Compositions and methods for purifying polyribonucleotides

Abstract

This disclosure relates to compositions and methods for separating and / or purifying polyribonucleotides. Polyribonucleotides can be separated from a mixture of polyribonucleotides and reagents that bind to aptamers on the polyribonucleotides. The method comprises (a) preparing a sample comprising a plurality of polyribonucleotides, a subset of which comprises linear polyribonucleotides having aptamers; (b) contacting the sample with reagents that bind to aptamers; and (c) separating the linear polyribonucleotides having aptamers bound to the reagents from the plurality of polyribonucleotides.

Description

Technical Field

[0001] Sequence Listing This application has been electronically filed in Extensible Markup Language (XML) format and includes a sequence listing, which is hereby incorporated by reference in its entirety. The XML copy created on October 30, 2023, is named 51509-067WO2_Sequence_Listing_10_27_23.XML and is 143,826 bytes in size.

Background Art

[0002] Polynucleotides are useful for various therapeutic and modification applications. Therefore, new compositions and methods for separating and purifying polynucleotides are needed.

Summary of the Invention

Means for Solving the Problems

[0003] In one aspect, the present disclosure features a method of separating a linear polynucleotide having an aptamer from a plurality of polynucleotides including a mixture of linear and circular polynucleotides. The method includes: (a) preparing a sample comprising a plurality of polynucleotides, a subset of which comprises a linear polynucleotide having an aptamer; (b) contacting the sample with a reagent that binds to the aptamer; and (c) separating the linear polynucleotide having the aptamer bound to the reagent from the plurality of polynucleotides.

[0004] In some embodiments, the linear polynucleotide having an aptamer is transcribed from deoxyribonucleotides encoding the linear polynucleotide comprising the aptamer.

[0005] In some embodiments, the method further includes generating a linear polyribonucleotide having an aptamer by attaching the aptamer to the linear polyribonucleotide.

[0006] In another embodiment, the present disclosure features a method for separating linear polyribonucleotides from a plurality of polyribonucleotides, including linear polyribonucleotides and cyclic polyribonucleotides. The method comprises: (a) preparing a sample comprising a plurality of polyribonucleotides, a subset of which comprises linear polyribonucleotides; (b) binding aptamers to the linear polyribonucleotides; (c) contacting the sample with a reagent that binds to the aptamers; and (d) separating the linear polyribonucleotides having aptamers bound to the reagent from the plurality of polyribonucleotides.

[0007] In some embodiments, the step of attaching an aptamer to a linear polyribonucleotide includes covalently attaching the aptamer to the 3' or 5' end of the linear polyribonucleotide.

[0008] In some embodiments, the step of attaching an aptamer to a linear polyribonucleotide includes hybridizing the aptamer to a region of the linear polyribonucleotide.

[0009] In some embodiments of any of the aforementioned models, the cyclic polyribonucleotide lacks an aptamer.

[0010] In some embodiments, the separation step includes recovering a portion of the sample that has not been bound by the reagent. For example, the portion of the sample that has not been bound by the reagent may contain cyclic polyribonucleotides.

[0011] The reagent may be, for example, a polypeptide, small molecule, lipid, carbohydrate, RNA, or metal.

[0012] In some embodiments, the reagent is a polypeptide. The polypeptide may be, for example, protein A, streptavidin, lambda peptide, or MS2 bacteriophage coat protein. The polypeptide may be selected from Table 1.

[0013] In some embodiments, the reagent is a small molecule. The small molecule may be, for example, biotin or tetracycline. In some embodiments, the small molecule is a metabolite or an amino acid. In some embodiments, the small molecule is selected from Table 2.

[0014] In some embodiments, the reagent is a carbohydrate.

[0015] In some embodiments, the aptamer comprises a nucleic acid sequence selected from any one of sequence numbers 1 to 124.

[0016] In some embodiments, the aptamer may contain a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with respect to the aptamers shown in Table 1. The reagent may be the corresponding reagent shown in Table 1 (e.g., any one of SEQ ID NOs: 1 to 66).

[0017] In some embodiments, the aptamer may contain a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with the aptamers shown in Table 2 (e.g., any one of SEQ ID NOs. 67 to 119). The reagent may be the corresponding reagent shown in Table 2.

[0018] In some embodiments, the aptamer may contain a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with respect to the aptamers shown in Table 3. The reagent may be the corresponding reagent shown in Table 3 (e.g., SEQ ID NO: 120 or 121).

[0019] In some embodiments, the aptamer may contain a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with respect to the aptamers shown in Table 4. The reagent may be the corresponding reagent shown in Table 4 (e.g., any one of SEQ ID NOs. 122-124).

[0020] In some embodiments, the separation step includes immobilizing the reagent.

[0021] In some embodiments, the reagent is conjugated to particles. The particles may include, for example, magnetic beads. In some embodiments, the reagent is conjugated to a resin containing multiple particles. The resin may include, for example, crosslinked poly[styrene-divinylbenzene], agarose, or SEPHAROSE®.

[0022] In some embodiments, the column includes a resin. The method may include contacting the sample with the column and recovering an eluate containing a portion of the sample that is not bound to the reagent from multiple polyribonucleotides in the sample.

[0023] In some embodiments, the method further comprises preparing a linear precursor polyribonucleotide prior to step (a) and cyclizing the linear precursor polyribonucleotide to produce a cyclic polyribonucleotide. The linear precursor may include a 5' self-splicing intron fragment and a 3' self-splicing intron fragment. The cyclic polyribonucleotide can be produced by self-splicing of the linear precursor. The 5' self-splicing intron fragment and the 3' self-splicing intron fragment may each be a group I or group II self-splicing intron fragment. In some embodiments, the cyclization of the cyclic polyribonucleotide is brought about by sprint ligation of the linear precursor.

[0024] In some embodiments, the cyclic polyribonucleotide includes an open reading frame (ORF). The ORF may encode a polypeptide. In some embodiments, the level of expression of the purified cyclic polyribonucleotide from the ORF is increased by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) compared to the level of expression from the ORF before isolation.

[0025] In some embodiments, the cyclic polyribonucleotide includes at least one internal ribosome entry site (IRES) (e.g., IRES). The ORF can be operably ligated to the IRES.

[0026] In some embodiments, the separation step further includes washing the polyribonucleotides having aptamers bound to the reagent one or more times.

[0027] In some embodiments, the separation step further includes eluting the polyribonucleotide having the aptamer from the reagent.

[0028] In some embodiments, the method includes preparing a plurality of reagents, each of which binds to a different aptamer region.

[0029] In some embodiments, the method involves preparing the reagent in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10) relative to the polyribonucleotide containing the aptamer region.

[0030] In some embodiments, the method separates at least 500 μg (e.g., at least 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, or more) of cyclic polyribonucleotides.

[0031] In some embodiments, cyclic polyribonucleotides in amounts ranging from 500 μg to 1,000 mg (e.g., 500 μg to about 1 mg, e.g., about 600 μg, 700 μg, 800 μg, 900 μg, or 1 mg; e.g., about 1 mg to about 10 mg, e.g., about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg; e.g., about 10 mg to about 100 mg, e.g., about 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg; e.g., about 100 mg to about 1,000 mg, e.g., 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg) are separated by the method.

[0032] In another embodiment, the disclosure features a population of polyribonucleotides produced by the method described herein. The population may include, for example, cyclic polyribonucleotides lacking aptamers, which may constitute at least 40% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) (moles / mol) of the total polyribonucleotides in the composition. In some embodiments, the population may contain less than 40% (e.g., less than 30%, 20%, 10%, or 5%) (moles / mol) of the total polyribonucleotides in the composition of linear polyribonucleotides.

[0033] In some embodiments, the total weight of polyribonucleotides in a population of polyribonucleotides is at least 500 μg (for example, at least 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, or more).

[0034] In some embodiments, the total weight of polyribonucleotides in a population of polyribonucleotides is 500 μg to 1,000 mg (e.g., 500 μg to about 1 mg, e.g., about 600 μg, 700 μg, 800 μg, 900 μg, or 1 mg, e.g., about 1 mg to about 10 mg, e.g., about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg, e.g., about 10 mg to about 100 mg, e.g., about 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg, e.g., about 100 mg to about 1,000 mg, e.g., 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1,000 mg).

[0035] In another embodiment, the present disclosure features a composition comprising a collection of polyribonucleotides described herein (e.g., produced by a method described herein) and a diluent, carrier, or excipient.

[0036] definition To facilitate understanding of this disclosure, several terms are defined below. Terms as defined herein have the meanings generally understood by those skilled in the art in the area relating to this disclosure. Terms such as “a,” “an,” and “the” are not intended to refer only to singular entities, but also to general classes, although specific examples may be used for illustrative purposes only. The term “or” is used to mean “and / or” unless expressly intended to refer only to substitutes, or unless the substitutes are mutually exclusive, but this disclosure supports definitions that refer only to substitutes and “and / or.” Terms herein are used to describe specific embodiments, but their use should not be construed as limiting, except as outlined in the claims.

[0037] When used herein, any value provided within a range of values ​​includes both the upper and lower limits, as well as any value that falls within the range of the upper and lower limits.

[0038] As used herein, the term "approximately" refers to a value within ±10% of the enumerated values.

[0039] As used herein, the term "aptamer" refers to a polynucleotide that specifically binds to a molecule (e.g., a reagent). An aptamer may be a part of a polyribonucleotide molecule. Typically, aptamers are 5 to 500 nucleotides long (e.g., 5 to 200, 5 to 150, 5 to 100, 5 to 50, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 20 to 200, 20 to 150, 20 to 100, or 20 to 50 nucleotides). Aptamers bind to their target through secondary structure rather than sequence homology.

[0040] 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 polyribonucleotides) to cells by covalent modification of cyclic polyribonucleotides, via a partially or fully encapsulated agent or a combination thereof. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type substances), nanoparticles (e.g., nanoparticles encapsulated or covalently bonded to cyclic polyribonucleotides), liposomes, fusosomes, in vitro differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently bonded to polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents).

[0041] As used herein, the terms “cyclic polyribonucleotide,” “cyclic RNA,” and “circRNA” are interchangeable and mean polyribonucleotide molecules having a structure without free ends (i.e., free 3' and / or 5' ends), such as polyribonucleotide molecules that form a cyclic or endless structure via covalent or non-covalent bonds. A cyclic polyribonucleotide may be, for example, a covalently closed polyribonucleotide.

[0042] As used herein, the terms “disease,” “disorder,” and “symptom” refer, respectively, to a suboptimal state of health, such as a condition that is or would normally be diagnosed or treated by a medical professional.

[0043] As used herein, the term “expression sequence” refers to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide. An exemplary expression sequence encoding a peptide or polypeptide comprises a plurality of nucleotide triads, each of which may encode an amino acid and is referred to as a “codon.”

[0044] "Heterogeneous" means occurring under circumstances other than those in which it occurs naturally. A "heterogeneous" polynucleotide sequence indicates that the polynucleotide sequence is used in a way other than that found in the natural genome of that sequence. For example, a "heterogeneous promoter" is used to drive the transcription of a sequence that is not naturally transcribed by that promoter; therefore, "heterogeneous promoter" sequences are often included in expression constructs using recombinant nucleic acid technology. The term "heterogeneous" is also used to refer to a given sequence that is placed in a relationship with another sequence that does not occur naturally; for example, heterogeneous coding or non-coding nucleotide sequences are commonly inserted into the genome by genome transformation technology, resulting in a genetically modified or recombinant genome.

