of identity with any nucleotide sequence other than the target in the genome of the subject (e.g., mammal) that is to be introduced as determined, for example, by a standard BLAST search.

[0252] In some embodiments, the polyribonucleotide comprises at least one miRNA (or miRNA precursor), e.g., 2, 3, 4, 5, 6, or more miRNAs or miRNA precursors. In some embodiments, the polyribonucleotide comprises a sequence encoding a miRNA (or a precursor thereof) having at least about 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, or 99% or 100% nucleotide sequence complementarity to a target sequence.

[0253] siRNAs and shRNAs resemble intermediates in the processing pathway of endogenous microRNA (miRNA) genes. In some embodiments, siRNAs can function as miRNAs and vice versa. Like siRNAs, microRNAs use RISC to downregulate target genes, but unlike siRNAs, most animal miRNAs do not cleave mRNAs. Instead, miRNAs reduce protein output through translational repression or polyA removal and mRNA degradation. Known miRNA binding sites are within the 3'UTR of mRNAs; 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 mature siRNAs and miRNAs are interchangeable, exogenous siRNAs downregulate mRNAs with seed complementary to the siRNA.

[0254] Lists of known miRNA sequences can be found in databases maintained by research organizations such as the Wellcome Trust Sanger Institute, Penn Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and the European Molecule Biology Laboratory, among others. Known effective siRNA sequences and cognate binding sites are also well represented in the relevant literature. RNAi molecules are easily designed and generated by techniques known in the art. In addition, computational tools exist that increase the chances of finding effective specific sequence motifs.

[0255] Protein Binding Sequences In some embodiments, the cyclic polyribonucleotide comprises one or more protein binding sites that allow proteins, such as ribosomes, to bind to internal sites within the RNA sequence.By designing a protein binding site, such as a ribosome binding site, into the cyclic polyribonucleotide, the cyclic polyribonucleotide can evade or reduce detection by the host's immune system by masking the cyclic polyribonucleotide from components of the host's immune system, regulating degradation, or regulating translation.

[0256] In some embodiments, the cyclic polyribonucleotide comprises at least one immunity protein binding site, e.g., to evade an immune response, e.g., 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 aids in masking the cyclic polyribonucleotide as exogenous. In some embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and aids in masking the cyclic polyribonucleotide as exogenous or foreign.

[0257] The traditional mechanism of ribosome association to linear RNA involves ribosome binding to the capped 5' end of the RNA. The first peptide bond is formed as soon as the ribosome moves from the 5' end to the start codon. According to the present disclosure, internal initiation of translation of a circular polyribonucleotide (i.e., cap-independent) does not require a free or capped end. Rather, the ribosome binds to an uncapped internal site, whereby the ribosome initiates polypeptide elongation at the start codon. In some embodiments, the circular polyribonucleotide comprises one or more RNA sequences that include a ribosome binding site, e.g., a start codon.

[0258] Natural 5'UTRs have characteristics that play a role in translation initiation. They contain 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 (SEQ ID NO:24), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". 5'UTRs are also known to form secondary structures involved in elongation factor binding.

[0259] In some embodiments, the cyclic polyribonucleotide encodes a protein binding sequence that binds to a protein. In some embodiments, the protein binding sequence targets or localizes the cyclic polyribonucleotide to a specific target. In some embodiments, the protein binding sequence specifically binds to an arginine-rich region of a protein.

[0260] In some 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, LIN28B , m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2, POLR2A, PRPF8, PTBP1, RBFO X2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRS Binding sites for proteins such as F1, 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.

[0261] Spacer sequence In some embodiments, the polyribonucleotides described herein comprise one or more spacer sequences. A spacer refers to any adjacent nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. A spacer may be present between any of the nucleic acid elements described herein. A spacer may also be present within the nucleic acid elements described herein.

[0262] The spacer may be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, each spacer region is at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. Each spacer region may be, for example, 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may comprise a polyA sequence. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may comprise a polyAC sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a poly-AG sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a poly-AT sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a random sequence.

[0263] Spacers may also be present within the nucleic acid regions described herein. For example, a polynucleotide cargo region may include one or more spacers. A spacer may separate regions within a polynucleotide cargo.

[0264] In some embodiments, the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 30 nucleotides in length. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the spacer sequence is 20-50 nucleotides in length. In certain embodiments, the spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.

[0265] The spacer sequence can be a polyA, polyAC, polyC, or polyU sequence.

[0266] In some embodiments, the spacer sequence can be polyAT, polyAC, polyAG, or a random sequence.

[0267] Spacer sequences can be used to separate the IRES from adjacent components to preserve the structure and function of the IRES or adjacent elements. Spacers can be specifically modified depending on the IRES. In some embodiments, RNA folding computer software such as RNAFold can be used to guide the design of various elements of the vector, including spacers.

[0268] In some embodiments, the polyribonucleotide comprises a 5' spacer sequence (e.g., between the 5' annealing region and the polyribonucleotide cargo). In some embodiments, the 5' spacer sequence is at least 10 nucleotides in length. In another embodiment, the 5' spacer sequence is at least 15 nucleotides in length. In further embodiments, the 5' spacer sequence is at least 30 nucleotides in length. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the 5' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the 5' spacer sequence is 20-50 nucleotides in length. In certain embodiments, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In one embodiment, the 5' spacer sequence is a polyA sequence. In another embodiment, the 5' spacer sequence is a polyAC sequence. In some embodiments, the 5' spacer sequence comprises a polyAG sequence. In some embodiments, the 5' spacer sequence comprises a polyAT sequence. In some embodiments, the 5' spacer sequence comprises a random sequence.

[0269] In some embodiments, the polyribonucleotide comprises a 3' spacer sequence (e.g., between the 3' annealing region and the polyribonucleotide cargo). In some embodiments, the 3' spacer sequence is at least 10 nucleotides in length. In another embodiment, the 3' spacer sequence is at least 15 nucleotides in length. In further embodiments, the 3' spacer sequence is at least 30 nucleotides in length. In some embodiments, the 3' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the 3' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the 3' spacer sequence is 20-50 nucleotides in length. In certain embodiments, the 3'spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In one embodiment, the 3'spacer sequence is a polyA sequence. In another embodiment, the 5'spacer sequence is a polyAC sequence. In some embodiments, the 5'spacer sequence comprises a polyAG sequence. In some embodiments, the 5'spacer sequence comprises a polyAT sequence. In some embodiments, the 5'spacer sequence comprises a random sequence.

[0270] In one embodiment, the polyribonucleotide comprises a 5' spacer sequence but does not comprise a 3' spacer sequence. In another embodiment, the polyribonucleotide comprises a 3' spacer sequence but does not comprise a 5' spacer sequence. In another embodiment, the polyribonucleotide does not comprise a 5' or 3' spacer sequence. In another embodiment, the polyribonucleotide does not comprise an IRES sequence. In a further embodiment, the polyribonucleotide does not comprise an IRES sequence, a 5' spacer sequence or a 3' spacer sequence.

[0271] In some embodiments, the spacer sequence is at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides, ribonucleotides, at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least about 200 ribonucleotides, at least about 250 ribonucleotides, at least about 300 ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 600 ribonucleotides, at least about 700 ribonucleotides, at least about 800 ribonucleotides, at least about 900 ribonucleotides, or at least about 100 ribonucleotides.

[0272] Bioreactor In some embodiments, any method of purifying polyribonucleotides (e.g., circular polyribonucleotides) described herein may be carried out in a bioreactor. A bioreactor refers to any container in which a chemical or biological process involving an organism or a biochemically active substance derived from such an organism is carried out. The bioreactor may be adapted for a cell-free method of producing or purifying circular RNA as described herein. The container for the bioreactor may include culture flasks, dishes, or bags, which may be single-use (disposable), autoclavable, or sterilizable. The bioreactor may be made of glass, or may be polymer-based, or may be manufactured from other materials.

[0273] Examples of bioreactors include, but are not limited to, stirred tank (e.g., well-mixed) and flat plate (e.g., plug flow) bioreactors, air-lift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibratory mixers, fluidized bed reactors, and membrane bioreactors. The mode of operation of a bioreactor may be a batch or continuous process. A bioreactor is continuous when the streams of reagents and products are continuously fed and removed from the system. A batch bioreactor may have a continuous recirculation flow but may not have a continuous feed of reagents or product withdrawal.

[0274] Some methods of the present disclosure are directed to large-scale production of polyribonucleotides. For large-scale production methods, the methods can be carried out in volumes ranging from 1 liter (L) to 50 L or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more). In some embodiments, the method can be performed with a volume of 5L to 10L, 5L to 15L, 5L to 20L, 5L to 25L, 5L to 30L, 5L to 35L, 5L to 40L, 5L to 45L, 10L to 15L, 10L to 20L, 10L to 25L, 20L to 30L, 10L to 35L, 10L to 40L, 10L to 45L, 10L to 50L, 15L to 20L, 15L to 25L, 15L to 30L, 15L to 35L, 15L to 40L, 15L to 45L, or 15L to 50L.

[0275] In some embodiments, the bioreactor may produce at least 1 g of RNA. In some embodiments, the bioreactor may produce 1-200 g of RNA (e.g., 1-10 g, 1-20 g, 1-50 g, 10-50 g, 10-100 g, 50-100 g, 50-200 g of RNA). In some embodiments, the amount produced is measured per liter (e.g., 1-200 g per liter), per batch or reaction (e.g., 1-200 g per batch or reaction), or per unit of time (e.g., 1-200 g per hour or day).

[0276] In some embodiments, two or more bioreactors can be utilized in series to increase production capacity (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 bioreactors can be used in series).

[0277] How to use In some embodiments, polyribonucleotides (e.g., circular polyribonucleotides) generated as described herein are used as effectors in therapy or agriculture.