[0045] When used herein, “increase fitness” or “promote fitness” of an organism means any desirable modification of any physiological activity or activity carried out by the organism as a result of administration of the peptide or polypeptide described herein, for example, one or more of the following desired effects, but not limited to: (1) increased tolerance to biological or abiotic stress; (2) increased yield or biomass; (3) alteration of flowering time; (4) increased resistance to pests or pathogens; (5) increased resistance to herbicides; (6) increased population of the organism (e.g., agriculturally important insects); (7) increased reproductive rate of the organism (e.g., insects, e.g., honeybees or silkworms); (8) increased motility of the organism (e.g., insects, e.g., honeybees or silkworms); (9) increased motility of the organism (e.g., insects, e.g., honeybees or silkworms); (10) increased population of the organism (e.g., insects, e.g., honeybees or silkworms); (11) increased motility of the organism (e.g., insects, e.g., honeybees or silkworms); (12) increased motility of the organism (e.g., honeybees or silkworms); (9) Increase in the body weight of the target organism (e.g., insects, e.g., bees or silkworms); (10) Increase in pollination by the target organism (e.g., insects, e.g., bees or silkworms) (e.g., the number of plants pollinated within a given period); (11) Increase in the production of by-products of the target organism (e.g., insects, e.g., bees or silkworms) (e.g., honey from bees or silk from silkworms); (12) Increase in the nutrient content of the target organism (e.g., insects) (e.g., proteins, fatty acids or amino acids); or (13) Increase in the resistance of the target organism to pesticides (e.g., neonicotinoids (e.g., imidacloprid) or organophosphate insecticides (e.g., phosphorothioates, e.g., fenitrothion)); (14) Enhancement of the health of the target organism or reduction of disease of the target organism, such as humans or non-human animals. The increase in host fitness can be determined by comparing it with the target organism to which the modifier has not been administered.Conversely, “decreased fitness” of the subject means any undesirable modification of any physiological activity or activity carried out by the subject organism as a result of administration of the peptides or polypeptides described herein, for example, one or more of the following intended effects, but not limited to: (1) decreased tolerance to biological or abiotic stress; (2) decreased yield or biomass; (3) alteration of flowering time; (4) decreased resistance to pests or pathogens; (5) decreased resistance to herbicides; (6) decrease in the population of the subject organism (e.g., agriculturally important insects); (7) decreased reproductive rate of the subject organism (e.g., insects, e.g., bees or silkworms); (8) decreased motility of the subject organism (e.g., insects, e.g., bees or silkworms); (9) decreased body weight of the subject organism (e.g., insects, e.g., bees or silkworms). (9) A decrease in the metabolic rate or activity of the target organism (e.g., insects, e.g., bees or silkworms); (10) A decrease in pollination by the target organism (e.g., insects, e.g., bees or silkworms) (e.g., the number of plants pollinated within a given period); (11) A decrease in the production of by-products (e.g., honey from bees or silk from silkworms) of the target organism (e.g., insects, e.g., bees or silkworms); (12) A decrease in the nutrient content (e.g., proteins, fatty acids or amino acids) of the target organism (e.g., insects); or (13) A decrease in the resistance of the target organism to pesticides (e.g., neonicotinoids (e.g., imidacloprid) or organophosphate insecticides (e.g., phosphorothioates, e.g., fenitrothion)); (14) A decrease in the health or disease of the target organism, such as humans or non-human animals. The decrease in host fitness can be determined by comparing it with the target organism to one to which the modifier has not been administered. It will be apparent to those skilled in the art that specific changes in the physiology, phenotype, or activity of an object, such as changes in the flowering period in plants, can be considered to increase or decrease the fitness of the object depending on the circumstances (for example, to adapt to changes in climate or other environmental conditions).For example, a delay in flowering (e.g., a decrease of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% in the number of plants in a population that flower on a given calendar date) can be considered a favorable adaptation to a later or cooler spring, and therefore may increase the fitness of the plants; conversely, a similar delay in flowering under the circumstances of an earlier or warmer spring may be considered to decrease the fitness of the plants.

[0046] As used herein, the term “intron fragment” refers to a portion of an intron, where a first intron fragment and a second intron fragment together form an intron, such as a catalytic intron. An intron fragment may also be a 5' portion of an intron (e.g., a 5' portion of a catalytic intron) or a 3' portion of an intron (e.g., a 3' portion of a catalytic intron), whereby the 5' and 3' intron fragments together form a functional intron, such as a catalytic self-splicing functional intron. The term “intron fragment” is intended to refer to an intron that has been divided into two parts. The term “intron fragment” is not intended to state, mean, or suggest that the two parts or halves are of equal length. The term “intron fragment” is used synonymously with the term “divided intron.”

[0047] As used herein, the term “impurity” refers to an undesirable substance present in a composition, for example, a pharmaceutical composition described herein. In some embodiments, the impurity is a process-related impurity. In some embodiments, the impurity is a product-related substance other than the desired product in the final composition, for example, an active pharmaceutical component, for example, a cyclic polyribonucleotide. As used herein, the term “process-related impurity” refers to a substance other than the linear polyribonucleotide described herein that is undesirable in the final composition, preparation, or product, and is used, present, or produced in the preparation of the composition, preparation, or product. In some embodiments, the process-related impurity is an enzyme used in the synthesis or cyclization of polyribonucleotides. As used herein, the term “product-related substance” refers to a substance or by-product produced during the synthesis of a composition, preparation, or product, or any intermediate thereof. In some embodiments, the product-related substance is a deoxyribonucleotide fragment. In some embodiments, the product-related substance is a deoxyribonucleotide monomer. In some embodiments, the product-related substance is one or more derivatives or fragments of the polyribonucleotides described herein, for example, one or more fragments of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acids, monoribonucleic acids, diribonucleic acids, or triribonucleic acids.

[0048] As used herein, the terms “linear polyribonucleotide,” “linear RNA,” and “linear polyribonucleotide molecule” are interchangeable and mean a polyribonucleotide molecule having 5' and 3' ends. One or both of the 5' and 3' ends may be free ends or may be ligated to another. A linear polyribonucleotide may be an uncyclized (e.g., pre-cyclization) polyribonucleotide and can be used as a starting material for cyclization, for example, by sprint ligation or by chemical, enzymatic, ribozyme, or splicing catalyst cyclization methods.

[0049] As used herein, the term “modified oligonucleotide” means an oligonucleotide containing a nucleotide having at least one modification to a sugar, nucleic acid base, or internucleotide bond.

[0050] As used herein, the term “modified ribonucleotide” means a ribonucleotide containing a nucleoside having at least one modification to a sugar, nucleic acid base, or nucleoside bond.

[0051] As used herein, the term “naked delivery” refers to a formulation for delivery to cells that does not rely on a carrier and does not use covalent modifications to the portion that would otherwise be used to facilitate delivery to cells. Naked delivery formulations do not contain any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation of cyclic polyribonucleotides is a formulation that contains cyclic polyribonucleotides without covalent modifications and does not contain a carrier.

[0052] As used herein, the terms “nicked RNA,” “nicked linear polyribonucleotide,” or “nicked linear polyribonucleotide molecule” are interchangeable and mean a polyribonucleotide molecule having 5' and 3' ends resulting from the nicking or degradation of circular RNA. “Nicked circular RNA” means circular RNA that has been nicked.

[0053] The term “optionally substituted X” is intended, as used herein, to be equivalent to “X which is optionally substituted” (e.g., “alkyl which is optionally substituted alkyl”). It is not intended to mean that the feature “X” (e.g., alkyl) is essentially optional. The term “optionally substituted” as used herein means having 0, 1, or more substituents (e.g., substituents of 0-25, 0-20, 0-10, or 0-5). For example, a C1 alkyl group, i.e., methyl, can be substituted with oxo to form a formyl group, and can be further substituted with -OH or -NH2 to form a carboxyl group or an amide group.

[0054] The term "pharmaceutical composition" is also intended to disclose that cyclic or linear polyribonucleotides contained within a pharmaceutical composition can be used for therapeutic treatment of the human or animal body.

[0055] As used herein, the term “polynucleotide” means a molecule comprising one or more nucleic acid subunits or nucleotides, and can be used interchangeably with “nucleic acid” or “oligonucleotide.” A polynucleotide may comprise one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or their variants. A nucleotide may comprise a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO3) groups. A nucleotide may comprise a nucleic acid base, a pentose (either ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. Polyribonucleotide, ribonucleic acid, or RNA may refer to a macromolecule comprising multiple ribonucleotides polymerized by phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0056] Polydeoxyribonucleotides, deoxyribonucleic acids, and DNA refer to macromolecules containing multiple deoxyribonucleotides polymerized via phosphodiester bonds. A nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide refers to a deoxyribonucleoside polyphosphate, such as deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTP, containing a detectable tag (e.g., a fluorophore), such as a luminescent tag or marker. A nucleotide may contain any subunits that can be incorporated into a growing nucleic acid chain. Such subunits may be specific to A, C, G, T, or U, or one or more complementary A, C, G, T, or U, or any other subunit complementary to purines (i.e., A or G, or their variants) or pyrimidines (i.e., C, T, or U, or their variants). In some cases, polynucleotides are deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or their derivatives or variants. In some cases, polynucleotides are, to name a few, small interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), small hairpin RNA (shRNA), nuclear small RNA (snRNA), messenger RNA (mRNA), mRNA precursor (pre-mRNA), antisense RNA (asRNA), and encompass both nucleotide sequences and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helical, or hairpin. In some cases, polynucleotide molecules are cyclic. Polynucleotides can have a variety of lengths. Nucleic acid molecules can have lengths of at least approximately 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 bases, 1 kilobase (kb), 2kb, 3kb, 4kb, 5kb, 10kb, 50kb, or more. Polynucleotides can be isolated from cells or tissues.Embodiments of polynucleotide sequences may include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0057] Embodiments of polynucleotides, for example, polyribonucleotides or polydeoxyribonucleotides, include polynucleotides containing 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-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylkeosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, These include 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylkeosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid(v), butoxosin, pseudouracil, keosin, 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, nucleotides include modifications to their phosphate moieties, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10, or more phosphate moieties) and modifications of thiol moieties (e.g., α-thiotriphosphate and β-thiotriphosphate).In some embodiments, nucleic acid molecules are also modified with base moieties, sugar moieties, or phosphate backbone (for example, with one or more atoms typically available to form hydrogen bonds with complementary nucleotides, or with one or more atoms typically not available to form hydrogen bonds with complementary nucleotides). In some embodiments, nucleic acid molecules include amine-modifying groups such as aminoallyl-dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP) to enable covalent bonding of amine-reactive moieties such as N-hydroxysuccinimide esters (NHS). Substitutes for standard DNA base pairs or RNA base pairs in oligonucleotides of this disclosure may offer higher bit density per cubic mm, higher safety (resistance to accidental or intentional synthesis of natural toxins), easier identification in photoprogrammed polymerases, or lower secondary structures. Such alternative base pairs compatible with natural and mutant polymerases for de novo or amplified 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, 8:612-4, which are incorporated herein by reference for all purposes.

[0058] As used herein, the term “polyribonucleotide cargo” includes any sequence comprising at least one polyribonucleotide. In embodiments, a polyribonucleotide cargo comprises one or more expression (or coding) sequences, each expression (or coding) sequence encoding a polypeptide. In embodiments, a polyribonucleotide cargo comprises one or more non-coding sequences, such as polyribonucleotides having a regulatory or catalytic function. In embodiments, a polyribonucleotide cargo comprises a combination of expression and non-coding sequences. In embodiments, a polyribonucleotide cargo comprises one or more polyribonucleotide sequences described herein, for example, one or more regulatory elements, internal ribosome entry site (IRES) elements, or spacer sequences.

[0059] When used interchangeably herein, the terms “polyA” and “polyA sequence” refer to an untranslated adjacent region of a nucleic acid molecule consisting of adenosine residues and having a length of at least 5 nucleotides. 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 on the 3' side (e.g., downstream) of an open reason frame (e.g., an open reason frame encoding a polypeptide), and the polyA sequence is located on the 3' side of a termination element (e.g., a stop codon) so that the polyA is not translated. In some embodiments, the polyA sequence is located on the 3' side of both the termination element and the 3' untranslated region.

[0060] When used herein, nucleic acid elements are "connected" or "linked" if they are arranged in a vector such that they can be transcribed to form linear polyribonucleotides and then cyclized into cyclic polyribonucleotides using the methods provided herein.

[0061] As used herein, the term “plant-modifying polypeptide” refers to a polypeptide that can modify a plant’s genetic characteristics (e.g., by increasing or decreasing gene expression, or otherwise altering the nucleotide sequence of DNA or RNA), epigenetic characteristics, or biochemical or physiological characteristics in a manner that results in a change in the plant’s physiology or phenotype, such as an increase or decrease in plant fitness.

[0062] As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together, most often by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologues, paralogs, fragments thereof, and other equivalents, variants, and analogs. Polypeptides may be single molecules or multimolecular complexes such as dimers, trimers, or tetramers. Polypeptides may also include single-chain or multi-chain polypeptides, such as antibodies or insulin, and may associate or link. The most common disulfide bonds are found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids.

[0063] As used herein, the terms “purify,” “refining,” and “purify” refer to one or more steps or processes of removing 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) 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 substance is 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.

[0064] 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 relation to nucleic acids (e.g., polyribonucleotides), the purity of isolated nucleic acids (e.g., circular RNA) can be expressed in relation to a population of nucleic acids that is free from 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 unit of the total mass of the isolated material and can be determined, for example, using pure circular RNA as a reference. The levels of purity found herein may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, greater than 95%, or greater than 99% (w / w). In some embodiments, the levels of contaminants, impurities, or by-products are less than or equal to about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (w / w). Purity can be determined by detecting the level of a specific analyte (e.g., circular RNA) or a specific 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 nucleic acid population, and by calculating the percentage (w / w) of the analyte relative to the total nucleic acid content (e.g., determined by assays known in the art).

[0065] As used herein, the phrase “substantially free of one or more impurities or by-products” means 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 minimum amount of one or more impurities or by-products, such as a sample containing a population of concentrated circular RNA. A minimum 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). In another example, a sample or concentrated population of circular RNA is substantially free of one or more impurities or by-products if one or more impurities or by-products are present in amounts 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). In another example, a sample or concentrated population of circular RNA is substantially free of one or more impurities or by-products if one or more impurities or by-products are present in amounts less than 10% (w / w) (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less). In another example, a sample or concentrated population of circular RNA is substantially free of one or more impurities or by-products if one or more impurities or by-products are present in amounts less than 5% (w / w) (e.g., 4%, 3%, 2%, 1% (w / w) or less). In yet another example, a sample or concentrated population of circular RNA is substantially free of one or more impurities or by-products if one or more impurities or by-products are present in amounts 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).

[0066] As used herein, “regulatory element” refers to a portion of a nucleic acid sequence or similar that alters the expression of an expression sequence within a cyclic or linear polyribonucleotide.

[0067] As used herein, the term “replica element” refers to a sequence and / or motif that is useful for replication or initiates the transcription of a cyclic polyribonucleotide.

[0068] As used herein, “spacer” refers to any adjacent nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.

[0069] As used herein, the term “sequence identity” is determined by the alignment of two peptide sequences or two nucleotide sequences using a global or local alignment algorithm. Sequences are said to be “substantially identical” or “essentially similar” if they share at least a certain minimum percentage of sequence identity when they are 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, GAP's default parameters are used: gap creation penalty = 50 (nucleotides) / 8 (protein) and gap expansion penalty = 3 (nucleotides) / 2 (protein). For nucleotides, the default score matrix used is nwsgapdna, and for proteins, the default score matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignment and sequence identity percentage scores are determined using computer programs such as the GCG Wisconsin Package, Version 10.3, or EmbossWin version 2.10.0 (using the "Needle" program), available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA. Alternatively, or in addition to these methods, percentage identity is determined by searching a database using algorithms such as FASTA or BLAST. Sequence identity refers to sequence identity across the entire length of the sequence.

[0070] As used herein, “structured” with respect to RNA refers to RNA sequences that are predicted by RNAFold software or similar prediction tools to form structures (e.g., hairpin loops) that have themselves or other sequences within the same RNA molecule.

[0071] As used herein, the term “subject” refers to living organisms such as animals, plants, or microorganisms. In embodiments, the subject is a vertebrate (e.g., mammals, birds, fish, reptiles, or amphibians). 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., monkeys, apes), an ungulate (e.g., cattle, buffalo, bison, sheep, goats, pigs, camels, llamas, alpacas, deer, horses, donkeys), a carnivore (e.g., dogs, cats), a rodent (e.g., rats, mice), or a rabbit (e.g., rabbits). In the embodiments, the subjects are birds such as members of avian taxa such as Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In the embodiments, the subjects are invertebrates such as arthropods (e.g., insects, spiders, crustaceans), nematodes, annelids, helminths, or mollusks. In the embodiments, the subjects are invertebrates that are agricultural pests of invertebrates or parasites of invertebrate or vertebrate hosts. In the embodiments, the subjects are plants such as angiosperms (which may be dicotyledonous or monocotyledonous) or gymnosperms (e.g., conifers, cycads, gnetophytes, ginkgo), ferns, horsetails, clubmosses, or mosses. In the embodiments, the subjects are eukaryotic algae (unicellular or multicellular). In the embodiments, the subjects are agricultural or horticulturally important plants such as furrow crops, fruit-bearing plants and trees, vegetables, trees, and ornamental plants, such as ornamental flowers, shrubs, trees, ground cover, and turf.

[0072] As used herein, “termination element” refers to a portion of a nucleic acid sequence, such as a cyclic or linear polyribonucleotide, that terminates the translation of an expression sequence.

[0073] As used herein, the term “total ribonucleotide molecules” means the total amount of any ribonucleotide molecules, including linear polyribonucleotide molecules, cyclic polyribonucleotide molecules, monomeric ribonucleotides, other polyribonucleotide molecules, their fragments, and their modified variations, as measured by the total mass of ribonucleotide molecules.

[0074] As used herein, the terms “to treat” and “to treat” refer to preventive or therapeutic treatment of a disease or disorder in a subject (e.g., infection, cancer, toxicity, or allergic reaction). The effects of treatment may include restoring, alleviating, reducing the severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of, stabilizing (i.e., preventing exacerbation of) the state of the disease or disorder, or preventing the transmission of the disease or disorder, compared to the state of the disease or disorder in the absence of therapeutic treatment. Embodiments include treating plants to control diseases or adverse conditions caused by or involving invertebrate pests or microbial (e.g., bacteria, fungi, or viruses) pathogens. Embodiments also include treating plants to enhance their natural defenses or immune capabilities to provide resistance to pressure from pests or pathogens.

[0075] As used herein, the term “translation initiation sequence” refers to a nucleic acid sequence that initiates translation of an expression sequence in a circular or linear polyribonucleotide.

[0076] As used herein, “therapeutic polypeptide” refers to a polypeptide that, when administered to or expressed in a subject, provides several therapeutic benefits. In embodiments, a therapeutic polypeptide is used to treat or prevent a disease, disorder, or condition in a subject by administering the therapeutic peptide to the subject or by expressing the therapeutic polypeptide in the subject. In other embodiments, the therapeutic polypeptide is expressed intracellularly and administered to a subject to provide therapeutic benefits.

[0077] As used herein, “vector” means a DNA fragment obtained from a higher organism’s virus, plasmid, or cell into which an exotic DNA fragment may or may be inserted for cloning or expression (for example, by PCR). In some embodiments, the vector may be stably maintained within an organism. The vector may include, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, or multiple cloning sites (MCS). The term includes linear DNA fragments (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 vector provided herein includes multiple cloning sites (MCS). In another embodiment, the vector provided herein does not include MCS.

[0078] As used herein, “translation efficiency” is the rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency can be expressed, for example, as the amount of protein or peptide produced per given amount of protein or peptide-encoding transcript within a given period of time in a given translation system, for example, in a cell-free translation system such as rabbit reticulocyte lysate.

[0079] As used herein, the term “yield” refers to the relative amount (w / w) of the 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). The yield can be expressed as a percentage. In connection with this disclosure, the amounts of the analyte (e.g., cyclic polyribonucleotides) in the starting material and the analyte obtained after the purification step can be measured using an assay (e.g., gel electrophoresis or spectrophotometric method). Using the methods of this disclosure, a yield of a concentrated population of cyclic polyribonucleotides of about 20% (w / w) or more can be obtained compared to the amount present in the starting material, e.g., a mixed population of polyribonucleotides. For example, the method can be used to obtain yields of purified cyclic polyribonucleotides of approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or higher. [Brief explanation of the drawing]

[0080] [Figure 1] This is a schematic diagram illustrating an exemplary method for separating cyclic polyribonucleotides from linear polyribonucleotides that contain an aptamer. The left side shows a linear polyribonucleotide containing an aptamer located near the 5' end of the polyribonucleotide; however, this disclosure specifically envisions an alternative embodiment where the aptamer is located at the 3' end of the linear polyribonucleotide. The linear polyribonucleotide is cyclized to produce a cyclic polyribonucleotide that does not contain an aptamer. A reagent conjugated to particles is added to the mixture. The reagent binds to the aptamer on the linear polyribonucleotide, while the cyclic polyribonucleotide is not bound by the reagent, thereby separating the aptamer-containing linear polyribonucleotide from the aptamer-deficient cyclic polyribonucleotide. [Figure 2]This schematic diagram illustrates an exemplary method for separating a cyclic polyribonucleotide from a linear polyribonucleotide that contains a region that hybridizes to an aptamer. The left side shows a linear polyribonucleotide containing a region located near the 5' end of the polyribonucleotide that hybridizes to the aptamer-containing polyribonucleotide; however, this disclosure specifically envisions an alternative embodiment where the aptamer hybridizes to a position located at the 3' end of the linear polyribonucleotide. The linear polyribonucleotide is cyclic, thereby producing a cyclic polyribonucleotide that does not contain the region that hybridizes to the aptamer-containing polyribonucleotide. The reagent conjugated to particles is added to the mixture. The reagent binds to the aptamer hybridized to the linear polyribonucleotide, while the cyclic polyribonucleotide is not bound by the reagent. This allows the linear polyribonucleotide hybridized to the aptamer to be separated from the cyclic polyribonucleotide that is not hybridized to the aptamer. [Figure 3] This gel shows linear byproducts 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, nick circular RNA, and spliced ​​introns. [Modes for carrying out the invention]

[0081] This disclosure describes compositions and methods for processing, for example, purifying, polyribonucleotides. Polyribonucleotides, such as linear or cyclic polyribonucleotides, can be used for various modification or therapeutic purposes. However, when polyribonucleotides are produced through certain biological reactions, various impurities, by-products, or incomplete products may be present. The present invention features methods useful for reducing or removing these impurities, by-products, or incomplete products from a sample in order to produce a composition having a desired polyribonucleotide composition, quantity, and / or purity, or a population containing multiple polyribonucleotides having a desired polyribonucleotide composition, quantity, and / or purity.

[0082] In certain embodiments, the method is useful for purifying polyribonucleotides that have undergone splicing. 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 cyclic polyribonucleotides (e.g., spliced) from linear polyribonucleotides, or linear polyribonucleotides from cyclic polyribonucleotides. Such purified compositions containing the desired polyribonucleotides may be useful for various downstream applications, such as the delivery of polynucleotide cargoes (e.g., encoding genes or proteins) to target cells. Compositions and methods are described in more detail below.

[0083] method The method described herein comprises separating aptamer-containing polyribonucleotides from a plurality of polyribonucleotides. The method comprises preparing a sample containing a plurality of polyribonucleotides. The plurality of polyribonucleotides comprises a mixture of linear polyribonucleotides and cyclic polyribonucleotides. A subset of the plurality of polyribonucleotides is linear polyribonucleotides. The method comprises contacting the sample with a reagent that binds to aptamers and separating the aptamer-containing linear polyribonucleotides bound to the reagent from the plurality of polyribonucleotides in the sample (Figure 1).

[0084] In some embodiments, the method includes generating a linear polyribonucleotide having an aptamer by attaching the aptamer to the linear polyribonucleotide (Figure 2). The cyclic polyribonucleotide may include an open reading frame (ORF) encoding a polypeptide.

[0085] In some embodiments, the methods described herein include separating linear polyribonucleotides from a plurality of polyribonucleotides. The plurality of polyribonucleotides includes a mixture of linear polyribonucleotides and cyclic polyribonucleotides. The method includes preparing a sample having a plurality of polyribonucleotides, a subset of which includes linear polyribonucleotides; binding an aptamer to the linear polyribonucleotides; and contacting the sample with a column containing a resin having a plurality of particles conjugated with a reagent that binds to the aptamer. The method further includes recovering an eluate containing a portion of the sample that is not bound to the reagent from the plurality of polyribonucleotides in the sample.

[0086] In some embodiments, the methods described herein include separating a linear polyribonucleotide from a plurality of polyribonucleotides. The plurality of polyribonucleotides includes a mixture of linear polyribonucleotides and cyclic polyribonucleotides. The method includes: cyclizing a linear precursor to form a cyclic polyribonucleotide; preparing a sample comprising a plurality of polyribonucleotides, a subset of which comprises a linear polyribonucleotide; binding an aptamer to the linear polyribonucleotide; and contacting the sample with a reagent that binds to the aptamer. The method further includes separating a linear polyribonucleotide having an aptamer bound to the reagent from the plurality of polyribonucleotides in the sample.

[0087] In some embodiments of any of the methods described herein, the aptamer is located at the 5' or 3' end of a polyribonucleotide (e.g., linear or cyclic polyribonucleotide). In some embodiments, the aptamer is located at the 3' end of the polyribonucleotide. In some embodiments, the aptamer does not contain a polyA sequence.

[0088] In some embodiments, the reagent is conjugated to particles (e.g., directly or indirectly). The particles may be, for example, magnetic beads. In some embodiments, the reagent is conjugated to a resin containing a plurality of particles. The resin may include, for example, crosslinked poly[styrene-divinylbenzene], agarose, or SEPHAROSE®. In some embodiments, the column contains the resin.

[0089] In some embodiments of any of the methods described herein, the separation includes immobilizing a reagent. The method may include, for example, immobilizing a reagent, particles, or a combination thereof.

[0090] 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, optical force, centrifugal force, or acoustic force, to the beads or particles.

[0091] As described herein, the method can be used to separate, for example, unspliced ​​polyribonucleotides from those that have been spliced. In some embodiments, the method described herein includes separating spliced ​​polyribonucleotides from unspliced ​​or partially spliced ​​polyribonucleotides. In some embodiments, the spliced ​​polyribonucleotides are cyclic polyribonucleotides. In some embodiments, the spliced ​​polyribonucleotides are linear polyribonucleotides. In some embodiments, the spliced ​​polyribonucleotides lack introns, for example, after a splicing event in the process (e.g., self-splicing). In some embodiments, the polyribonucleotides having introns are linear polyribonucleotides.

[0092] In some embodiments, the method further includes washing the conjugated polyribonucleotide having the aptamer one or more times (e.g., two, three, four, five, or more times). Washing may be performed after the contact step and / or after the separation step.

[0093] In some embodiments, the method further includes performing a first elution step to release a conjugated polyribonucleotide containing an aptamer from the polyribonucleotide having the aptamer. The first elution step may include adding a first buffer and / or heating the sample to, for example, at least 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, or higher.

[0094] In some embodiments, the method further includes carrying out a second elution step. The second elution step may include adding a second buffer and / or heating the sample to, for example, at least 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, or higher. In some embodiments, the second buffer includes a denaturant, such as formamide or urea. The second buffer may include, for example, about 40% to about 60% formamide (e.g., about 40%, 45%, 50%, 55%, or 60% formamide).

[0095] In some embodiments, the method includes incubating the sample with the reagent for at least 10 minutes (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 minutes, or longer).

[0096] In some embodiments, the method includes recovering a portion of the sample that has not been bound with the reagent.