[0278] For example, polyribonucleotides purified by the methods described herein may be administered to a subject (e.g., in a pharmaceutical, veterinary, or agricultural composition). In some embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In embodiments, the subject is an avian, such as a member of an avian taxonomic group such as Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate, such as an arthropod (e.g., insects, arachnids, crustaceans), nematodes, annelids, worms, or mollusks. In embodiments, the subject is an invertebrate that is an invertebrate agricultural pest or a parasite on an invertebrate or vertebrate host. In embodiments, the subject is a plant, such as an angiosperm (which may be dicotyledonous or monocotyledonous) or gymnosperm (e.g., conifers, cycads, Gnetophytes, ginkgo), fern, horsetail, club moss, or bryophyte. In embodiments, the subject is a eukaryotic alga (unicellular or multicellular). In embodiments, the subject is an agriculturally or horticulturally important plant, such as row crops, fruit-bearing plants and trees, vegetables, trees, and ornamentals, e.g., ornamental flowers, shrubs, trees, ground covers, and turf.

[0279] In some embodiments, the disclosure provides a method of modifying a subject by providing a composition or preparation described herein to the subject. In some embodiments, the composition or preparation is or comprises a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or preparation is or comprises a eukaryotic or prokaryotic cell that contains a nucleic acid described herein.

[0280] In some embodiments, the disclosure provides a method of treating a condition in a subject in need thereof by providing to the subject a composition or preparation described herein. In some embodiments, the composition or preparation is or comprises a nucleic acid molecule (e.g., a DNA molecule or polyribonucleotide described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or preparation is or comprises a eukaryotic or prokaryotic cell that contains a nucleic acid described herein.

[0281] In some embodiments, the present disclosure provides methods of providing a polyribonucleotide (e.g., a circular polyribonucleotide) to a subject by providing to a subject a eukaryotic or prokaryotic cell that contains a polynucleotide described herein.

[0282] preparation In some embodiments of the present disclosure, the polyribonucleotides described herein (e.g., cyclic polyribonucleotides) can be formulated in a composition, e.g., an agricultural, veterinary, or pharmaceutical composition, for delivery to a cell, a plant, an invertebrate, a non-human vertebrate, or a human subject. In some embodiments, the polyribonucleotides are formulated in a pharmaceutical composition. In some embodiments, the composition comprises a polyribonucleotide and a diluent, a carrier, an adjuvant, or a combination thereof. In certain embodiments, the composition comprises a polyribonucleotide described herein and a carrier or a diluent without any carrier. In some embodiments, a composition comprising a polyribonucleotide and a diluent without any carrier is used for naked delivery of a polyribonucleotide (e.g., cyclic polyribonucleotide) to a subject.

[0283] The pharmaceutical composition may optionally include one or more additional active substances, e.g., therapeutic and / or prophylactic active substances. The pharmaceutical composition may optionally include an inert substance that serves as a vehicle or medium for the composition described herein (e.g., a cyclic polyribonucleotide, e.g., a composition that includes any one of the inert ingredients approved by the United States Food and Drug Administration (FDA) and listed in the inactive ingredient database). The pharmaceutical composition of the present invention may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceuticals can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersion agents, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersing agents, granulating agents, disintegrating agents, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof.

[0284] Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans that make the compositions suitable for administration to various animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or implement such modifications with no more than routine (if possible) experimentation. Subjects to which the pharmaceutical compositions are intended to be administered include, but are not limited to, humans and / or other primates; mammals, including commercially important mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially important birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0285] The preparations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, forming and / or packaging the product.

[0286] In some embodiments, the reference standard for the amount of linear polyribonucleotide molecules present in the preparation is 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL of linear polyribonucleotide molecules. , 200ng / mL, 300ng / mL, 400ng / mL, 500ng / mL, 600ng / mL, 1μg / ml, 10μg / mL, 50μg / mL, 100μg / mL, 200g / mL, 300μg / mL, 400μg / mL, 500μg / mL, 600μg / mL, 700μg / mL, 800μg / mL, 900μg / mL, 1mg / mL, 1.5mg / ml, or 2mg / ml or less.

[0287] In some embodiments, a reference standard for the amount of circular polyribonucleotide molecules present in a preparation is at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 100% (w / w), 110% (w / w), 120% (w / w), 130% (w / w), 140% (w / w), 150% (w / w), 160% (w / w), 170% (w / w), 180% (w / w), 190% (w / w), 200% (w / w), 21% (w / w), 22% (w / w), 23% (w / w), 24% (w / w), 25% (w / w), 26% (w / w), 27% (w / w), 28% (w / w), 29% (w / w), 30% (w / w), 31% (w / w), 32% (w / w), 33% (w / w), 34% (w / w), 35% (w / w), 36% (w / w), 37% (w / w), 38% (w / w), 39% (w / w), 40% (w / w), 40% (w / w), 40% (w / w), 40% (w / w), 40% (w / w), 40% (w / w 94%(w / w), 95%(w / w), 96%(w / w), 97%(w / w), 98%(w / w), 99%(w / w), 99.1%(w / w), 99.2%(w / w), 99.3%(w / w), 99.4%(w / w), 99.5%(w / w), 99.6%(w / w), 99.7%(w / w), 99.8%(w / w), 99.9%(w / w) or 100%(w / w) of the molecule.

[0288] In some embodiments, a reference standard for the amount of linear polyribonucleotide molecules present in a preparation is less than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) or less linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical preparation.

[0289] In some embodiments, a reference standard for the amount of nicked polyribonucleotide molecules present in a preparation is no more than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), or 15% (w / w) of nicked polyribonucleotide molecules of total ribonucleotide molecules in the pharmaceutical preparation.

[0290] In some embodiments, the reference standard for the amount of combination of nicks and linear polyribonucleotide molecules present in the preparation is 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) or less of combination of nicks and linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical preparation. In some embodiments, the pharmaceutical preparation is an intermediate pharmaceutical preparation of the final circular polyribonucleotide formulation. In some embodiments, the pharmaceutical preparation is a bulk drug or active pharmaceutical ingredient (API). In some embodiments, the pharmaceutical preparation is a formulation for administration to a subject.

[0291] In some embodiments, the preparation of circular polyribonucleotides is further processed (before, during or after reduction of linear RNA) to substantially remove DNA, protein contaminants, impurities or by-products (e.g., cellular proteins such as host cell proteins or process impurity proteins), endotoxins, mononucleotide molecules, and / or process-related impurities.

[0292] salt In some cases, the compositions or pharmaceutical compositions provided herein include one or more salts. To control osmolality, physiological salts such as sodium salts can be included in the compositions provided herein. Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride, and the like. In some cases, the compositions are formulated with one or more pharma- ceutically acceptable salts. The one or more pharma-ceutically acceptable salts can include those of inorganic ions, such as, for example, sodium, potassium, calcium, and magnesium ions. Such salts can include salts of inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid. Polyribonucleotides can exist in either linear or cyclic form.

[0293] Buffer / pH The compositions or pharmaceutical compositions provided herein may include one or more buffers, such as Tris buffer, borate buffer, succinate buffer, histidine buffer (e.g., with aluminum hydroxide adjuvant), or citrate buffer, etc. The buffers are in some cases within the range of 5-20 mM.

[0294] The compositions or pharmaceutical compositions provided herein can have a pH between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8. The compositions or pharmaceutical compositions can have a pH of about 7. Polyribonucleotides can exist in either linear or circular form.

[0295] Detergents / Surfactants The compositions or pharmaceutical compositions provided herein may contain, depending on the intended route of administration, one or more detergents and / or surfactants, such as polyoxyethylene sorbitan ester surfactants (commonly referred to as "Tween®"), such as polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), such as linear EO / PO block copolymers, sold under the trade name DOWFAX™; octoxynols, which may vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, such as octoxynol-9 (Triton® X-100, or t-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL®); CA-630 / NP-40; phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol (known as Brij® surfactants), such as triethylene glycol monolauryl ether (Brij® 30); and sorbitan esters (commonly known as “SPAN®”) such as sorbitan trioleate (Span® 85) and sorbitan monolaurate, octoxynol (such as octoxynol-9 (Triton® X-100) or t-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide ("CTAB"), or sodium deoxycholate. One or more detergents and / or surfactants may be included only in trace amounts. In some examples, the composition may contain less than 1 mg / ml each of octoxynol-10 and polysorbate 80. Non-ionic surfactants may be used herein. Surfactants may be classified by their "HLB" (hydrophilic / lipophilic balance). In some examples, the surfactant has an HLB of at least 10, at least 15, and / or at least 16.The polyribonucleotide may be included in a linear or circular form.

[0296] Diluent In some embodiments, the compositions of the present disclosure comprise a polyribonucleotide and a diluent. In some embodiments, the compositions of the present disclosure comprise a linear polyribonucleotide and a diluent.

[0297] The diluent may be a non-carrier excipient. The non-carrier excipient serves as a vehicle or medium for the composition, such as the cyclic polyribonucleotide described herein. The non-carrier excipient serves as a vehicle or medium for the composition, such as the linear polyribonucleotide described herein. Non-limiting examples of non-carrier excipients include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonicity agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The non-carrier excipient may be any of the inactive ingredients listed in the Inactive Ingredient Database that are approved by the United States Food and Drug Administration (FDA) and do not exhibit cell-penetrating effects. A non-carrier excipient can be, for example, any inactive ingredient suitable for administration to a non-human animal suitable for veterinary use. Modifications of compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood and a veterinary pharmacologist of ordinary skill can design and / or make such modifications with only routine experimentation, if any.

[0298] In certain embodiments, polyribonucleotides (e.g., cyclic polyribonucleotides) can be delivered as naked delivery formulations, such as those that include a diluent. Naked delivery formulations deliver polyribonucleotides to cells without a carrier and without modification or partial or complete encapsulation of polyribonucleotides, capped polyribonucleotides, or complexes thereof.