[0097] In some embodiments, the method involves preparing a plurality of reagents, each of which is conjugated to a different aptamer or a different portion within an aptamer. Each reagent can be conjugated to, for example, particles, such as magnetic particles or beads.

[0098] In some embodiments, the method involves preparing the reagent in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 5:1, 2:1, 1:2, 1:5, or 1:10) relative to a polyribonucleotide (e.g., a polyribonucleotide having an aptamer).

[0099] In some embodiments, the method includes preparing a sample of particles, such as beads, such as magnetic beads. The particles may be present in a container, such as a microcentrifuge tube, or packed into a column. The particles may be conjugated with a reagent. The method may also include running a mixture of polyribonucleotides through a column containing the particles. In such a case, the polyribonucleotides conjugated by the reagent will be bound to the column. In some embodiments, the particles are directly conjugated to a reagent, for example, that is designed to conjugate to a polyribonucleotide aptamer.

[0100] In some embodiments, for example, when using magnetic particles, the method may include pelletizing the magnetic particles in a container (e.g., a microcentrifuge tube) by, for example, providing permanent magnets.

[0101] In some embodiments, the method described herein concentrates a desired amount of polyribonucleotide in a sample. For example, the method can concentrate a desired amount of polyribonucleotide (e.g., spliced, e.g. circular) 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 before purification.

[0102] In some embodiments, the purification method yields cyclic polyribonucleotides having less than 50% (mol / mol) of linear polyribonucleotides (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% (mol / mol)).

[0103] In some embodiments, at least 500 μg (e.g., at least 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, or more) of linear polyribonucleotides having aptamers is separated by the method described herein.

[0104] Method of joining Methods described herein include ligating an aptamer to a linear polynucleotide. For example, the method may include ligating an aptamer to the 3' or 5' end of a linear polyribonucleotide. In some embodiments, the method includes ligating an aptamer to the 3' or 5' end of a linear polyribonucleotide, where the aptamer is not located at the 3' or 5' end of the linear polyribonucleotide. In embodiments, the polyribonucleotide containing the aptamer is ligated to the end, and the aptamer is ligated to the end of the linear polyribonucleotide, while, for example, after ligation, the adjacent region forms a new 5' or 3' end of the linear polyribonucleotide. Ligation can be carried out by ligating the aptamer to the linear polyribonucleotide. In some embodiments, the linear polyribonucleotide contains a portion of the aptamer, and the ligation step includes ligating the remaining portion of the aptamer.

[0105] Aptamers or polyribonucleotides containing aptamers may be conjugated according to any available techniques, including, but not limited to, chemical and enzymatic methods.

[0106] Such enzymatic methods include, for example, preparing a ligase (e.g., RNA ligase) that binds to the free ends of linear RNA, such as the 3' end of a linear polyribonucleotide and the 5' end of an aptamer, or the 5' end of a linear polyribonucleotide and the 3' end of an aptamer.

[0107] In one example, either the 5' or 3' end of a linear polyribonucleotide may encode a ligase ribozyme sequence such that, during in vitro transcription, the resulting linear polyribonucleotide contains an active ribozyme sequence capable of ligating the 5' or 3' end of the linear polyribonucleotide to an aptamer. Ligase ribozymes can be derived from group I introns, hepatitis delta virus, hairpin ribozymes, or selected by SELEX (phylogenetic evolution of ligands by exponential enrichment).

[0108] In another example, an aptamer can be linked to a linear polyribonucleotide using at least one non-nucleic acid moiety. For example, at least one non-nucleic acid moiety may react with a region or feature near the 5' or 3' end of the linear polyribonucleotide in order to bind to it. In another example, at least one non-nucleic acid moiety may be located at, linked to, or near the 5' or 3' end of the linear polyribonucleotide. The non-nucleic acid moiety may be homologous or heterologous. In a non-limiting example, the non-nucleic acid moiety may be a bond such as a hydrophobic bond, an ionic bond, a biodegradable bond, or a cleavable bond. In another non-limiting example, the non-nucleic acid moiety may be a ligation moiety. In yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or peptide moiety, e.g., an aptamer or non-nucleic acid linker as described herein.

[0109] In another example, linear polyribonucleotides can be spliced ​​into aptamers. In some embodiments, both the linear polyribonucleotide and the aptamer contain a loop E sequence for ligating. In another embodiment, the linear polyribonucleotide and the aptamer may contain cyclic introns, e.g., 5' and 3' splice junctions, or cyclic catalytic introns, e.g., group I, group II, or group III introns. Non-limiting examples of group I intron self-splicing sequences include the self-splicing reordered intron-exon sequence from the T4 bacteriophage gene td, and the Tetrahymena intervening sequence (IVS) rRNA.

[0110] In another example, aptamers may be bound to linear polyribonucleotides by non-nucleic acid moieties that attract between atoms at, near, or linked to the 5' and 3' ends of the linear polyribonucleotide, or between atoms on the molecular surface. Linear polyribonucleotides may be bound to aptamers by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugations, hyperconjugations, and antibonds.

[0111] In another example, the linear polyribonucleotide may contain a ribozyme RNA sequence near its 5' end, and the aptamer may contain a ribozyme RNA sequence near its 3' end, or vice versa. The ribozyme RNA sequence can be covalently ligated to the peptide when the sequence is exposed to the remainder of the ribozyme. Peptides covalently ligated to the ribozyme RNA sequence near their 5' and 3' ends can associate with each other, thereby allowing the aptamer to be ligated to the linear polyribonucleotide. A non-limiting list of ribozymes used in the linear primary constructs or linear polyribonucleotides of the present invention, or an incomplete list of methods for incorporating or covalently ligating peptides, is provided in U.S. Patent Application Publication No. 20030082768, the contents of which are incorporated herein by reference in whole.

[0112] In yet another example, aptamers can be attached to linear polyribonucleotides using ligation chemical methods. Such methods may include, but are not limited to, click chemistry (e.g., alkyne and azido-based methods, or clickable bases), olefin metathesis, phosphoramide ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.

[0113] Methods of circularization This disclosure provides a method for cyclizing polyribonucleotides, for example, from linear precursors. Cyclization can be carried out using methods including, for example, recombinant techniques or chemical synthesis. For example, the DNA molecules used to generate RNA circles may include DNA sequences of naturally occurring original nucleic acid sequences, modified versions thereof, or DNA sequences encoding synthetic polypeptides not normally found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using a variety of techniques, but are not limited to classical mutagenesis and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of nucleic acid sequences, synthesis of oligonucleotide mixtures and ligation of mixed groups to "construct" mixtures of nucleic acid molecules, and combinations thereof.

[0114] Cyclic polyribonucleotides can be prepared according to any available techniques, including, but not limited to, chemical and enzymatic synthesis. In some embodiments, linear primary constructs or linear RNA can be cyclized or concatemerized to produce the circRNAs described herein. The mechanism of cyclization or concatemerization may occur, for example, through chemical, enzymatic, sprint ligation, or ribozyme-catalyzed methods. The newly formed 5'-3' bond may be an intramolecular or intermolecular bond. For example, sprint ligases such as Splint® ligase can be used for sprint ligation. According to this method, a single-stranded polynucleotide (sprint), such as single-stranded DNA or RNA, can be designed to hybridize with both ends of a linear polyribonucleotide, so that the two ends can be juxtaposed during hybridization with a single-stranded sprint. Therefore, sprint ligases can produce circRNAs by catalyzing the ligation of the two juxtaposed ends of a linear polyribonucleotide. In some embodiments, DNA or RNA ligases may be used in the synthesis of cyclic polynucleotides. In non-limiting examples, the ligase may be a circ ligase or a cyclic ligase.

[0115] In another example, either the 5' or 3' end of a linear polyribonucleotide may encode a ligase ribozyme sequence such that, during in vitro transcription, the resulting linear circRNA contains an active ribozyme sequence capable of ligating the 5' end of the linear polyribonucleotide to the 3' end of the linear polyribonucleotide. The ligase ribozyme can be derived from group I introns, hepatitis delta virus, hairpin ribozymes, or selected by SELEX (phylogenetic evolution of ligands by exponential enrichment).

[0116] In another example, a linear polyribonucleotide may be cyclized or concatemerized by using at least one non-nucleic acid moiety. For example, at least one non-nucleic acid moiety may react with a region or feature near the 5' or 3' end of the linear polyribonucleotide to cyclize or concatemerize the linear polyribonucleotide. In another example, at least one non-nucleic acid moiety may be located at, linked to, or near the 5' or 3' end of the linear polyribonucleotide. The non-nucleic acid moiety may be homologous or heterologous. In a non-limiting example, the non-nucleic acid moiety may be a bond such as a hydrophobic bond, an ionic bond, a biodegradable bond, or a cleavable bond. In another non-limiting example, the non-nucleic acid moiety may be a ligation moiety. In yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or peptide moiety, for example, an aptamer or non-nucleic acid linker as described herein.

[0117] In another example, linear polyribonucleotides may be cyclized or concatemerized by self-splicing. In some embodiments, the linear polyribonucleotide may include a loop E sequence for self-ligating. In another embodiment, the linear polyribonucleotide may include self-cyclic introns, such as 5' and 3' splice junctions, or self-cyclic catalytic introns such as group I, group II, or group III introns. Non-limiting examples of group I intron self-splicing sequences include a reversed intron-exon sequence of self-splicing derived from the T4 bacteriophage gene td, the intervening sequence (IVS) rRNA of Tetrahymena, or the pretRNA-Leu gene of the cyanobacterium Anabaena.

[0118] In another example, linear polyribonucleotides may be cyclized or concatemerized by non-nucleic acid moieties that attract between atoms, between atoms, or between atoms attached to the 5' and 3' ends of the linear polyribonucleotide, or between atoms attached to the molecular surface. One or more linear polyribonucleotides may be cyclized or concatemerized by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugations, hyperconjugations, and antibonds.

[0119] In another example, the linear polyribonucleotide may contain ribozyme RNA sequences near the 5' and 3' ends. The ribozyme RNA sequences may be covalently ligated to the peptide when the sequence is exposed to the rest of the ribozyme. Peptides covalently ligated to the ribozyme RNA sequences near the 5' and 3' ends may associate with each other, thereby causing cyclization or concatemerization of the linear polyribonucleotide. In yet another example, peptides covalently ligated to the ribozyme RNA near the 5' and 3' ends may, after being ligated using various methods known in the art, such as protein ligation, cause cyclization or concatemerization of the linear primary construct or linear mRNA. A non-limiting list of ribozymes used in the linear primary constructs or linear polyribonucleotides of the present invention, or an incomplete list of methods for incorporating or covalently linking peptides, is provided in U.S. Patent Application Publication No. 20030082768, the contents of which are incorporated herein by reference in their entirety.

[0120] In yet another example, cyclic polyribonucleotides can be generated using cyclic chemical methods. Such methods may include, but are not limited to, click chemistry (e.g., alkyne and azido-based methods, or clickable bases), olefin metathesis, phosphoramide ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.

[0121] Methods for producing cyclic polyribonucleotides as described herein are described, for example, in Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); in Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012).

[0122] Various methods for synthesizing cyclic polyribonucleotides are described elsewhere (see, for example, U.S. Patent No. 6,210,931, U.S. Patent No. 5,773,244, U.S. Patent No. 5,766,903, U.S. Patent No. 5,712,128, U.S. Patent No. 5,426,180, U.S. Patent Application Publication No. 20100137407, International Publication No. 1992001813, International Publication No. 2010084371, and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015); the contents of each of these are incorporated herein by reference in their entirety).

[0123] reagent The methods described herein use reagents that bind to aptamers on polyribonucleotides. The reagents may be, for example, polypeptides, small molecules, lipids, carbohydrates, RNA, or metals.

[0124] In some embodiments, the reagent is a polypeptide. The polypeptide may be, for example, protein A, streptavidin, lambdapeptide, or MS2 bacteriophage coated protein. In some embodiments, the polypeptide is a polypeptide selected from Table 1.

[0125] In some embodiments, the reagent is a small molecule. The small molecule may be, for example, one selected from Table 2. In some embodiments, the small molecule is biotin or tetracycline. In some embodiments, the small molecule is a metabolite or an amino acid.

[0126] In some embodiments, the reagent is a carbohydrate.

[0127] In some embodiments, the reagent is a lipid.

[0128] In some embodiments, the reagent is RNA. In some embodiments, the RNA is selected from Table 3.

[0129] In some embodiments, the reagent is a metal. The metal may be, for example, nickel, cobalt, cadmium, zinc, or manganese. In some embodiments, the metal is selected from Table 4.

[0130] Aptamer The polyribonucleotide aptamers described herein are designed to bind to reagents. In some embodiments, the aptamers may contain modified nucleotides (e.g., having modified phosphates, sugars, or bases). In some embodiments, the aptamers contain a portion designed to bind to a reagent and a portion that does not bind to a reagent (e.g., a terminal region).

[0131] The aptamer may be, for example, at least 5 nucleotides in length (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 aptamer has a length of, for example, 5-200, 10-200, 10-150, 10-100, 10-50, 20-200, 20-150, 20-100, 20-50, 5-100, 5-95, 10-90, 10-80, 12-60, 15-50, 15-40, 15-30, 18-30, 20-25, or 20-22 nucleotides.