[0299] A naked delivery formulation is a formulation that does not contain a carrier, in which the polyribonucleotide (e.g., a cyclic polyribonucleotide) does not have a covalent modification attached to a moiety that aids in delivery to a cell, or is without partial or complete encapsulation of the polyribonucleotide. In some embodiments, the polyribonucleotide that does not have a covalent modification attached to a moiety that aids in delivery to a cell is a polyribonucleotide that is not covalently attached to a protein, small molecule, particle, polymer, or biopolymer. The polyribonucleotide that does not have a covalent modification attached to a moiety that aids in delivery to a cell does not contain a modified phosphate group. For example, the polyribonucleotide that does not have a covalent modification attached to a moiety that aids in delivery to a cell does not contain a phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoroamidate, phosphorodiamidate, alkyl or aryl phosphonate, or phosphotriester.

[0300] In some embodiments, the naked delivery formulation does not include any or all of a transfection reagent, a cationic carrier, a carbohydrate carrier, a nanoparticle carrier, or a protein carrier. In certain embodiments, the naked delivery formulation is selected from the group consisting of phytoglycogen octenyl succinate, phytoglycogen β-dextrin, anhydrous modified phytoglycogen β-dextrin, lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-β-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3 -(2-Hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N,N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

[0301] In some embodiments, the naked delivery formulation comprises a non-carrier excipient. In some embodiments, the non-carrier excipient comprises an inactive ingredient that does not exhibit a cell-penetrating effect. In some embodiments, the non-carrier excipient comprises a buffer, such as PBS. In some embodiments, the non-carrier excipient is a solvent, a non-aqueous solvent, a diluent, a suspending aid, a surfactant, an isotonicity agent, a thickener, an emulsifier, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersant, a granulating agent, a disintegrant, a binder, a buffer, a lubricant, or an oil.

[0302] In some embodiments, the naked delivery formulation comprises a diluent. The diluent can be a liquid diluent or a solid diluent. In some embodiments, the diluent is an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of buffers include 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate. Examples of isotonicity agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.

[0303] Carrier In some embodiments, the compositions of the present disclosure comprise a cyclic polyribonucleotide and a carrier. In some embodiments, the compositions of the present disclosure comprise a linear polyribonucleotide and a carrier.

[0304] In certain embodiments, the compositions comprise a circular polyribonucleotide as described herein in a vesicle or other membrane-based carrier. In certain embodiments, the compositions comprise a linear polyribonucleotide as described herein in a vesicle or other membrane-based carrier.

[0305] In other embodiments, the composition comprises a cyclic polyribonucleotide in or through a cell, vesicle, or other membrane-based carrier. In other embodiments, the composition comprises a linear polyribonucleotide in or through a cell, vesicle, or other membrane-based carrier. In one embodiment, the composition comprises a cyclic polyribonucleotide in a liposome or other similar vesicle. In one embodiment, the composition comprises a linear polyribonucleotide in a liposome or other similar vesicle. Liposomes are spherical vesicular structures composed of a mono- or multi-membrane lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible, non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB) ​​(see, e.g., for review, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679).

[0306] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers. Methods for the preparation of multilamellar vesicle lipids are known in the art (see, for example, U.S. Pat. No. 6,693,086, the teachings of which are incorporated herein by reference for their related preparation of multilamellar vesicle lipids). When lipid membranes are mixed with aqueous solution, vesicle formation can occur spontaneously, but it can also be promoted by applying force in the form of shaking, by using homogenizers, sonicators, or extrusion devices (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155 / 2011 / 469679 for review). Extruded lipids can be prepared by extrusion through reduced size filters as described in Templeton et al., Nature Biotechnol., 15:647-52, 1997, the teachings of which regarding the preparation of extruded lipids are incorporated herein by reference.

[0307] In certain embodiments, the compositions of the present disclosure include polyribonucleotides and lipid nanoparticles, such as lipid nanoparticles, as described herein. In certain embodiments, the compositions of the present disclosure include linear polyribonucleotides and lipid nanoparticles. Lipid nanoparticles are another example of carriers that provide a biocompatible and biodegradable delivery system for the polyribonucleotide molecules described herein. Lipid nanoparticles are another example of carriers that provide a biocompatible and biodegradable delivery system for the linear polyribonucleotide molecules described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the properties of SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLN consists of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, the two components increase drug encapsulation efficiency, facilitate surface modification, and prevent leakage of water-soluble drugs. For a review, see, for example, Li et al. 2017, Nanomaterials 7, 122; doi:10.3390 / nano7060122.

[0308] Further non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), protein carriers (e.g., proteins covalently bound to polyribonucleotides or proteins covalently bound to linear polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin. Non-limiting examples of cationic carriers include lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)-2-hydroxypropyl]-2-hydroxypropyl-2-hydroxypropyl, and the like. N,N-diisopropyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HC1), diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). Non-limiting examples of protein carriers include human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

[0309] Exosomes may also be used as drug delivery vehicles for the compositions or preparations described herein. Exosomes may also be used as drug delivery vehicles for the linear polyribonucleotide compositions or preparations described herein. For review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-96, doi.org / 10.1016 / j.apsb.2016.02.001.

[0310] Ex vivo differentiated red blood cells may also be used as a carrier for the compositions or preparations described herein. Ex vivo differentiated red blood cells may also be used as a carrier for the linear polyribonucleotide compositions or preparations described herein. See, for example, WO 2015 / 073587; WO 2017 / 123646; WO 2017 / 123644; WO 2018 / 102740; WO 2016 / 183482; WO 2015 / 153102; WO 2018 / 151829; WO 2018 / 009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28):10131-10136; U.S. Pat. No. 9,644,180; Huang et al. 2017. Nature Communications 8:423; Shi et al. 2014. Proc Natl Acad Sci USA. See USA.111(28):10131-10136.

[0311] For example, fusosome compositions as described in WO 2018 / 208728 may also be used as carriers for delivering the polyribonucleotide molecules described herein. For example, fusosome compositions as described in WO 2018 / 208728 may also be used as carriers for delivering the linear polyribonucleotide molecules described herein.

[0312] Virosomes and virus-like particles (VLPs) can also be used as carriers for delivering the polyribonucleotide molecules described herein to target cells. Virosomes and virus-like particles (VLPs) can also be used as carriers for delivering the linear polyribonucleotide molecules described herein to target cells.

[0313] Plant nanovesicles and plant messenger packs (PMPs), for example as described in International Patent Publication Nos. WO 2011 / 097480, WO 2013 / 070324, WO 2017 / 004526, or WO 2020041784, may also be used as carriers for delivering the compositions or preparations described herein. Plant nanovesicles and plant messenger packs (PMPs) may also be used as carriers for delivering the linear polyribonucleotide compositions or preparations described herein.

[0314] Microbubbles can also be used as carriers for delivering the polyribonucleotide molecules described herein.Microbubbles can also be used as carriers for delivering the linear polyribonucleotide molecules described herein.See, for example, U.S. Pat. No. 7,115,583;Beeri, R. et al., Circulation.2002 Oct 1;106(14):1756-1759;Bez, M. et al., Nat Protoc.2019 Apr;14(4):1015-1026;Hernot, S. et al., Adv Drug Deliv Rev.2008 Jun 30;60(10):1153-1166;Rychak, JJ et al., Adv Drug Deliv Rev.2014 Jun;72:82-93.In some embodiments, the microbubbles are albumin-coated perfluorocarbon microbubbles.

[0315] The carrier comprising polyribonucleotides described herein may comprise a plurality of particles. The particles may have a median article size of 30-700 nanometers (e.g., 30-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 100-500, 50-500, or 200-700 nanometers). The size of the particles may be optimized to favor deposition of the payload comprising polyribonucleotides into cells. Deposition of polyribonucleotides into specific cell types may favor different particle sizes. For example, the particle size may be optimized for deposition of polyribonucleotides into antigen-presenting cells. The particle size may be optimized for deposition of polyribonucleotides into dendritic cells. Additionally, the particle size may be optimized for deposition of polyribonucleotides into draining lymph node cells.

[0316] Lipid Nanoparticles The compositions, methods, and delivery systems provided by the present disclosure may use any suitable carrier or delivery modality, including, in certain embodiments, lipid nanoparticles (LNPs) as described herein. The lipid nanoparticles, in certain embodiments, include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or amphoteric lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers as described in Table 5 of International Publication No. WO2019217941, which is incorporated herein by reference in its entirety); and one or more sterols (e.g., cholesterol).

[0317] Lipids (e.g., lipid nanoparticles) that can be used in nanoparticle formation include, for example, those described in Table 4 of WO2019217941, which is incorporated by reference - for example, a lipid-containing nanoparticle can include one or more of the lipids in Table 4 of WO2019217941. The lipid nanoparticle can include additional elements, such as a polymer, for example, a polymer described in Table 5 of WO2019217941, which is incorporated by reference.

[0318] In certain embodiments, the conjugated lipid, if present, is PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradecyl)glycerol), PEG-glyceryl-2,3-diamino-2-(trimethylsilyl)-1,1-dimethylethyl)-2,2-dimethylethyl)-1,1-dimethylethyl)-2,3-dimethylethyl ... The polyoxyalkylene glycols may include one or more of the following: N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, N-(carbonyloxypropyl-l-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those listed in Table 2 of WO2019051289 (incorporated by reference), as well as combinations of the above.

[0319] In some embodiments, sterols that may be incorporated into the lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009 / 127060 or U.S. Patent Publication No. 2010 / 0130588, which are incorporated by reference. Further exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, which are incorporated by reference herein.

[0320] In some embodiments, the lipid particles include an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits particle aggregation, and a sterol. The amounts of these components can be independently varied to achieve desired properties. For example, in some embodiments, the lipid nanoparticles include an ionizable lipid in an amount of about 20 mol% to about 90 mol% of the total lipid (in other embodiments, it can be 20-70% (mol), 30-60% (mol), or 40-50% (mol); about 50 mol% to about 90 mol% of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount of about 5 mol% to about 30 mol% of the total lipid, a conjugated lipid in an amount of about 0.5 mol% to about 20 mol% of the total lipid, and a sterol in an amount of about 20 mol% to about 50 mol% of the total lipid. The ratio of total lipid to nucleic acid can be varied as needed. For example, the ratio of total lipid to nucleic acid (mass or weight) can be about 10:1 to about 30:1.