[0132] The aptamer may have a GC content of, for example, 30-70%, such as 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. The aptamer may have a melting temperature (Tm) of, for example, about 45°C to about 75°C, such as 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.

[0133] In some embodiments, the aptamer comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 1 to 124. For example, the aptamer may comprise a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 124.

[0134] In some embodiments, the aptamer may contain a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with respect to the aptamers shown in Table 1. The reagent may be the corresponding reagent shown in Table 1 (e.g., any one of SEQ ID NOs: 1 to 66).

[0135] In some embodiments, the aptamer may contain a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with the aptamers shown in Table 2 (e.g., any one of SEQ ID NOs. 67 to 119). The reagent may be the corresponding reagent shown in Table 2.

[0136] In some embodiments, the aptamer may contain a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with respect to the aptamers shown in Table 3. The reagent may be the corresponding reagent shown in Table 3 (e.g., SEQ ID NO: 120 or 121).

[0137] In some embodiments, the aptamer may contain a nucleic acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with respect to the aptamers shown in Table 4. The reagent may be the corresponding reagent shown in Table 4 (e.g., any one of SEQ ID NOs. 122-124).

[0138] [Table 1-1]

[0139] [Table 1-2]

[0140] [Table 1-3]

[0141] [Table 1-4]

[0142] [Table 1-5]

[0143] [Table 2-1]

[0144] [Table 2-2]

[0145] [Table 2-3]

[0146] [Table 2-4]

[0147] [Table 3]

[0148] [Table 4]

[0149] Those skilled in the art will understand that many aptamer sequences and their corresponding reagents are well known in the art and accessible through any suitable database. For example, many aptamer sequences and their corresponding reagents can be found in the Aptagen database (aptagen.com) or the Registry of Standard Biological Parts (parts.igem.org / DNA / Aptamer). Other databases are well known to those skilled in the art. The aptamer sequences and their corresponding reagents listed in each of the aforementioned and other known databases are incorporated herein by reference in their entirety.

[0150] The methods described herein may include attaching an aptamer to a polyribonucleotide. Those skilled in the art will understand that a portion of the aptamer may be present on the polyribonucleotide, and the method may include attaching a second portion of the aptamer to the polyribonucleotide to form a complete aptamer.

[0151] particle The reagents described herein may be conjugated (e.g., directly or indirectly) to particles, such as magnetic particles or beads. In some embodiments, the reagent is conjugated to a plurality of particles. In some embodiments, the particles are conjugated to a plurality of reagents.

[0152] Magnetic particles include at least one component that is responsive to magnetic force. Magnetic particles may be entirely magnetic or may contain non-magnetic components. Magnetic particles may be magnetic beads, for example, substantially spherical magnetic beads. Magnetic particles may be entirely magnetic or may contain one or more magnetic cores surrounded by one or more additional materials, such as one or more functional groups and / or modifications for binding to one or more target molecules. In some examples, 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.

[0153] The particles, for example, magnetic particles or beads, may be porous, non-porous, hollow, solid, semi-solid, semi-fluid, fluid, and / or a combination thereof. In some cases, the particles, for example, beads, may be soluble or degradable. In some cases, the particles, for example, beads, may not be degradable. In some embodiments, the beads are composed of crosslinked agarose, for example, SEPHAROSE®.

[0154] Particles, such as magnetic particles or beads, may contain natural and / or synthetic materials. For example, particles, such as beads, may contain natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include proteins and sugars, such as deoxyribonucleic acid, gums, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ispacula, acacia, agar, gelatin, shellac, karaya gum, xanthan gum, corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginates, or their natural polymers. Examples of synthetic polymers include acrylic, nylon, silicone, spandex, viscose rayon, polycarboxylic acid, 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 thereof (e.g., copolymers). Beads may also be formed from non-polymer materials, including lipids, micelles, ceramics, glass ceramics, material composites, metals, other inorganic materials, and others.

[0155] Crosslinking may be permanent or reversible, depending on the specific crosslinking agent used. Reversible crosslinking may allow the polymer to linearize or dissociate under appropriate conditions. In some cases, reversible crosslinking may also allow for the reversible bonding of materials bonded to the surface of beads.

[0156] The particles, such as beads or magnetic particles, may be of uniform or non-uniform size. In some cases, the diameter of the particles, such as 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, particles, such as beads, may have diameters of less than approximately 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, particles, such as beads, may have diameters in the range of approximately 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.

[0157] The particles may be of any suitable shape. Examples of particle shapes, such as magnetic particles or beads, include, but are not limited to, spherical, aspherical, oval, rectangular, amorphous, annular, cylindrical, and variations thereof.

[0158] 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 a reagent to particles (e.g., beads), an aptamer to a linear polyribonucleotide, or any combination or variation thereof. In some embodiments, the aptamer is conjugated to a linear polyribonucleotide by a chemical linker. In some embodiments, the reagent is conjugated to particles by a chemical linker. The particles may be, for example, magnetic particles or beads. The beads may be, for example, cross-linked agarose, e.g., SEPHAROSE® beads. In some embodiments, the reagent is directly conjugated to particles (e.g., beads, e.g., magnetic beads or cross-linked agarose, e.g., SEPHAROSE® beads).

[0159] A chemical linker provides space, rigidity, and / or flexibility between, for example, a reagent and particles or between an aptamer and a linear polyribonucleotide. In some embodiments, the linker may be a bond, such as a covalent bond, such as an amide bond, disulfide bond, CO bond, CN bond, NN bond, CS bond, or any type of bond resulting from a chemical reaction, such as a chemical conjugation. In some embodiments, the linker has 250 or fewer 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- (containing 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 Contains 0, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 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 linker skeleton consists of 250 or fewer 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 Includes ~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). The linker's "backbone" refers to the atoms in the linker that together form the shortest path from one part of the conjugate to the other part of the conjugate. The atoms within the linker's framework are directly involved in linking one part of the conjugate to the other part of the conjugate.For example, a hydrogen atom bonded to a carbon within the backbone of a linker is not considered to be directly involved in connecting one portion of a conjugate to the other portion of the conjugate.

[0160] In some embodiments, the linker may include a synthetic group derived from, for example, a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). The 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., a 1-25 amino acid, 1-10 amino acid, 1-9 amino acid, 1-8 amino acid, 1-7 amino acid, 1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid, 1-2 amino acid, or 1 amino acid sequence). In some embodiments, the linker is one or more optionally substituted C1-C 20 alkylene, one or more optionally substituted C1-C 20 heteroalkylene (e.g., a PEG unit), one or more optionally substituted C2-C 20 alkenylene (e.g., C2 alkenylene), one or more optionally substituted C2-C 20 heteroalkenylene, one or more optionally substituted C2-C 20 alkynylene, one or more optionally substituted C2-C 20 heteroalkynylene, one or more optionally substituted C3-C 20 cycloalkylene (e.g., cyclopropylene, cyclobutylene), one or more optionally substituted C2-C 20 heterocycloalkylene, one or more optionally substituted C4-C 20 cycloalkenylene, one or more optionally substituted C4-C 20 heterocycloalkenylene, one or more optionally substituted C8-C 20 cycloalkynylene, one or more optionally substituted C8-C 20 heterocycloalkynylene, one or more optionally substituted C5-C 15 arylene (e.g., C6 arylene), one or more optionally substituted C3-C 15Heteroallylenes (e.g., imidazole, pyridine), O, S, NRi (Ri is H, optionally substituted C1-C) 20 Alkyl, optionally substituted C1-C 20 Heteroalkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Heteroalkenyls, optionally substituted C2-C 20 Alkinyl, optionally substituted C2-C 20 Heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C2-C 20 Heterocycloalkyl, optionally substituted C4-C 20 Cycloalkenyls, optionally substituted C4-C 20 Heterocycloalkenyls, optionally substituted C8-C 20 Cycloalkynyl, optionally substituted C8-C 20 Heterocycloalkynyl, optionally substituted C5-C 15 Aryl or optionally substituted C3-C 15 It may also contain heteroaryl compounds, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino compounds.

[0161] Covalent conjugation of two or more components in a linker-based conjugate 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, but not limited to, maleimides and cysteines, amines and activated carboxylic acids, thiols and maleimides, activated sulfonic acids and amines, isocyanates and amines, azides and alkynes, and alkenes and tetrazines. Site-specific conjugation to polypeptides can be achieved using techniques known in the art.

[0162] resin In some embodiments, the methods described herein include using a resin having a plurality of particles conjugated to a reagent that binds to an aptamer. The method may also include using a column containing the resin. The method may also include recovering an eluate (e.g., not bound to the resin) from a plurality of polyribonucleotides in the sample, containing a portion of the sample that is not bound to the reagent. In some embodiments, the resin includes crosslinked poly[styrene-divinylbenzene], agarose, or SEPHAROSE®.

[0163] The compositions and methods of the present invention may utilize a surface linked to a reagent designed to bind to an aptamer. The resin surface refers to a portion of a support structure (e.g., a substrate) that is accessible to contact one or more reagents. The shape, form, material, and modification of the resin surface can be selected from a variety of options depending on the application. In one embodiment, the resin surface is SEPHAROSE®. In one embodiment, the resin surface is agarose.

[0164] The surface of the resin may be substantially flat or planar. Alternatively, the surface of the resin may be circular or contoured. Examples of contours that may be included on the surface of the resin include wells, depressions, columns, dams, and channels.

[0165] Exemplary materials that can be used as resin surfaces include, but are not limited to, acrylic, carbon (e.g., graphite, carbon fiber), cellulose (e.g., cellulose acetate), ceramics, controlled-pore glass, cross-linked polysaccharides (e.g., agarose or SEPHAROSE®), gels, glass (e.g., modified or functionalized glass), gold (e.g., atomically flat Au(l11)), graphite, inorganic glass, inorganic polymers, latex, metal oxides (e.g., SiO2, TiO2, stainless steel), metalloids, metals (e.g., atomically flat Au(l11)), mica, molybdenum disulfide, and nanomaterials (e.g., highly oriented thermal Examples include graphite-dissolved (HOPG) nanosheets, nitrocellulose, NYLON®, optical fiber bundles, organic polymers, paper, plastics, polyacryloyl morpholide, poly(4-methylbutene), polyethylene terephthalate, poly(vinyl butyrate), polybutylene, polydimethylsiloxane (PDMS), polyethylene, polyformaldehyde, polymethacrylate, polypropylene, polysaccharides, polystyrene, polyurethane, polyvinylidene fluoride (PVDF), quartz, rayon, resins, rubber, semiconductor materials, silica, silicon (e.g., surface silicon oxide), sulfides, and TEFLON®. A single material or a mixture of several different materials can form resins useful in the present invention.

[0166] In some embodiments, the surface of the resin contains a polymer.

[0167] In some embodiments, the surface of the resin includes SEPHAROSE®. An example is shown below (where n is any positive integer): [ka]

[0168] In some embodiments, the surface of the resin contains agarose. An example is shown below (where n is any positive integer): [ka] Structure of agarose: D-galactose and 3,6-anhydro-α-galactopyranose repeating units.

[0169] In some embodiments, the surface of the resin contains a polystyrene-based polymer. A schematic diagram of the synthesis of a polystyrene-divinylbenzene copolymer is shown below: [ka]

[0170] In some embodiments, the resin surface comprises an acrylic-based polymer. Poly(methyl methacrylate) is an example shown below (where n is any positive integer): [ka]

[0171] In some embodiments, the resin surface contains a dextran-based polymer. Examples of dextran are shown below: [ka]

[0172] In some embodiments, the surface of the resin contains silica. Examples are shown below: [ka]

[0173] In some embodiments, the surface of the resin contains polyacrylamide. An example of crosslinking with NN-methylenebisacrylamide is shown below: [ka]

[0174] In some embodiments, the surface of the resin includes a tentacle-based, for example, methacrylate-based phase.

[0175] Several surfaces known in the art are suitable for use in the method of the present invention. Suitable surfaces may include, but are not limited to, borosilicate glass, agarose, SEPHAROSE®, magnetic beads, polystyrene, polyacrylamide, membranes, silica, semiconductor materials, silicon, organic polymers, ceramics, glass, metals, plastic polycarbonates, polycarbonates, polyethylene, polyethylene glycol terephthalate, polymethyl methacrylate, polypropylene, polyvinyl acetate, polyvinyl chloride, polyvinylpyrrolidinone, and soda-lime glass.

[0176] In one embodiment, the surface of the resin is modified to include channels, patterns, layers, or other stereoconfigurations (e.g., patterned surfaces). The surface may be in the form of beads, boxes, columns, cylinders, discs, dishes (e.g., glass dishes, Petri dishes), fibers, films, filters, microtiter plates (e.g., 96-well microtiter plates), multi-blade sticks, nets, pellets, plates, rings, rods, rolls, sheets, slides, sticks, trays, tubes, or vials. The surface may be a single individual body (e.g., a single tube, a single bead), any number of surface bodies (e.g., a rack of 10 tubes, several beads), or a combination thereof (e.g., a tray containing multiple microtiter plates, a column filled with beads, a microtiter plate filled with beads).

[0177] In some embodiments, the surface may include a membrane-based resin matrix. In some embodiments, the resin surface may include a porous resin or a non-porous resin. Examples of porous resins include additional agarose-based resins (e.g., cyanogen bromide-activated SEPHAROSE® (GE); WorkBeads® 40 ACT and WorkBeads 40 / 10000ACT (Bioworks)), methacrylates (e.g., Tosoh's 650M derivative), polystyrene-divinylbenzene (Life Tech's Poros medium / GE's source medium), fractogel, polyacrylamide, silica, controlled-pore glass, dextran derivatives, acrylamide derivatives, additional polymers, and combinations thereof.

[0178] In some embodiments, the surface may include one or more pores. In some embodiments, the pore diameter may be 300 to 8,000 angstroms, for example, 500 to 4,000 angstroms.

[0179] The resins described herein contain a plurality of particles. Examples of particle sizes include 5 μm to 500 μm, 20 μm to 300 μm, and 50 μm to 200 μm. In some embodiments, the particle size may be 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, or 200 μm.

[0180] The reagents may be immobilized, coated, bound, fixed, adhered, or bonded to any of the surface forms described herein (e.g., beads, boxes, columns, cylinders, disks, dishes (e.g., glass dishes, Petri dishes), fibers, films, filters, microtiter plates (e.g., 96-well microtiter plates), multi-blade sticks, nets, pellets, plates, rings, rods, rolls, sheets, slides, sticks, trays, tubes, or vials).

[0181] In one embodiment, the surface is modified to include chemical modification sites that can be used to bond reagents to separate sites or locations on the surface (e.g., by covalent or non-covalent bonds). The chemical modification sites include, for example, adding a pattern of chemical functional groups that can be used to covalently bond reagents, including amino groups, carboxyl groups, oxo groups, and thiol groups, and generally also including corresponding reactive functional groups. Examples of surface functionalization include amino derivatives, thiol derivatives, aldehyde derivatives, formyl derivatives, azide derivatives (click chemistry), biotin derivatives, alkyne derivatives, hydroxyl derivatives, activated hydroxyl or derivatives, carboxylic acid derivatives, activated carboxylic acid derivatives, activated carbonates, activated esters, NHS esters (succinimidyl), NHS carbonates (succinimidyl), imide esters or derivatives (derivated), cyanogen bromide derivatives, maleimide derivatives, and haloacetyl derivatives. Examples include iodoacetamide / iodoacetyl derivatives, epoxide derivatives, streptavidin derivatives, tresyl derivatives, dienes / conjugate diene derivatives (Diels-Alder reaction), alkene derivatives, substituted phosphate derivatives, bromohydrins / halohydrins, substituted disulfides, pyridyl disulfide derivatives, aryl azides, acyl azides, azulactones, hydrazide derivatives, halobenzene derivatives, nucleoside derivatives, branched / polyfunctional linkers, dendrimer-functionalized linkers, nucleoside derivatives, or any combination thereof.

[0182] In some embodiments, the bonding capacity of the linked surfaces may be at least 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, or more.

[0183] In some embodiments, the resin-containing column is designed to conjugate at least 500 μg (e.g., at least 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, or more) of polyribonucleotides to, for example, an aptamer. In some embodiments, the column is designed to conjugate 500 μg to 1,000 mg of polyribonucleotides to, for example, an aptamer.

[0184] composition As described herein, the present invention is characterized by a composition comprising a population of polyribonucleotides produced by the method described herein. The population may include, for example, cyclic polyribonucleotides lacking aptamers, the cyclic polyribonucleotides 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) (moles / 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 less than 1%) (moles / mol) of linear polyribonucleotides in the composition.

[0185] In other embodiments, the population comprises, for example, a polyribonucleotide in a first configuration having an aptamer and a polyribonucleotide in a second configuration having an aptamer, wherein the polyribonucleotide in the first configuration constitutes at least 1% of the total polyribonucleotides in the composition, 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) (moles / moles).

[0186] In some embodiments described herein, the present invention features a composition comprising a mixture of polyribonucleotides. A first subset of the mixture comprises cyclic polyribonucleotides lacking aptamers, and a second subset of the polyribonucleotides comprises linear polyribonucleotides having aptamers. The first subset comprises at least 1%, for example, at least 5%, for example, at least 10%, at least 20%, at least 30%, or at least 40% (for example, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) (moles / moles) of the total polyribonucleotides in the composition. In some embodiments, the linear polyribonucleotides comprise various different linear polyribonucleotide species, for example, each containing an aptamer.

[0187] In some embodiments described herein, the present invention relates to a composition comprising a polyribonucleotide having an aptamer and a reagent designed to bind to the aptamer, wherein the reagent is conjugated to particles, for example, via a linker.

[0188] In some embodiments of any of the compositions described herein, the linear polyribonucleotide comprises an intron or a portion thereof. The aptamer may be located at the 5' or 3' end of the intron or a portion thereof.

[0189] In some embodiments, the polyribonucleotide may be a modified polyribonucleotide.

[0190] In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the 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 on a mass basis. Purity can be measured by any one of several analytical techniques known to those skilled in the art, such as the use of separation techniques including, but not limited to, chromatography (using columns, paper, gel, HPLC, UHPLC, etc., or by IC, SEC, reversed phase, anion exchange, mixed mode, etc.) or electrophoresis (urea PAGE, tip-based, polyacrylamide gel, RNA, capillary, c-IEF, etc.), with or without the presence 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 staining or radioactive decay for detection. Alternatively, purity can be measured without the use of separation techniques by mass spectrometry, microscopy, circular dichroism (CD) spectroscopy, UV or UV-vis spectrophotometric analysis, fluorescence measurement (e.g., Qubit), ribonuclease H analysis, surface plasmon resonance (SPR), or methods using silver or dye staining or radioactive decay for detection.

[0191] 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 the cyclic polyribonucleotide molecules. Percentages can be measured by any one of several analytical techniques known to those skilled in the art, such as the use of separation techniques including, but not limited to, chromatography (using columns, paper, gel, HPLC, UHPLC, etc., or by IC, SEC, reversed phase, anion exchange, mixed mode, etc.) or electrophoresis (urea PAGE, tip-based, polyacrylamide gel, RNA, capillary, c-IEF, etc.), with or without the presence 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 staining or radioactive decay for detection. Alternatively, purity can be measured without the use of separation techniques by mass spectrometry, microscopy, circular dichroism (CD) spectroscopy, UV or UV-vis spectrophotometric analysis, fluorescence measurement (e.g., Qubit), ribonuclease H analysis, surface plasmon resonance (SPR), or methods using silver or dye staining or radioactive decay for detection.

[0192] In one embodiment, the cyclic polyribonucleotide preparation (for example, the cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) contains 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, It has cyclic polyribonucleotide concentrations of 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, 1,000 μg / mL, 5,000 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. In one embodiment, the cyclic polyribonucleotide preparation (for example, a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) is substantially mononucleotide-free, or has a mononucleotide content of 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, 1,000 μg / mL, 5,000 μg / mL, 10,000 μg / mL, or 100,000 μg / mL or less.In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) has a mononucleotide content from a detection limit of 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, 1,000 μg / mL, 5,000 μg / mL, 10,000 μg / mL, or 100,000 μg / mL.

[0193] In one embodiment, the cyclic polyribonucleotide preparation (e.g., the cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) is, on a mass basis, 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), and 1% (w / w) of the total nucleotides. The mononucleotide content is less than or equal to 2%(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), or any percentage in between, where the total nucleotide content is the total mass of deoxyribonucleotide molecules and ribonucleotide molecules.

[0194] In one embodiment, the cyclic polyribonucleotide preparation (for example, the cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) is available in concentrations 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, 50 μg / mL, 100 μg / mL, and 200 g. The linear RNA-containing substances include those containing 1 μg / 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, 700 mg / mL, 750 mg / mL, 800 ng / mL, 850 ng / mL, 900 ng / mL, 950 ng / mL, or 1 μg / mL or less, for example, linear RNA-compatible substances or RNA fragments.In one embodiment, the cyclic polyribonucleotide preparation (e.g., the cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) can be up to 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, 650 mg / mL, 700 mg / mL, 700 ng / mL, 750 mg / mL, 800 ng / mL, 8 The product contains linear RNA-containing substances, such as linear RNA counterparts or RNA fragments, from detection limits of 50 ng / mL, 900 ng / mL, 950 ng / mL, 1 μg / mL, 10 μg / mL, 50 μg / mL, 500 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.

[0195] In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) has a nic RNA content of 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 less, or a percentage between these. In one embodiment, the cyclic polyribonucleotide preparation (for example, a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) has a nic RNA content as low as 0, or is substantially free of nic RNA.

[0196] In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) has a combined content of linear RNA and nick RNA of 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 less, or percentages between these. In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) has a combined content of nic RNA and linear RNA that is as low as 0, or is substantially free of nic RNA and linear RNA.

[0197] 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) contains linear RNA-containing material, such as linear RNA counterparts or RNA fragments, below the detection limit of analytical methods such as mass spectrometry, UV spectroscopy or fluorescence detection, light scattering techniques, surface plasmon resonance (SPR), HPLC, or separation methods including HPLC, chip or gel-based electrophoresis, either before or after separation, detection methods using silver or dye staining or radioactive decay, or methods used in the presence or absence of microscopy, visual methods or spectrophotometers.

[0198] In one embodiment, the cyclic polyribonucleotide preparation (for example, a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) has linear RNA in amounts of 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.

[0199] In some embodiments, the linear polyribonucleotide molecule of the cyclic polyribonucleotide preparation includes a linear counterpart or fragment thereof of the cyclic polyribonucleotide molecule. In some embodiments, the linear polyribonucleotide molecule of the cyclic polyribonucleotide preparation includes a linear counterpart (e.g., a pre-cyclization version). In some embodiments, the linear polyribonucleotide molecule of the cyclic polyribonucleotide preparation includes a non-corresponding counterpart or fragment thereof to the cyclic polyribonucleotide. In some embodiments, the linear polyribonucleotide molecule of the cyclic polyribonucleotide preparation includes a non-corresponding counterpart to the cyclic polyribonucleotide. In some embodiments, the linear polyribonucleotide molecule includes a combination of a cyclic polyribonucleotide counterpart and a cyclic polyribonucleotide non-corresponding counterpart or fragment thereof. In some embodiments, the linear polyribonucleotide molecule includes a combination of a cyclic polyribonucleotide counterpart and a cyclic polyribonucleotide non-corresponding counterpart. In some embodiments, the linear polyribonucleotide molecular fragment is a fragment having a length of 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, or any number of nucleotides in between.

[0200] 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, as measured, for example, 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 in between. 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 greater than about 1.8, as measured, for example, by a spectrophotometer. In some embodiments, the A260 / A280 absorbance ratio is approximately 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 higher.

[0201] 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) is substantially free of impurities or by-products. In various embodiments, the level of at least one impurity or by-product in the composition containing the 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 before purification or treatment to remove the impurities or by-products. 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) compared to the composition before 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) compared to the composition before purification or treatment to remove the impurity or by-product. In some embodiments, the cyclic polyribonucleotide preparation (e.g., the cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) is further substantially free of process-related impurities or by-products.In some embodiments, 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 fragments, and monodeoxyribonucleotide molecules. In some embodiments, the pharmaceutical preparation contains protein contaminants, impurities, or by-products as protein (e.g., cellular proteins such as host cell proteins) in amounts of 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 less than 500 ng per milligram (mg) of cyclic polyribonucleotide molecules.

[0202] In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) is substantially free of DNA-containing material, for example, the template DNA or cellular DNA (e.g., host cell DNA) has a DNA content as low as 0, or 1 pg / mL, 10 pg / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, It has a DNA content of 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, 1,000 μg / mL, 5,000 μg / mL, 10,000 μg / mL, or 100,000 μg / mL or less.

[0203] In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) is substantially free of DNA-containing material, has a DNA content of approximately 0, or on a mass basis contains 0.001% (w / w), 0.01% (w / w), 0.1% (w / w), 1% (w / w), 2% (w / w), 3% (w / w) of the total nucleotides. The DNA content is 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), and 50%(w / w) or less, where total nucleotide molecules refer to the total mass of deoxyribonucleotide-containing substances and ribonucleotide molecules. In one embodiment, a cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide preparation) is measured by quantitative liquid chromatography-mass spectrometry (LC-MS) after total DNA digestion with an enzyme that digests nucleosides (the DNA content is calculated inversely from the calibration curve for each base (i.e., A, C, G, T) when measured by LC-MS), and contains substantially no DNA, or a DNA content of approximately 0. The DNA content is such that it contains 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), or 50%(w / w) of the total nucleotides on a mass basis.

[0204] In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) contains protein contaminants, impurities, or by-products in amounts of 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 or less (e.g., cellular proteins (CPs), e.g., enzymes, product-related proteins, e.g., carrier proteins). In some embodiments, the cyclic polyribonucleotide (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide) contains protein (e.g., cellular protein (CP), product-related proteins such as enzymes, etc.) contaminants, impurities, or by-products from detection limits of up to 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.

[0205] In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) contains 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 less than 500 ng of protein (e.g., cellular protein (CP), product-related proteins such as enzymes) contaminants, impurities, or by-products per milligram (mg) of cyclic polyribonucleotide. In one embodiment, the cyclic polyribonucleotide (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of a cyclic polyribonucleotide) contains protein (e.g., cellular protein (CP), product-related proteins such as enzymes, etc.) contaminants, impurities, or by-products from detection levels of up to 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 per milligram (mg) of the cyclic polyribonucleotide.