[0321] In certain embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can range from about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and nucleic acid can be adjusted to obtain a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the total lipid content of the lipid nanoparticle formulation can range from about 5 mg / ml to about 30 mg / mL.

[0322] Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of the compositions described herein, e.g., the nucleic acids described herein (e.g., RNA (e.g., circular polyribonucleotides, linear polyribonucleotides)), include the following: [ka] In certain embodiments, an LNP comprising formula (i) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0323] [ka] In certain embodiments, an LNP comprising formula (ii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0324] [ka] In certain embodiments, an LNP comprising formula (iii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0325] [ka] In certain embodiments, an LNP comprising formula (v) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0326] [ka] In certain embodiments, an LNP comprising formula (vi) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0327] [ka] In certain embodiments, an LNP comprising formula (viii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0328] [ka] In certain embodiments, an LNP comprising formula (ix) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0329] [ka] During the ceremony, X 1 But, O, NR 1 or a direct bond, X 2 is C2-5 alkylene, and X 3 is C(=O) or a direct bond, and R 1 is H or Me, and R 3 is C1-3 alkyl, R 2 is C1-3 alkyl, or R 2 The nitrogen atom to which it is attached and X 2 together with 1 to 3 carbon atoms of X to form a 4-, 5-, or 6-membered ring, or 1 But NR 1 and R 1 and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 But R 3 and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring; Y 1 is C2-12 alkylene, and Y 2 but, [ka] is selected from n is 0 to 3, R 4is C1-15 alkyl, Z 1 is C1-6 alkylene or a direct bond, Z 2 but [ka] (in either orientation) or absent, with the proviso that Z 1 is a direct bond, and Z 2 If is non-existent; R 5 is C5-9 alkyl or C6-10 alkoxy, R 6 is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, R 7 is H or Me, or a salt thereof, with the proviso that R 3 and R 2 is a C2 alkyl group, and X 1 is O and X 2 is a linear C3 alkylene, and X 3 But C(=0) and Y 1 is a linear Ce alkylene, (Y 2 )nR 4 but, [ka] and R 4 is a linear C5 alkyl; Z 1 is C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If H, then R 5 and R 6 But Cx is not alkoxy.

[0330] In certain embodiments, an LNP comprising formula (xii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell. [ka] In certain embodiments, an LNP comprising formula (xi) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0331] [ka] In certain embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv).

[0332] [ka] In certain embodiments, an LNP comprising formula (xv) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0333] [ka] In some embodiments, an LNP comprising a formulation of formula (xvi) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0334] [ka]

[0335] In certain embodiments, the lipid compounds used to form lipid nanoparticles for delivery of the compositions described herein, e.g., the nucleic acids described herein (e.g., RNA (e.g., circular polyribonucleotides, linear polyribonucleotides)), are made by one of the following reactions: [ka]

[0336] In some embodiments, LNPs comprising formula (xxi) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxi) are LNPs described by WO2021113777 (e.g., lipids of formula (1), such as lipids in Table 1 of WO2021113777). [ka] During the ceremony, Each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)- * or -C(O)O- * In the formula, * " refers to the point of attachment to R1 or R3; R1 and R3 are each independently oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyl linear or branched C9-C alkyl groups optionally substituted with one or more substituents selected from the group consisting of oxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl;20 Alkyl or C9-C 20 alkenyl; and R2 is [ka] is selected from the group consisting of:

[0337] In some embodiments, LNPs comprising formula (xxii) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxii) are LNPs described by WO2021113777 (e.g., lipids of formula (2), such as lipids in Table 2 of WO2021113777). [ka] During the ceremony, each n is independently an integer from 1 to 15; R1 and R2 each independently represent [ka] is selected from the group consisting of: R3 is [ka] is selected from the group consisting of:

[0338] In some embodiments, LNPs comprising formula (xxiii) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxiii) are LNPs described by WO2021113777 (e.g., lipids of formula (3), such as lipids in Table 3 of WO2021113777). [ka] During the ceremony, X is -O-, -S-, or -OC(O)-. * is selected from the group consisting of * indicates the point of attachment to R1; R1 is, [ka] is selected from the group consisting of R2 is [ka] is selected from the group consisting of:

[0339] In some embodiments, the compositions described herein (e.g., nucleic acids (e.g., circular polyribonucleotides, linear polyribonucleotides) or proteins) are provided in LNPs that include an ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), e.g., as described in Example 1 of U.S. Pat. No. 9,867,888, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadecanedioate (LP01), e.g., as synthesized in Example 13 of WO 2015 / 095340, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., as synthesized in Examples 7, 8, or 9 of U.S. Patent Publication No. 2012 / 0027803, which is incorporated herein by reference in its entirety. In one embodiment, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, as synthesized in Examples 14 and 16 of WO 2010 / 053572, which is incorporated by reference in its entirety.In certain embodiments, the ionizable lipid is the imidazole cholesterol ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,14,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., structure (I) from WO 2020 / 106946, which is incorporated herein by reference in its entirety.

[0340] In some embodiments, the ionizable lipid may be a cationic lipid, an ionizable cationic lipid, for example, a cationic lipid that may exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that may be easily protonated. In some embodiments, the cationic lipid is a lipid that may be positively charged, for example, under physiological conditions. Exemplary cationic lipids include one or more amine groups with a positive charge. In some embodiments, the lipid particles include cationic lipids in a combination with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structured lipids (e.g., sterols), PEG, cholesterol, and polymer-conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. Exemplary cationic lipids disclosed herein may have an effective pKa greater than 6.0. In some embodiments, the lipid nanoparticle may include a second cationic lipid that has a different effective pKa (e.g., higher than the first effective pKa) than the first cationic lipid. The lipid nanoparticles can include 40-60 mol percent cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and a therapeutic agent, such as a nucleic acid described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) encapsulated within or associated with the lipid nanoparticles. In some embodiments, the nucleic acid is formulated simultaneously with the cationic lipids. The nucleic acid can be adsorbed to the surface of the LNP, e.g., LNPs including cationic lipids. In some embodiments, the nucleic acid can be encapsulated within the LNP, e.g., LNPs including cationic lipids. In some embodiments, the lipid nanoparticles can include a targeting moiety, e.g., coated with a targeting agent. In some embodiments, the LNP formulation is biodegradable.In some embodiments, lipid nanoparticles comprising one or more lipids described herein, e.g., formula (i), (ii), (vii) and / or (ix), encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of the RNA molecules.

[0341] Exemplary ionizable lipids that may be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of International Publication No. WO2019051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, one or more of the following formulas: X of US2016 / 0311759; I of US20150376115 or US2016 / 0376224; I, II, or III of US20160151284; I, IA, II, or IIA of US20170210967; No. Ic; U.S. Patent Application Publication No. 2013 / 0178541, No. A; U.S. Patent Application Publication No. 2013 / 0303587 or U.S. Patent Application Publication No. 2013 / 0123338, No. I; U.S. Patent Application Publication No. 2015 / 0141678, No. I; U.S. Patent Application Publication No. 2015 / 0239926, No. II, III, IV, or V; U.S. Patent Application Publication No. 2017 / 0119904, No. I; WO 2017 / 117528, No. I or II; U.S. Patent Application Publication No. 2012 / 0149894 A; U.S. Patent Application Publication No. 2015 / 0057373 A; WO 2013 / 116126 A; U.S. Patent Application Publication No. 2013 / 0090372 A; U.S. Patent Application Publication No. 2013 / 0274523 A; U.S. Patent Application Publication No. 2013 / 0274504 A; U.S. Patent Application Publication No. 2013 / 0053572 A; WO 2013 / 0 16058, A; WO 2012 / 162210, A; U.S. Patent Application Publication No. 2008 / 042973, I; U.S. Patent Application Publication No. 2012 / 01287670, I, II, III, or IV; U.S. Patent Application Publication No. 2014 / 0200257, I or II; U.S. Patent Application Publication No. 2015 / 0203446, I, II, or III; U.S. Patent Application Publication No. 2015 / 0005363, I or III;U.S. Patent Application Publication No. 2014 / 0308304, I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV; U.S. Patent Application Publication No. 2013 / 0338210; WO 2009 / 132131, I, II, III, or IV; U.S. Patent Application Publication No. 2012 / 01011478, A; U.S. Patent Application Publication No. 2012 / 0027796, I or XXXV; U.S. Patent Application Publication No. 2012 / 0058144, XIV or XVII; U.S. Patent Application Publication No. 2012 / 0058144, XIV or XVII; No. 2013 / 0323269; U.S. Patent Application Publication No. I of U.S. Patent Application Publication No. 2011 / 0117125; U.S. Patent Application Publication No. I, II, or III of U.S. Patent Application Publication No. 2011 / 0256175; U.S. Patent Application Publication No. I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII of U.S. Patent Application Publication No. 2012 / 0202871; U.S. Patent Application Publication No. I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of U.S. Patent Application Publication No. 2011 / 0076335; U.S. Patent Application Publication No. 2006 / 008378 I or II of the specification; I of U.S. Patent Application Publication No. 2013 / 0123338; ​​I or XAYZ of U.S. Patent Application Publication No. 2015 / 0064242; XVI, XVII, or XVIII of U.S. Patent Application Publication No. 2013 / 0022649; I, II, or III of U.S. Patent Application Publication No. 2013 / 0116307; I, II, or III of U.S. Patent Application Publication No. 2013 / 0116307; I or II of U.S. Patent Application Publication No. 2010 / 0062967; U.S. Patent Application Publication No. 2013 / 0189351 I-X of US Patent Application Publication No. 2014 / 0039032; V of US Patent Application Publication No. 2018 / 0028664; I of US Patent Application Publication No. 2016 / 0317458; I of US Patent Application Publication No. 2013 / 0195920; 5, 6, or 10 of US Patent Application Publication No. 10,221,127; III-3 of WO 2018 / 081480; I-5 or I-8 of WO 2020 / 081938; 18 or 25 of US Patent Application Publication No. 9,867,888;US2019 / 0136231A;WO2020 / 219876II;US2012 / 0027803A1;US2019 / 0240349OF-02;US10,086,013A23;cKK-E12 / A6 of Miao et al (2020);C12-200 of WO2010 / 053572;7C1 of Dahlman et al (2017);Whitehead et al al., 304-O13 or 503-O13; TS-P4C2 of U.S. Pat. No. 9,708,628; I of WO 2020 / 106946; I of WO 2020 / 106946; and (1), (2), (3) or (4) of WO 2021 / 113777. Exemplary lipids further include any one of the lipids in Tables 1 to 16 of WO 2021 / 113777.