[0206] In some embodiments, cyclic polyribonucleotide preparations (e.g., cyclic polyribonucleotide pharmaceutical preparations or compositions or intermediates in the production of cyclic polyribonucleotide preparations) have low levels of endotoxins or are endotoxin-free, as measured, for example, by the horseshoe crab hemocyte extract (LAL) test. In some embodiments, pharmaceutical preparations or compositions or intermediates in the production of cyclic polyribonucleotides contain or are endotoxin-free at levels of less than 20 EU / kg (weight), 10 EU / kg, 5 EU / kg, or 1 EU / kg, as measured by the horseshoe crab hemocyte extract test. In some embodiments, cyclic polyribonucleotide compositions have low levels of nucleases or ligases or are endotoxin-free.

[0207] In some embodiments, the cyclic polyribonucleotide preparation (e.g., the cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) is approximately 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) It contains at least one enzyme, such as polymerase, such as RNA polymerase, in amounts of 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), and 1%(w / w).

[0208] In one embodiment, the cyclic polyribonucleotide preparation (e.g., a cyclic polyribonucleotide pharmaceutical preparation or composition or intermediate in the production of the cyclic polyribonucleotide preparation) is sterile or substantially microorganism-free, for example, the composition or preparation supports the growth of fewer than 100 viable microorganisms when tested under sterile conditions, and the composition or preparation is USP <71> The composition or preparation meets the standards of the USP <85> The criteria are met. In some embodiments, the pharmaceutical preparation contains bioburden of less than 100 CFU / 100ml, 50 CFU / 100ml, 40 CFU / 100ml, 30 CFU / 100ml, 20 CFU / 100ml, 10 CFU / 100ml, or less than 1 CFU / 100ml before sterilization.

[0209] In some embodiments, the cyclic polyribonucleotide preparation can be further purified using techniques known in the art, such as column chromatography or pH control and vial inactivation, to remove impurities or by-products.

[0210] In some embodiments, the total weight of polyribonucleotides in the composition is at least 500 μg (e.g., at least 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg or more). In some embodiments, the total weight of polyribonucleotides in the polyribonucleotide population is 500 μg to 1,000 mg.

[0211] Polynucleotides The present invention features polyribonucleotides used in separation and / or purification methods 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 produced from linear polyribonucleotides (for example, by splicing the compatible ends of linear polyribonucleotides). In some embodiments, linear polyribonucleotides are transcribed from a deoxyribonucleotide template (e.g., a vector, a linearized vector, or cDNA). Accordingly, the present invention features linear deoxyribonucleotides, cyclic deoxyribonucleotides, linear polyribonucleotides, and cyclic polyribonucleotides and compositions thereof that are useful in the production of polyribonucleotides.

[0212] Linear polyribonucleotides The present invention is characterized by a linear polyribonucleotide that may comprise one or more of the following: a 3' intron fragment; a 3' splice site; a 3' exon; a polyribonucleotide cargo; a 5' exon; a 5' splice site; and a 5' intron fragment. In some embodiments, the 3' intron fragment corresponds to the 3' portion of a catalytic group I intron, e.g., from the cyanobacterial Anabaena pretRNA-Leu gene, Tetrahymena prerRNA, T4 phage td gene, or a variant thereof. In some embodiments, the 5' intron fragment corresponds to the 5' portion of a catalytic group I intron, e.g., from the cyanobacterial Anabaena pretRNA-Leu gene, Tetrahymena prerRNA, T4 phage td gene, or a variant thereof.

[0213] The linear polyribonucleotide may contain additional elements, for example, outside or between the above elements. For example, any of the above elements may be separated by a spacer sequence as described herein. The aptamers described herein may be located within any region of the linear polyribonucleotide described herein.

[0214] In some embodiments, the linear polyribonucleotide comprises, in the following 5' to 3' order: aptamer, first cyclic sequence (e.g., first intron fragment); polyribonucleotide cargo; and second cyclic sequence (e.g., second intron fragment). In some embodiments, the linear polyribonucleotide comprises, in the following 5' to 3' order: aptamer, 3' intron fragment; 3' splice site; 3' exon; polyribonucleotide cargo; 5' exon; 5' splice site; and 5' intron fragment.

[0215] In some embodiments, the linear polyribonucleotide comprises, in the following 5' to 3' order: a first cyclic sequence (e.g., a first intron fragment); a polyribonucleotide cargo; a second cyclic sequence (e.g., a second intron fragment); and an aptamer. In some embodiments, the linear polyribonucleotide comprises, in the following 5' to 3' order: a 3' intron fragment; a 3' splice site; a 3' exon; a polyribonucleotide cargo; a 5' exon; a 5' splice site; a 5' intron fragment; and an aptamer.

[0216] In certain embodiments, the foregoing provides a method for generating linear polyribonucleotides by performing transcription (e.g., in vitro transcription in a cell-free system) using a deoxyribonucleotide provided herein (e.g., a vector, linearization vector, or cDNA) as a template (e.g., a vector, linearization vector, or cDNA provided herein, in which the RNA polymerase promoter is located upstream of the region encoding the linear polyribonucleotide).

[0217] A deoxyribonucleotide template can be transferred to generate linear polyribonucleotides containing the components described herein. Upon expression, the linear polyribonucleotides can be spliced ​​to produce splicing-compatible polyribonucleotides, which can then be spliced ​​to produce cyclic polyribonucleotides for later use, for example.

[0218] In some embodiments, the linear polyribonucleotides are 50-20,000, for example, 300-20,000 (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, The length of a ribonucleotide is 800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000. A linear polyribonucleotide may have a length of, 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.

[0219] Cyclic polyribonucleotide In some embodiments, the present invention is characterized by a cyclic polyribonucleotide. In embodiments, the cyclic polyribonucleotide includes a splice junction linking the 5' and 3' exons. In embodiments, the cyclic polyribonucleotide lacks an intron after splicing, for example. In embodiments, the cyclic polyribonucleotide lacks an aptamer after splicing, for example.

[0220] In embodiments, the cyclic polynucleotide further comprises a polyribonucleotide cargo. In embodiments, the polyribonucleotide cargo comprises an expression (or coding) sequence, a non-coding sequence, or a combination of an expression (or coding) sequence and a non-coding sequence. In embodiments, the polyribonucleotide cargo comprises an expression (or coding) sequence encoding a polypeptide. In embodiments, the polyribonucleotide comprises at least one IRES (e.g., IRES) operably linked to the expression sequence encoding the polypeptide. In some embodiments, the cyclic polyribonucleotide further comprises a spacer region between at least one IRES and a 5' exon fragment or a 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 some embodiments, the spacer region includes a poly-A sequence. In some embodiments, the spacer region includes poly-AC, poly-AG, poly-AU, or other heterogeneous or random sequences.

[0221] In some embodiments, the cyclic polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

[0222] In some embodiments, the cyclic polyribonucleotide is large enough to accommodate at least one binding site to a ribosome. In some embodiments, the size of the cyclic polyribonucleotide is long enough to encode a useful polypeptide, and thus lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, 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 1,400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, and at least 100 nucleotides can be produced.

[0223] In some embodiments, the cyclic polyribonucleotide comprises one or more elements described herein. In some embodiments, the elements are separated from each other by spacer sequences. In some embodiments, the elements are 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 1,000 nucleotides, up to about 1 kb, at least about 1,000 nucleotides, or any amount of nucleotides in between. In some embodiments, one or more elements are adjacent to each other and, for example, lack spacer elements.

[0224] In some embodiments, the cyclic polyribonucleotide comprises one or more repeating elements. In some embodiments, the cyclic polyribonucleotide comprises one or more modifications as described herein. In one embodiment, the cyclic polyribonucleotide comprises at least one nucleoside modification. In one embodiment, up to 100% of the nucleosides of the cyclic polyribonucleotide are modified. In one embodiment, at least one nucleoside modification is a uridine modification or an adenosine modification.

[0225] As a result of cyclization, cyclic polyribonucleotides may possess certain characteristics that distinguish them from linear polyribonucleotides. For example, cyclic polyribonucleotides may contain more accessible aptamers than linear polyribonucleotides. In some embodiments, cyclic polyribonucleotides are less susceptible to exonuclease degradation compared to linear polyribonucleotides. As such, cyclic polyribonucleotides are more stable than linear polyribonucleotides, particularly when incubated in the presence of exonucleases. The increased stability of cyclic polyribonucleotides compared to linear polyribonucleotides makes them more useful as cell transformation reagents for polypeptide production and easier and longer-lasting storage than linear polyribonucleotides. The stability of exonuclease-treated cyclic polyribonucleotides can be tested using methods standard in the art to determine whether RNA degradation has occurred (e.g., by gel electrophoresis). Furthermore, unlike linear polyribonucleotides, cyclic polyribonucleotides are less susceptible to dephosphorylation when incubated with phosphatases such as bovine intestinal phosphatase.

[0226] Polyribonucleotide cargo The polyribonucleotide cargo described herein comprises any sequence containing at least one polyribonucleotide. In some embodiments, the polyribonucleotide cargo comprises an expression (or coding) sequence, a non-coding sequence, or an expression (or coding) sequence and a non-coding sequence. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide. In some embodiments, the polyribonucleotide cargo comprises an IRES operably ligated to an expression sequence encoding a polypeptide. In some embodiments, the polyribonucleotide cargo comprises an expression sequence encoding a polypeptide having a biological effect on a subject.

[0227] The polyribonucleotide cargo may contain, 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 1 to 20,000 nucleotides, 1 to 10,000 nucleotides, 1 to 5,000 nucleotides, 100 to 20,000 nucleotides, 100 to 10,000 nucleotides, 100 to 5,000 nucleotides, 500 to 20,000 nucleotides, 500 to 10,000 nucleotides, 500 to 5,000 nucleotides, 1,000 to 20,000 nucleotides, 1,000 to 10,000 nucleotides, or 1,000 to 5,000 nucleotides.

[0228] In embodiments, the polyribonucleotide cargo comprises one or more expression (or coding) sequences, each of which codes for a polypeptide. In embodiments, the polyribonucleotide cargo comprises one or more non-coding sequences. In embodiments, the polyribonucleotide consists entirely of non-coding sequences. In embodiments, the polyribonucleotide cargo comprises a combination of expression sequences and non-coding sequences.

[0229] In some embodiments, polyribonucleotides prepared as described herein are used as therapeutic agents or effectors in agriculture. For example, cyclic polyribonucleotides prepared by the methods described herein (e.g., the cell-free method described herein) can be administered to a subject (e.g., in pharmaceutical, animal, or agricultural compositions). In another example, cyclic polyribonucleotides prepared by the methods described herein (e.g., the cell-free method described herein) can be delivered to cells.

[0230] In some embodiments, the polyribonucleotide comprises any feature, or any combination of features, as disclosed in PCT International Publication No. 2019 / 118919, which is incorporated herein by reference in whole.

[0231] In some embodiments, the polyribonucleotide cargo includes an open reading frame. In some embodiments, the open reading frame is operably linked to an IRES. In some embodiments, the open reading frame encodes RNA or a polypeptide. In some embodiments, the open reading frame encodes a polypeptide, and the polyribonucleotide (e.g., cyclic polyribonucleotide) results in increased polypeptide expression (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) compared to, for example, a linear polyribonucleotide encoding the polypeptide. In some embodiments, increasing the purity of polyribonucleotides, such as cyclic 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) compared to populations of cyclic and linear polyribonucleotides.

[0232] Polypeptide expression sequence In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargo of cyclic polyribonucleotides) comprise one or more expression (or coding) sequences, each expression (coding) sequence encoding a polypeptide. In some embodiments, the cyclic polyribonucleotide comprises two, three, four, five, six, seven, eight, nine, ten or more expression sequences.

[0233] Each coding polypeptide may be linear or branched. Polypeptides may have lengths of approximately 5 to 40,000 amino acids, 15 to 35,000 amino acids, 20 to 30,000 amino acids, 25 to 25,000 amino acids, 50 to 20,000 amino acids, 100 to 15,000 amino acids, 200 to 10,000 amino acids, 500 to 5,000 amino acids, 1,000 to 2,500 amino acids, or any range in between. In some embodiments, the polypeptide has a useful 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.

[0234] Polypeptides included herein may include naturally occurring polypeptides or polypeptides that do not exist in nature. In some cases, polypeptides may be functional fragments or variants of a reference polypeptide (e.g., enzymatic activity fragments or variants of an enzyme). For example, a polypeptide may be a functionally active variant of any of the polypeptides described herein, having 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 with the sequence of a polypeptide described herein or a naturally occurring polypeptide, either across a specific region or across the entire sequence. In some cases, the polypeptide may have at least 50% identity with the protein of interest (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more).

[0235] Some examples of polypeptides, but not limited to, include fluorescent tags or markers, antigens, therapeutic polypeptides, plant-modified polypeptides, or polypeptides for agricultural use.

[0236] Therapeutic polypeptides may include hormones, neurotransmitters, growth factors, enzymes (e.g., oxidoreductase, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, lysosomal enzymes, desaturases), cytokines, antigen-binding polypeptides (e.g., antigen-binding antibodies or antibody-like fragments, e.g., single-chain antibodies, nanobodies, or other polypeptides containing Ig heavy or light chains), Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, interferons, interleukins, and thrombolytic agents.