[0342] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,3lZ)-heptatriaconta-6,9,28,3l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), as described in, for example, Example 9 of WO2019051289A9 (incorporated herein in its entirety). In some embodiments, the ionizable lipid is lipid ATX-002, as described in, for example, Example 10 of WO2019051289A9 (incorporated herein in its entirety). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13,16-diene-1-amine (compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is compound 6 or compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated herein by reference in its entirety).

[0343] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl 16-O-dimethyl-1, ... PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diercoyl phosphatidylcholine (DEPC), palmitoyl oleoyl ... dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine phatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Further exemplary lipids include, in certain embodiments, but are not limited to, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, which is incorporated herein by reference. Such lipids include, in certain embodiments, plant lipids that have been shown to improve hepatic transfection with mRNA (e.g., DGTS).

[0344] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO 2017 / 099823 or US 2018 / 0028664, the entire contents of which are incorporated herein by reference.

[0345] In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II, or IV of US Patent Publication No. 2018 / 0028664 (herein incorporated by reference in its entirety). The non-cationic lipid may, for example, comprise 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to neutral lipid is in the range of about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0346] In some embodiments, the lipid nanoparticles do not contain any phospholipids.

[0347] In some embodiments, the lipid nanoparticles may further comprise components such as sterols to provide membrane integrity. One exemplary sterol that may be used in the lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2 , 4'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO 2009 / 127060 and U.S. Patent Publication No. 2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0348] In some embodiments, components that provide membrane integrity, such as sterols, may comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%). In some embodiments, such components comprise 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0349] In some embodiments, the lipid nanoparticles may contain polyethylene glycol (PEG) or conjugated lipid molecules. These are generally used to inhibit lipid nanoparticle aggregation and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as a (methoxypolyethylene glycol)-conjugated lipid.

[0350] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradecanoyloxy)propyl-1-methylethyl)-2-propanediol (DMG ... -0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Further exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. No. 5,885,613, U.S. Pat. No. 6,287,591, U.S. Patent Application Publication No. 2003 / 0077829, and the like. No. 2003 / 0077829, U.S. Patent Application Publication No. 2005 / 0175682, U.S. Patent Application Publication No. 2008 / 0020058, U.S. Patent Application Publication No. 2011 / 0117125, U.S. Patent Application Publication No. 2010 / 0130588, U.S. Patent Application Publication No. 2016 / 0376224, U.S. Patent Application Publication No. 2017 / 0119904, U.S. Patent Application Publication No. 2018 / 0028664, and WO 099823, all of which are incorporated by reference in their entireties. In some embodiments, the PEG-lipid is a compound of formula III, III-aI, III-a-2, III-b-1, III-b-2, or V of U.S. Patent Application Publication No. 2018 / 0028664, the entire contents of which are incorporated herein by reference. In some embodiments, the PEG-lipid is of formula II of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224, both of which are incorporated herein by reference in their entireties.In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB ( PEG-lipids may be one or more of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 3,4-ditetradecoxylbenzyl-[ω]-methyl-poly(ethylene glycol) ether, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, ... [ka] The present invention includes a structure selected from the following:

[0351] In some embodiments, lipids conjugated with molecules other than PEG can be used in place of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (GPL) conjugates can be used in place of or in addition to PEG-lipids.

[0352] Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9, all of which are incorporated herein by reference in their entireties.

[0353] In some embodiments, the PEG or conjugated lipid may comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEG or conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. The molar ratios of ionizable lipid, non-cationic lipid, sterol, and PEG / conjugated lipid may be varied as needed. For example, the lipid particles may comprise 30-70% ionizable lipid per mole or total weight of the composition, 0-60% cholesterol per mole or total weight of the composition, 0-30% non-cationic lipid per mole or total weight of the composition, and 1-10% conjugated lipid per mole or total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid per mole or total weight of the composition, 40-50% cholesterol per mole or total weight of the composition, and 10-20% non-cationic lipid per mole or total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid per mole or total weight of the composition, 20-40% cholesterol per mole or total weight of the composition, and 5-10% non-cationic lipid per mole or total weight of the composition and 1-10% conjugated lipid per mole or total weight of the composition. The composition may contain 60-70% ionizable lipid per mole or total weight of the composition, 25-35% cholesterol per mole or total weight of the composition, and 5-10% non-cationic lipid per mole or total weight of the composition. The composition may also contain up to 90% ionizable lipid per mole or total weight of the composition and 2-15% non-cationic lipid per mole or total weight of the composition.Formulations may also be used that contain, for example, 8-30% ionizable lipid per mole or total weight of the composition, 5-30% non-cationic lipid per mole or total weight of the composition, and 0-20% cholesterol per mole or total weight of the composition; 4-25% ionizable lipid per mole or total weight of the composition, 4-25% non-cationic lipid per mole or total weight of the composition, 2-25% cholesterol per mole or total weight of the composition, 10-35% conjugated lipid per mole or total weight of the composition, and 5% cholesterol per mole or total weight of the composition; or The lipid nanoparticle formulation may comprise 2-30% ionizable lipid per mole or total weight of the composition, 2-30% non-cationic lipid per mole or total weight of the composition, 1-15% cholesterol per mole or total weight of the composition, 2-35% conjugated lipid per mole or total weight of the composition, and 1-20% cholesterol per mole or total weight of the composition; or up to 90% ionizable lipid per mole or total weight of the composition and 2-10% non-cationic lipid per mole or total weight of the composition, or 100% cationic lipid per mole or total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol, and PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol, and PEGylated lipid in a molar ratio of 60:38.5:1.5.

[0354] In one embodiment, the lipid particle comprises an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, where the molar ratio of lipids is in the range of 20-70 mole percent for the ionizable lipid with a target of 40-60, the molar percent of the non-cationic lipid is in the range of 0-30 with a target of 0-15, the molar percent of the sterol is in the range of 20-70 with a target of 30-50, and the molar percent of the PEGylated lipid is in the range of 1-6 with a target of 2-5.

[0355] In one embodiment, the lipid particles comprise an ionizable lipid / non-cationic lipid / sterol / conjugated lipid molar ratio of 50:10:38.5:1.5.

[0356] In one aspect, the disclosure provides a lipid nanoparticle formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0357] In some embodiments, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be included in the lipid nanoparticles of the present invention. In other words, the lipid nanoparticles may contain other compounds in addition to the nucleic acid or at least a second nucleic acid different from the first nucleic acid. The other additional compounds may be selected from the group consisting of, but are not limited to, small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0358] In some embodiments, the LNPs comprise a biodegradable, ionizable lipid, hi some embodiments, the LNPs comprise (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate (also referred to as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,l2Z)-octadeca-9,l2-dienoate)) or another ionizable lipid. See, e.g., the lipids in WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086, and the references provided therein. In certain embodiments, the terms cationic and ionizable with respect to LNP lipids are synonymous, e.g., ionizable lipids are cationic depending on the pH.

[0359] In certain embodiments, the average LNP diameter of an LNP formulation can be tens of nm to hundreds of nm, e.g., as measured by dynamic light scattering (DLS). In certain embodiments, the average LNP diameter of an LNP formulation can be about 40 nm to about 150 nm, e.g., about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In certain embodiments, the average LNP diameter of the LNP formulation may be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the average LNP diameter of the LNP formulation may be about 80 nm. In certain embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In certain embodiments, the average LNP diameter of the LNP formulation is in the range of about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

[0360] LNPs may be relatively homogeneous in some cases. Polydispersity index may be used to indicate the homogeneity of LNPs, e.g., the size distribution of lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow size distribution. LNPs may have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of LNPs may be about 0.10 to about 0.20.

[0361] The zeta potential of LNPs can be used to indicate the electrokinetic potential of a composition. In some embodiments, the zeta potential can represent the surface charge of LNPs. Lipid nanoparticles with relatively low positive or negative charges are generally desirable, since more highly charged species can undesirably interact with cells, tissues, and other elements in the body. In certain embodiments, the zeta potential of the LNP can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0362] The efficiency of protein and / or nucleic acid encapsulation represents the amount of protein and / or nucleic acid encapsulated or otherwise associated with the LNP after preparation compared to the initial amount provided. It is desirable for the encapsulation efficiency to be high (e.g., near 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after disintegrating the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence can be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of proteins and / or nucleic acids may be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0363] The LNPs may optionally include one or more coatings. In some embodiments, the LNPs may be formulated into capsules, films, or tablets with coatings. The capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0364] Additional exemplary lipids, formulations, methods, and characterizations of LNPs are taught by WO 2020 / 061457 and WO 2021 / 113777, each of which is incorporated by reference in its entirety. Additional exemplary lipids, formulations, methods, and characterizations of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021). doi.org / 10.1038 / s41578-021-00358-0, which is incorporated by reference in its entirety (see, e.g., exemplary lipids and lipid derivatives in Figure 2 of Hou et al.).

[0365] In certain embodiments, in vitro or ex vivo cell lipofection is performed using LIPOFECTAMINE® MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using GenVoy_ILM ionizable lipid mixture (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), which is incorporated herein by reference in its entirety.

[0366] LNP formulations optimized for delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910 (both incorporated by reference) and are useful for delivery of circular polyribonucleotides and linear polyribonucleotides as described herein.

[0367] Additional specific LNP formulations useful for delivery of nucleic acids (e.g., circular polyribonucleotides, linear polyribonucleotides) are described in U.S. Pat. Nos. 8,158,601 and 8,168,775 (both of which are incorporated by reference), including the formulations used in patisiran, which is sold under the name ONPATTRO.

[0368] In embodiments, a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) encoding at least a portion (e.g., an antigenic portion) of a protein or polypeptide described herein is formulated in an LNP: (a) the LNP comprises a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid, (b) the LNP has an average particle size of 80 nm to 160 nm, and (c) the polyribonucleotide. In embodiments, the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) that has been formulated in the LNP is a vaccine.

[0369] A representative dose of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) LNP may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg / kg (RNA). In some embodiments, the dose of the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) antigen compositions described herein is 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg. EXAMPLES

[0370] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as their invention.

[0371] Example 1: Linear RNA pull-down method to enrich for circular RNA This example describes the design of a method to remove linear RNA by-products from circular RNA generated by self-splicing.

[0372] When circular RNA is generated by self-splicing, several major linear impurities or by-products can be present in the in vitro transcription (IVT) mixture: unspliced ​​linear RNA, partially spliced ​​linear RNA, nicked circular RNA, and spliced ​​introns (Figure 2).

[0373] The oligomers are designed to have a complementary sequence to an intron region, a sequence that is absent in the circular RNA product and present only in the linear by-product as part of the linear RNA or spliced-out form (Figures 2 and 3). The oligomers are designed to have a biotin attached by a TEG linker that can bind with high affinity to streptavidin protein. Once the oligomers bind to the linear by-product containing the intron sequence, the by-product can be captured by streptavidin beads and the circular RNA can be enriched in the unbound fraction.

[0374] Example 2: Linear RNA pull-down specifically captures linear by-products and enriches circular RNA This example demonstrates the enrichment of circular RNA by capturing linear by-products via oligo-streptavidin interactions.

[0375] In this example, constructs were designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the open reading frame (ORF), exon fragment 1 (E1), and the 5' half of the catalytic intron. The ORF was either Gaussia luciferase (Gluc, in Figure 4) or firefly luciferase (Fluc, in Figure 5). The length of the linear RNA was approximately 1.4 Kb for the Gluc ORF and approximately 2.6 Kb for the Fluc ORF.

[0376] For linear RNA pull-down (LP) to remove linear by-products containing intron sequences, six different oligomers to the intron region were designed: four oligomers to the 3' half-intron and two oligomers to the 5' half-intron. Each oligomer was 23 nucleotides and had a biotin linked by TEG.

[0377] Linear RNA was synthesized by in vitro transcription from a DNA template using T7 RNA polymerase in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 minutes. Synthesized linear RNA was purified with an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; no further or additional reactions were required.

[0378] Self-spliced ​​RNA (200 pmol) was mixed with the same amount of each oligomer (200 pmol of each oligomer, 1.2 nmoles of oligomer in total) in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) in a total of 500 μl (final RNA concentration was 400 nM). To test the effect of RNA:oligomer ratio on LP efficiency, 2x (800 nM) and 5x more oligomer (2 μM) were used. As a negative control, RNA without oligomer was used. The RNA-oligomer mixture was incubated for 30 min at room temperature (RT) and then mixed with 100 μl of streptavidin-SEPHAROSE® (Sigma). The mixture was incubated for 1 h at RT on a mix rotor, and the unbound fraction was collected by spinning down the streptavidin-SEPHAROSE® beads by centrifugation. The beads were washed 3 times with 1x binding buffer. The RNA bound to the beads was eluted by heating the resin at 75° C. for 10 min in the presence of 1x binding buffer ("Eluted" in Figures 4 and 5). The RNA still bound to the beads was eluted by heating the beads at 95° C. for 5 min in the presence of 95% formamide ("Resin" in Figures 4 and 5). The concentration of unbound and eluted RNA was measured by Qubit, and 200 ng of RNA was separated by urea polyacrylamide gel electrophoresis (urea PAGE), stained with a gel stain, and visualized using an image processing system.

[0379] For both 1.4 Kb (Figure 4) and 2.6 Kb (Figure 5) RNAs, a significant enrichment of circular RNAs in the unbound fractions was observed compared to the input (approximately 90% enrichment for 1.4 Kb RNA (Figure 4) and 50% enrichment for 2.6 Kb RNA (Figure 5)). The majority of the RNA bound to the resin was linear RNA containing intron regions (linear RNA, Figures 4 and 5), indicating that the oligomers specifically captured intron-containing RNA. Increasing the oligomer concentration did not affect the enrichment yield of circular RNA for both 1.4 Kb and 2.6 Kb RNAs.

[0380] Example 3: A single oligomer can capture linear RNA by-products and enrich for circular RNA This example demonstrates the enrichment of circular RNA by capturing linear by-products via oligo-streptavidin interactions. In this example, the linear RNA contained an extended region at the 5' end of the 3' half-intron that had been spliced ​​out during self-splicing.

[0381] In this example, a construct was designed containing the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. The construct contained an extended sequence of 36 nucleotides at the 5' end.

[0382] To pull down linear by-products containing the extended sequence, oligomers were designed to the extended region: the oligomers were 36 nucleotides and had a biotin linked by TEG.

[0383] Linear RNA was synthesized by in vitro transcription from DNA template using T7 RNA polymerase in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 minutes. Synthesized linear RNA was purified with RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; no further / additional reaction was required.

[0384] Self-spliced ​​RNA (200 pmoles) was mixed with 400 pmoles of oligo in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) (final RNA concentration was 400 nM, oligomer concentration was 800 nM). To test the effect of RNA:oligomer ratio on linear RNA pull-down efficiency, 2.5x (1 μM) or more than 5x (2 μM) oligomer was used. As a negative control, RNA without oligomer was used. The RNA-oligomer mixture was incubated at RT for 30 min and then mixed with 100 μl of streptavidin-SEPHAROSE® (Sigma). The mixture was incubated for 1 h at RT on a mix rotor and the unbound fraction was collected by spinning down the SEPHAROSE® beads by centrifugation. The beads were washed 3 times with 1x binding buffer. The RNA bound to the resin was eluted by heating the resin in the presence of 1× binding buffer at 75° C. for 10 min ("Eluted" in FIG. 6). The RNA still bound to the beads was eluted by heating the resin in the presence of 95% formamide at 95° C. for 5 min ("Resin" in FIG. 6). The concentration of unbound and eluted RNA was measured by Qubit, and 200 ng of RNA was separated by urea PAGE, stained with a gel stain, and visualized by using an imaging system.

[0385] Enrichment of circular RNA in the unbound fraction was significant compared to the input (50% enrichment at 800 nM oligo (Figure 6)). The majority of RNA bound to the resin was linear RNA with an extended region, indicating that the oligomer specifically captures linear RNA with an extended region (linear RNA, Figure 6). Increasing oligomer concentration significantly affected the enrichment yield of circular RNA, from 50% for the 800 nM oligomer to 96% for the 4 μM oligomer.

[0386] Example 4: Comparing batch purification and column packing methods This example demonstrates two different approaches to enriching circular RNA by capturing linear by-products via oligo-streptavidin interactions: one method was to incubate the RNA-oligo mixture with streptavidin-SEPHAROSE® beads in a tube (batch method), and the other method was to pre-pack the beads into a column and allow the RNA-oligomer mixture to pass through by gravity (column method).

[0387] In this example, a construct was designed containing the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron.

[0388] To pull down linear by-products containing intron sequences, six different oligomers against the intron region were designed: an oligomer against the 3' half-intron and two oligomers against the 5' half-intron. Each oligomer was 23 nucleotides and had a biotin linked by TEG.

[0389] Linear RNA was synthesized by in vitro transcription from DNA template using T7 RNA polymerase in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 minutes. Synthesized linear RNA was purified with RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; no further / additional reaction was required.

[0390] Self-spliced ​​RNA (1 nmole) was mixed with 2 nmoles of each oligo in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) in a total of 2.5 ml (final RNA concentration was 400 nM, oligomer concentration was 800 nM per oligomer). The RNA-oligomer mixture was incubated at RT for 30 min in the presence or absence of heating at 75°C for 3 min. As a negative control, an RNA mixture without oligomer was used. For the batch purification method, 500 μl of streptavidin-SEPHAROSE® beads were added to the mixture. The mixture was then incubated on a mix rotor for 1 h at RT, and the unbound fraction was collected by spinning down the SEPHAROSE® beads by centrifugation. For column purification, 500 μl of beads were pre-packed into an empty column and the RNA-oligomer mixture was placed on the packed resin. The RNA-oligomer mixture was passed through the resin by gravity and the flow-through was collected. The RNA concentration of the unbound or flow-through was measured by Qubit, and 200 ng of RNA was separated by urea PAGE, stained with a gel stain, and visualized using an imaging system.

[0391] No significant difference in enrichment of circular RNA compared to input was found between batch and column purification methods (see, e.g., lanes 3 and 4 vs. lanes 6 and 7, respectively) (Figure 7). Heating and cooling the RNA-oligomers did not increase the enrichment of circular RNA. This data indicates that the linear RNA pull-down (LP) method is scalable through column packing of SEPHAROSE® beads.

[0392] Example 5: Sequential linear RNA pull-down can further enrich circular RNAs when circularization efficiency is low This example demonstrates the enrichment of circular RNA by sequential linear RNA pull-down.

[0393] In this example, a construct was designed containing the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron.

[0394] To pull down linear by-products containing intron sequences, six different oligomers to the intron region were designed: four to the 3' half-intron and two to the 5' half-intron. Each oligomer was 23 nucleotides and had a biotin linked by TEG.

[0395] Linear RNA was synthesized by in vitro transcription from DNA template using T7 RNA polymerase in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 minutes. Synthesized linear RNA was purified with RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; however, no further / additional reaction was required. Self-spliced ​​RNA (1 nmole) was mixed with 2 nmoles of each oligo in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) in a total of 2.5 ml (final RNA concentration was 400 nM, oligomer concentration was 800 nM per each oligomer). The RNA-oligomer mixture was incubated at RT for 30 min. For column purification, 500 μl of beads were pre-packed in an empty column and placed on top of the loaded resin with the RNA-oligomer mixture. The RNA-oligomer mixture was passed through the resin by gravity and the flow-through was collected. The collected flow-through was mixed with additional oligomer (800 nM final) and then passed through freshly pre-packed resin. The flow-through was collected and the first and second round concentrations of the flow-through were measured by Qubit. 200 ng of RNA was separated by urea PAGE, stained with gel stain and visualized using an image processing system.

[0396] The first round of linear RNA pull-down resulted in a 130% enrichment of circular RNA (Figure 8, 1st LP; circular RNA purity from 23% to 54%). The second round of pull-down resulted in additional enrichment, then ultimately achieved >200% enrichment (Figure 8, 2nd LP; circular RNA purity from 23% to 72%).

[0397] Example 6: Optimization of linear RNA pull-down with different salt concentrations This example demonstrates the effect of salt concentration in the binding buffer on circular RNA enrichment by capturing linear by-products via oligo-streptavidin interactions.

[0398] In this example, constructs were designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. The ORF was either hEPO (hEPO, in FIG. 9A) or SARS-CoV-2 spike protein (spike, in FIG. 9B). The length of the linear RNA was about 1.2 Kb for the hEPO ORF and about 4.5 Kb for the SARS-CoV-2 spike ORF (it runs with a 2 Kb ladder on a 6% urea PAGE even though the RNA size is 4.5 Kb).

[0399] To pull down linear by-products containing intron sequences, six different oligomers to the intron region were designed: four to the 3' half-intron and two to the 5' half-intron. Each oligomer was 23 nucleotides and had a biotin linked by TEG.

[0400] Linear RNA was synthesized by in vitro transcription from a DNA template using T7 RNA polymerase in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 minutes. Synthesized linear RNA was purified with an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; no further or additional reactions were required.

[0401] Self-spliced ​​RNA (1 nmole) was mixed with 2 nmoles of each oligo in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) in a total of 2.5 ml (final RNA concentration was 400 nM and oligomer concentration was 800 nM per oligomer). To test the effect of salt concentration on circular RNA enrichment, NaCl concentrations of 500 mM, 1000 mM and 2000 mM were also tested. The RNA-oligomer mixture was incubated at RT for 30 min. For column purification, 500 μl of beads were pre-packed in an empty column and placed on the resin loaded with the RNA-oligomer mixture. The RNA-oligomer mixture was passed through the resin by gravity and the flow-through was collected. The flow-through was collected and the concentration of RNA in the flow-through was measured by Qubit. 200 ng of RNA was separated by urea PAGE, stained with gel stain and visualized using an image processing system.

[0402] For 1.2 Kb RNA, no significant difference was observed for circular RNA enrichment under different salt conditions (Figure 9A). However, circular RNA enrichment of 4.5 Kb RNA increased when salt concentration was increased (Figure 9B). At 150 mM NaCl, 60% enrichment was achieved (from 20% circular RNA purity from input to 32% circular RNA purity), whereas at 2000 mM NaCl, 90% enrichment was achieved (from 20% circular RNA purity from input to 38% circular RNA purity). This data indicates that higher salt can improve circular RNA enrichment of long RNAs in linear RNA pull-down (LP).

[0403] Example 7: Linear RNA pull-down mediated circular RNA enrichment enhances circular RNA expression after downstream purification This example demonstrates enhanced expression from circular RNA that has been purified by linear RNA pull-down.

[0404] In this example, constructs were designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing an ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. The ORF was either Gaussia luciferase (Gluc, in the case of Figure 10) or hEPO (in the case of Figure 11).

[0405] To pull down linear by-products containing intron sequences, six different oligomers to the intron region were designed: four oligomers to the 3' half-intron and two oligomers to the 5' half-intron. Each oligomer was 23 nucleotides and had a biotin linked by TEG.

[0406] Linear RNA was synthesized by in vitro transcription from DNA template using T7 RNA polymerase in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 minutes. Synthesized linear RNA was purified with RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; no further / additional reaction was required.

[0407] Self-spliced ​​RNA (1 nmole) was mixed with 2 nmoles of each oligo in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) in a total of 2.5 ml (final RNA concentration was 400 nM and oligomer concentration was 800 nM per each oligomer). The RNA-oligomer mixture was incubated at RT for 30 min. For column purification, 500 μl of beads were pre-packed in an empty column and placed on top of the packed resin with the RNA-oligomer mixture. The RNA-oligomer mixture was passed through the resin by gravity and the flow-through was collected. The collected flow-through was buffer exchanged with water by Amicon ultracentrifugal filter (100K cut-off).

[0408] The enriched circular RNA was purified in different downstream steps. As a control, in vitro transcribed RNA without enrichment was purified in parallel.

[0409] In the case of circular RNA encoding Gluc, four different purification methods were tested. The first method was column purification, where concentrated circular RNA was column purified (Monarch). The second method was gel purification. Concentrated circular RNA encoding Gluc was purified by urea PAGE, eluted with buffer (0.5 M sodium acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNAse-free water. The third method was reversed-phase chromatography (RP). Circular RNA encoding Gluc was purified by reversed-phase chromatography, and fractions were buffer exchanged with sodium citrate and then water in an Amicon ultracentrifugal filter (100K cutoff). The fourth method was anion exchange chromatography (AEX). Circular RNA encoding Gluc was purified by anion exchange chromatograph (AEX), and fractions were buffer exchanged with water in an Amicon ultracentrifugal filter (100K cutoff). In comparison, in vitro transcribed RNA was subjected to RP and AEX purification.

[0410] For the hEPO-encoding circular RNA, four different purification methods were tested. The first method used only buffer-exchanged circular RNA without purification (BE). The second method was gel purification. Concentrated hEPO-encoding circular RNA was purified by urea PAGE, eluted with buffer (0.5 M sodium acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated, and resuspended in RNAse-free water. The third method was reversed-phase chromatography (RP). The hEPO-encoding circular RNA was purified by reversed-phase chromatography, and fractions were buffer-exchanged with sodium citrate and then water on an Amicon ultracentrifugal filter (100K cutoff). The fourth method was anion-exchange chromatography (AEX). The hEPO-encoding circular RNA was purified by anion-exchange chromatograph (AEX), and fractions were buffer-exchanged with water on an Amicon ultracentrifugal filter (100K cutoff). As a comparison, in vitro transcribed RNA was subjected to gel purification, RP and AEX purification.

[0411] To compare the expression of Gluc from enriched (LP) or non-enriched (IVT) circular RNA, 0.1 pmole of purified circular RNA was transfected into HeLa cells (10,000 cells / well in a 96-well plate) using LIPOFECTAMINE® MessengerMAX transfection reagent (Invitrogen). At 6, 24 or 48 hours, the cell medium was collected. To measure Gluc activity, 10 μl of the collected cell medium was transferred to a white 96-well plate and a bioluminescence reporter assay system was used according to the manufacturer's instructions (Pierce Gaussia Luciferase Flash Assay Kit, 16158, Thermo Scientific). The plate was read in a luminometer (Promega).

[0412] To compare the expression of hEPO from concentrated (LP) or non-concentrated (IVT) circular RNA, 0.25 pmoles of purified circular RNA was transfected into HeLa cells (10,000 cells / well in 96-well plates) using LIPOFECTAMINE® MessengerMAX transfection reagent (Invitrogen). At 24 or 48 hours, cell culture medium was collected. The amount of secreted hEPO protein was measured by hEPO ELISA kit (Invitrogen) according to the manufacturer's instructions.

[0413] For both Gluc and hEPO RNA, circular RNAs purified via linear RNA pull-down (LP) showed better expression than circular RNAs purified using the same method without enrichment (e.g., LP-RP showed more than 4-fold higher expression for Gluc and 2-fold higher expression for hEPO than IVT-RP). This data indicates that enrichment of circular RNAs using the methods described herein enhances expression.

[0414] Example 8: Linear RNA pull-down with reduced numbers of oligomers enriches for circular RNA This example demonstrates the enrichment of circular RNA by capturing linear by-products through oligo-streptavidin interactions with fewer oligomers.

[0415] In this example, the construct was designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. In this example, the ORF is hEPO, and the length of the linear RNA carrying the hEPO ORF is approximately 1.4 Kb.

[0416] For linear pull-down to remove linear by-products containing intron sequences, six different oligomers for the intron region were designed: four oligomers for the 3' half-intron (#1-#4), and two oligomers for the 5' half-intron (#5 and #6) (Figure 12). Each oligomer was 23 nucleotides and had a biotin linked by TEG. In this example, the pull-down efficiency was tested when the number of oligomers was reduced.

[0417] In the first test, oligomers against the 3' half-intron were tested (#1-#4). Linear RNA was synthesized by in vitro transcription with T7 RNA polymerase from a DNA template in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 min. Synthesized linear RNA was purified with an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; no further or additional reactions were required.

[0418] Self-spliced ​​RNA (200 picomoles) was mixed with each oligomer (#1-#4) for the 3' half-intron at two different concentrations (2.4 μM or 4.8 μM) in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) in a total of 500 μl (final RNA concentration was 400 nM). As a negative control, RNA without oligomers was used. For positive control, a mixture of all six oligomers (#1-#6) was used. The RNA-oligomer mixture was incubated for 30 min at room temperature (RT). For column purification, 500 μl of resin was pre-filled in an empty column and the RNA-oligomer mixture was placed on the filled resin. The RNA-oligomer mixture was passed through the resin by gravity and the flow-through was collected. The concentration of the flow-through RNA was measured by Qubit, and 200 ng of RNA was separated by urea PAGE, stained with gel staining solution, and visualized using an imaging system.

[0419] The #1 and #2 oligomers, at both 2.4 μM and 4.8 μM concentrations, showed similar enrichment efficiency (FIG. 13A, 6% gel) and removal of the 3' half-intron (FIG. 13B, 10% gel) compared to the positive control when using all six oligomers in the pull-down.

[0420] In the second test, oligo combinations for both the 3' half-intron (#1 and #2) and the 5' half-intron (#5 and #6) were tested. Linear RNA was synthesized by in vitro transcription from a DNA template using T7 RNA polymerase in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 min. The synthesized linear RNA was purified with an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; no further or additional reactions were required.

[0421] Self-spliced ​​RNA (200 pmol) was mixed with #1 oligomer combined with #5 or #6 oligo, or #2 oligomer combined with #5 or #6 oligo in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) in a total of 500 μl (final RNA concentration was 400 nM) (final oligomer concentration is 2.4 μM, respectively). As a negative control, RNA without oligomer was used. As further controls, only #1 oligo or only #5 oligomer was used. For positive control, a mixture of all six oligomers (#1-#6) was used. The RNA-oligomer mixture was incubated for 30 min at room temperature (RT). For column purification, 500 μl of resin was pre-packed in an empty column and the RNA-oligomer mixture was placed on the packed resin. The RNA-oligomer mixture was passed through the resin by gravity and the flow-through was collected. The concentration of the flow-through RNA was measured by Qubit and 200 ng of RNA was separated by urea PAGE, stained with a gel stain and visualized using an imaging system.

[0422] The combination of two oligos for the 3' half-intron or 5' half-intron showed similar circular RNA enrichment efficiency (Figure 14A, 6% gel) and better intron removal than using a single oligomer for the 3' half-intron (Figure 14B, 10% gel). The combination of #1 and #5 oligos showed similar circular RNA enrichment efficiency (Figure 14A) and intron removal efficiency (Figure 14B) compared to the positive control when using a total of six oligomers for pulldown. These data indicate that the use of two oligomers has equivalent circular RNA enrichment and intron removal efficiency to the use of six oligomers.

[0423] Example 9: Linear RNA pull-down mediated circular RNA enrichment using reduced numbers of oligomers results in the expression of circular RNA This example demonstrates expression from circular RNA that has been purified by linear RNA pull-down using two oligomers.

[0424] In this example, the construct was designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the hEPO ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. Linear RNA was synthesized by in vitro transcription from a DNA template using T7 RNA polymerase in the presence of 7.5 mM NTPs. Template DNA was removed by treatment with deoxyribonuclease for 20 minutes. The synthesized linear RNA was purified with an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurred during transcription; no additional reaction was required.

[0425] For linear pull-down to remove linear by-products containing intron sequences, six different oligomers (#1 to #6) against the intron region were designed as described in Example 8.

[0426] Self-spliced ​​RNA (200 pmol) was mixed with #1 oligomer and #5 or #6 oligomer, or #2 oligomer and #5 or #6 oligomer in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) in a total of 500 μl (final RNA concentration was 400 nM) (final oligomer concentration is 2.4 μM, respectively). As a negative control, RNA without oligomer was used. As another control, only #1 oligomer or only #5 oligomer was used. For positive control, a mixture of all six oligomers (#1-#6) was used. The RNA-oligomer mixture was incubated for 30 min at room temperature (RT). For column purification, 500 μl of resin was pre-packed in an empty column and the RNA-oligomer mixture was placed on the packed resin. The RNA-oligomer mixture was passed through the resin by gravity and the flow-through was collected. The collected flow-through was buffer exchanged with water through an Amicon ultracentrifugal filter (100K cutoff).

[0427] To compare the expression of hEPO from enriched (LP) circular RNA with different oligomers, 0.25 pmoles of purified circular RNA was transfected into HeLa cells (10,000 cells / well in 96-well plates) using LIPOFECTAMINE® MessengerMAX transfection reagent (Invitrogen). At 24 hours, cell culture medium was collected. The amount of secreted hEPO protein was measured by hEPO ELISA kit (Invitrogen) according to the manufacturer's instructions.

[0428] Circular RNAs purified via linear pull-down (LP) using two oligomers showed comparable expression to circular RNAs purified via LP using all six oligomers (#1-#6) (Figure 15). In particular, the data show that expression of circular RNAs obtained from circular RNAs purified via LP using a combination of two oligomers (i.e., an oligomer against the 3' half-intron and an oligomer against the 5' half-intron) is comparable in expression to that observed from circular RNAs purified via LP using all six oligomers.

[0429] Example 10: Linear RNA pull-down with a single oligomer targeting both the 3' half-intron and the 5' half-intron This example demonstrates the enrichment of circular RNA by capturing linear by-products via oligo-streptavidin interactions with a single oligomer that targets both the 3' and 5' half-introns.

[0430] In this example, the construct was designed to contain the 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing the ORF, exon fragment 1 (E1), and the 5' half of the catalytic intron. The ORF was hEPO and the linear RNA was approximately 1.4 Kb in length.

[0431] In this example, two oligomers were designed: oligo5-Bio_1A5 consists of the #1 oligo (oligomer for the 3' half-intron described in Example 8) followed by the #5 oligo (oligomer for the 5' half-intron described in Example 8); oligo5-Bio_5A1 consists of the #5 oligo followed by the #1 oligo. Both the 5-Bio_1A5 and 5-Bio_5A1 oligomers have eight nucleotides of a...

Claims

1. 1. A method for separating a linear polyribonucleotide comprising a target region from a plurality of polyribonucleotides comprising a mixture of linear and cyclic polyribonucleotides, comprising: (a) providing a sample comprising the plurality of polyribonucleotides, a subset of which comprises the linear polyribonucleotide comprising the target region; (b) contacting the sample with an oligonucleotide that hybridizes to the target region; (c) separating the linear polyribonucleotide comprising the target region hybridized to the oligonucleotide from the plurality of polyribonucleotides in the sample; A method comprising:

2. The method of claim 1 , wherein the cyclic polyribonucleotide lacks the target region.

3. 1. A method for separating a polyribonucleotide comprising a target region from a plurality of polyribonucleotides, comprising: (a) providing a sample comprising the plurality of polyribonucleotides, a subset of which comprises the target region, wherein the target region is not located at the 3' or 5' end of the polyribonucleotide comprising the target region; (b) contacting the sample with an oligonucleotide that hybridizes to the target region; (c) separating the polyribonucleotides comprising the target region hybridized to the oligonucleotides from the plurality of polyribonucleotides in the sample; A method comprising:

4. 1. A method for separating a linear polyribonucleotide comprising a target region from a plurality of circular polyribonucleotides comprising said target region, comprising: (a) providing a sample comprising said plurality of polyribonucleotides; (b) contacting the sample with an oligonucleotide that hybridizes to the target region of the linear polyribonucleotide with a first binding affinity and to the target region of the circular polynucleotide with a second binding affinity different from the first binding affinity; (c) separating the linear polyribonucleotide comprising the target region hybridized to the oligonucleotide from the plurality of circular polyribonucleotides in the sample; A method comprising:

5. 1. A method for separating polyribonucleotides in a first location that includes a target region from a plurality of polyribonucleotides in a second location that includes said target region, comprising: (a) providing a sample comprising said plurality of polyribonucleotides; (b) contacting the sample with an oligonucleotide that hybridizes to the target region of the polynucleotide in the first configuration with a first binding affinity and to the target region of the polynucleotide in the second configuration with a second binding affinity different from the first binding affinity; (c) separating the polyribonucleotides at the first location that include the target region hybridized to the oligonucleotides from the plurality of polyribonucleotides at the second location in the sample; A method comprising:

6. The method of any one of claims 1 to 5, wherein step (c) comprises immobilizing the oligonucleotide.

7. 6. The method of any one of claims 1 to 5, wherein the oligonucleotide is conjugated to a particle or a first capture agent, optionally by a linker, optionally wherein the linker is triethylene glycol and / or the chemical linker is conjugated to the 3' end or the 5' end of the oligonucleotide.

8. The method of claim 7, further comprising preparing a second capture agent that binds to the first capture agent, and optionally, the first capture agent comprises an antigen and the second capture agent comprises an antibody or an antigen-binding fragment thereof.

9. 9. The method of claim 8, wherein the second capture agent is conjugated to a particle, optionally the particle being a magnetic particle or a bead.

10. The method of any one of claims 1 to 5, wherein the polyribonucleotide comprising the target region comprises an intron or a portion thereof.

11. 11. The method of claim 10, wherein the target region comprises an intron or a portion thereof, or the target region is located 5' or 3' to the intron or a portion thereof.

12. The method of claim 11, wherein the method comprises separating spliced ​​polyribonucleotides from unspliced ​​or partially spliced ​​polyribonucleotides, and optionally, the spliced ​​polyribonucleotides are cyclic polyribonucleotides.

13. 13. The method of claim 12, wherein the spliced ​​polyribonucleotide lacks an intron or a portion thereof, or wherein the method enriches the amount of the spliced ​​polyribonucleotide by at least 50% compared to the sample.

14. 6. The method of any one of claims 1 to 5, further comprising washing the captured polyribonucleotides comprising the target region one or more times and / or performing a first elution step to release the captured polyribonucleotides comprising the target region.

15. The method of any one of claims 1 to 5, wherein step (b) comprises recovering a portion of the sample that is not bound by the oligonucleotide.

16. 6. The method of any one of claims 1 to 5, comprising providing a plurality of oligonucleotides, each oligonucleotide hybridizing to a different target region.

17. 6. The method of any one of claims 1 to 5, wherein the oligonucleotides have at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementarity to equal length portions of the target region.

18. 6. A population of polyribonucleotides produced by the method of any one of claims 1 to 5, optionally comprising cyclic polyribonucleotides lacking a target region, wherein the cyclic polyribonucleotides constitute at least 40% (mol / mol) of the total polyribonucleotides in the composition.

19. A composition comprising a polyribonucleotide comprising a target region and an oligonucleotide designed to hybridize to said target region, wherein said oligonucleotide is conjugated to a first capture agent.

20. 20. The composition of claim 19, further comprising a polyribonucleotide lacking the target region.

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