[0237] Polypeptides for agricultural use may be bacteriocins, lysins, antibacterial polypeptides, antifungal polypeptides, root nodule C-rich peptides, bacteriocyte regulatory peptides, peptide toxins, pesticidal polypeptides (e.g., insecticidal polypeptides or nematicidal polypeptides), antigen-binding polypeptides (e.g., antigen-binding antibodies or antibody-like fragments, such as single-chain antibodies, nanobodies or polypeptides containing other Ig heavy or light chains), enzymes (e.g., nucleases, amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones, and transcription factors.

[0238] In some cases, the polynucleotide expresses a non-human protein.

[0239] In some embodiments, the polynucleotide expresses an antibody, such as an antibody fragment, or a portion thereof. In some embodiments, the antibody expressed by the circular polynucleotide can be any isotype, such as IgA, IgD, IgE, IgG, IgM. In some embodiments, the circular polynucleotide expresses a portion of an antibody, such as a light chain, heavy chain, Fc fragment, CDR (complementary determining region), Fv fragment, or Fab fragment, a further portion thereof. In some embodiments, the circular polynucleotide expresses one or more portions of an antibody. For example, the circular polynucleotide can contain two or more expression sequences, each of which expresses a portion of the antibody, and the sum of which can constitute the antibody. In some cases, the circular polynucleotide contains one expression sequence encoding the heavy chain of an antibody and another expression sequence encoding the light chain of the antibody. In some cases, when the circular polynucleotide is expressed in a cellular or cell-free environment, the light and heavy chains can undergo appropriate modification, folding, or other post-translational modification to form a functional antibody.

[0240] In the embodiment, the polypeptide comprises multiple polypeptides, for example, multiple copies of a single polypeptide sequence, or multiple different polypeptide sequences. In the embodiment, the multiple polypeptides are linked together by linker amino acids or spacer amino acids.

[0241] In embodiments, the polynucleotide cargo contains 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 helps translocate folded proteins containing such Tat signal peptides across the lipid bilayer. See also, 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.

[0242] In embodiments, the polynucleotide cargo includes a sequence encoding a cell-permeable peptide (CPP). Hundreds of CPP sequences have been described; see, for example, the CPPsite database of publicly available cell-permeable peptides at crdd[dot]osdd[dot]net / raghava / cppsite / . Examples of commonly used CPP sequences are poly-arginine sequences, such as octoarginine or nonoarginine, which can be fused to the C-terminus of a CGI peptide.

[0243] In embodiments, the polynucleotide cargo includes a sequence encoding a self-assembling peptide; see, for example, Miki et al. (2021) Nature Communications, 21:3412, DOI:10.1038 / s41467-021-23794-6.

[0244] In some embodiments, the expression (or coding) sequence includes a polyA sequence (for example, at the 3' end of the expression sequence). In some embodiments, the length of the polyA sequence is greater than 10 nucleotides. In one embodiment, the polyA sequence is longer than 15 nucleotides (for example, at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides or more). In some embodiments, the polyA sequence is designed in accordance with the description of the polyA sequence in sections

[0202] to

[0204] of International Publication No. 2019 / 118919A1, which is incorporated herein by reference in whole. In some embodiments, the expression sequence lacks the polyA sequence (for example, at the 3' end of the expression sequence).

[0245] In some embodiments, cyclic polyribonucleotides contain, lack, or have modified polyA to modulate one or more characteristics of the cyclic polyribonucleotide. In some embodiments, cyclic polyribonucleotides lacking or having modified polyA improve one or more functional properties, such as immunogenicity (e.g., levels of one or more markers of an immune or inflammatory response), half-life, and / or expression efficiency.

[0246] Therapeutic polypeptides In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of cyclic polyribonucleotides) include at least one expression sequence encoding a therapeutic polypeptide. The therapeutic polypeptide is a polypeptide that, when administered to or expressed within a subject, provides several therapeutic benefits. The administration of or expression of the therapeutic polypeptide within a subject can be used to treat or prevent a disease, disorder, or condition or its symptoms. In some embodiments, the cyclic polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten or more therapeutic polypeptides.

[0247] In some embodiments, the polyribonucleotide comprises an expression sequence encoding a therapeutic protein. This protein can treat a disease in a target that requires it. In some embodiments, the therapeutic protein may compensate for a mutated, downexpressed, or absent protein in a target that requires it. In some embodiments, the therapeutic protein may target, interact with, or bind to cells, tissues, or viruses in a target that requires it.

[0248] Therapeutic polypeptides may be polypeptides that can be secreted from cells or that can be localized in the cytoplasm, nucleus, or membrane compartment of cells.

[0249] Therapeutic polypeptides include hormones, neurotransmitters, growth factors, enzymes (e.g., oxidoreductase, 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, e.g., single-chain antibodies, nanobodies, or other polypeptides containing Ig heavy or light chains), 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, e.g., 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), secretory proteins, gene-editing proteins (e.g., CRISPR-Cas, TALEN, or Zn fingers), or gene-writing proteins. It may also be a protein (see, for example, International Publication No. 2020 / 047124, which is incorporated herein by reference in its entirety).

[0250] 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 a polyribonucleotide (e.g., a cyclic polyribonucleotide) may be any isotype such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, the polyribonucleotide expresses a portion of the antibody, e.g., a light chain, heavy chain, Fc fragment, CDR (complementarity-determining region), Fv fragment, or Fab fragment, or a further portion thereof. In some embodiments, the polyribonucleotide expresses one or more portions of the antibody. For example, the polyribonucleotide may contain two or more expression sequences, each of which may express a portion of the antibody, and their sum may constitute the antibody. In some cases, the polyribonucleotide includes one expression sequence encoding the heavy chain of the antibody and another expression sequence encoding the light chain of the antibody. When the polyribonucleotide is expressed subcellularly, the light and heavy chains may undergo appropriate modifications, folding, or other post-translational modifications to form a functional antibody.

[0251] In some embodiments, polyribonucleotides (e.g., cyclic polyribonucleotides) prepared as described herein are used as therapeutic agents or effectors in agriculture. For example, polyribonucleotides prepared by the methods described herein can be administered to a subject (e.g., in pharmaceutical, animal, or agricultural compositions). In embodiments, the subject is a vertebrate (e.g., mammal, bird, fish, reptile, or 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., monkey, ape), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horse, donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or rabbit (e.g., rabbit). In the embodiments, the subjects are birds such as members of avian taxa such as Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In the embodiments, the subjects are invertebrates such as arthropods (e.g., insects, spiders, crustaceans), nematodes, annelids, helminths, or mollusks. In the embodiments, the subjects are invertebrates that are agricultural pests of invertebrates or parasites of invertebrate or vertebrate hosts. In the embodiments, the subjects are plants such as angiosperms (which may be dicotyledonous or monocotyledonous) or gymnosperms (e.g., conifers, cycads, Gnetophytes, ginkgo), ferns, horsetails, clubmosses, or mosses. In the embodiments, the subjects are eukaryotic algae (unicellular or multicellular). In the embodiments, the subjects are agricultural or horticulturally important plants such as furrow crops, fruit-bearing plants and trees, vegetables, trees, and ornamental plants, such as ornamental flowers, shrubs, trees, ground cover, and turf.

[0252] Plant-modified polypeptides In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of polyribonucleotides) include at least one expression (or coding) sequence encoding a plant-modified polypeptide. A plant-modified polypeptide refers to a polypeptide that can modify the genetic characteristics of a plant (e.g., by increasing or decreasing gene expression, or by otherwise modifying the nucleotide sequence of DNA or RNA), modify epigenetic characteristics, or modify 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 plant's fitness. In some embodiments, the 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 may modify the physiology or phenotype of various plants, increase the fitness of various plants, or result in such changes in one or more specific plants (e.g., a specific species or genus of plants).

[0253] Examples of polypeptides that can be used herein include enzymes (e.g., recombinant enzymes, helicases, integrases, ribonucleases, deoxyribonucleases, or ubiquitinated proteins), pore-forming proteins, signaling ligands, cell-permeable peptides, transcription factors, receptors, antibodies, nanobodies, gene-editing proteins (e.g., CRISPR-Cas endonucleases, TALENs, or Zn fingers), riboproteins, protein aptamers, or chaperones.

[0254] Agricultural polypeptides In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargo of polyribonucleotides) include at least one expression (or coding) sequence encoding an agricultural polypeptide. The 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 covering) or to the plant environment (e.g., by soil drenching or granular soil application) to result 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 commensal to the interior or surface of a plant or non-human animal host, the alteration resulting in an increase in the host's fitness. 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 in contact with non-human vertebrates, invertebrates, microorganisms, or plant cells.

[0255] 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.

[0256] Examples of polypeptide embodiments useful for agricultural applications include bacteriocins, lysants, antimicrobial peptides, nodule C-rich peptides, and microbial 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. Agriculturally useful polypeptide embodiments include peptide toxins, such as those naturally produced by entomopathogenic fungi (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, such as antimicrobial or antifungal polypeptides for controlling plant diseases, or pesticidal polypeptides (e.g., insecticidal polypeptides or nematode-killing polypeptides) for controlling invertebrate pests such as insects or nematodes. Embodiments of agriculturally useful polypeptides also include antibodies, nanobodies, and their fragments, such as 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 also include transcription factors, such as plant transcription factors; see, for example, the "AtTFDB" database, publicly available at agris-knowledgebase[dot]org / AtTFDB / , which lists families of transcription factors identified in the model plant Arabidopsis thaliana. Examples of agriculturally useful polypeptides include nucleases, such as exonucleases or endonucleases (e.g., Cas nucleases such as Cas9 or Cas12a).Examples of agriculturally useful polypeptides further include cell-permeable peptides, enzymes (e.g., amylase, cellulase, peptidase, lipase, chitinase), and peptide pheromones (e.g., yeast conjugation pheromones, invertebrate reproductive and larval signaling pheromones; see, e.g., Altstein (2004) Peptides, 25:1373-76).

[0257] Internal ribosome entry site In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of polyribonucleotides) include one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRESs are operably ligated to one or more expression (or coding) sequences (e.g., each IRES is operably ligated to one or more expression (or coding) sequences). In embodiments, the IRESs are located between a heterologous promoter and the 5' end of a coding sequence.

[0258] IRES elements suitable for inclusion within polyribonucleotides include RNA sequences capable of associating with eukaryotic ribosomes. In some embodiments, the IRES elements are 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.

[0259] In some embodiments, the IRES element is derived from the DNA of organisms including, but not limited to, viruses, mammals, and Drosophila. Such viral DNA can be derived from, but is not limited to, encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA, along with 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.

[0260] In some embodiments, if present, the IRES sequence may be associated with Taura syndrome virus, triatmavirus, Tyler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, wheat aphid virus, reticuloendotheliosis virus, human poliovirus 1, brown marmorated stink bug enterovirus, Cassirim wasp 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, cerebrospinal herbicide virus (EMCV), Drosophila C virus, and Crucifer tobamo) virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black queen bee larva virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, 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 PDG F2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Salivirus, Cosavirus, Parechovirus, Drosophila hairless, yeast (S. cerevisiae) TFIID, yeast (S.The IRES sequence is for cerevisiae)YAP1, human c-src, human FGF-1, salpicomavirus, turnip crinkle virus, an aptamer for eIF4G, or coxsackievirus B3 (CVB3) or coxsackievirus A (CVB1 / 2). In yet another embodiment, the IRES is the IRES sequence of coxsackievirus B3 (CVB3). In yet another embodiment, the IRES is the IRES sequence of encephalomyocarditis virus.

[0261] In some embodiments, the polyribonucleotide includes at least one IRES adjacent to at least one (e.g., two, three, four, five, or more) expression sequences. In some embodiments, the IRESs are adjacent to both sides of at least one (e.g., two, three, four, five, or more) expression sequences. In some embodiments, the polyribonucleotide includes one or more IRES sequences on one or both sides of each expression sequence, resulting in the separation of the resulting peptides and / or polypeptides.

[0262] In some embodiments, the polyribonucleotide cargo includes an IRES. For example, the polyribonucleotide cargo may include a circular RNA IRES, for example, described in Chen et al. MOL. CELL 81(20):4300-18, 2021, which is incorporated herein by reference in its entirety.

[0263] Adjustment element In some embodiments, the polyribonucleotide described herein (e.g., a polyribonucleotide cargo of polyribonucleotides) comprises one or more regulatory elements. In some embodiments, the polyribonucleotide comprises regulatory elements, such as sequences that regulate the expression of an expression sequence within the polyribonucleotide.

[0264] The regulatory element may include a sequence located adjacent to the expression sequence encoding the expression product. The regulatory element may be operably linked to the adjacent sequence. The regulatory element may increase the amount of the product expressed compared to the amount expressed in the absence of the regulatory element. Furthermore, one regulatory element may increase the amount or number of products expressed for multiple expression sequences linked side by side. Thus, one regulatory element can promote the expression of one or more expression sequences. Multiple regulatory elements are well known to those skilled in the art.

[0265] In some embodiments, the regulatory element is a translation modulator. A translation modulator can regulate the translation of an expression sequence in a polyribonucleotide. A translation modulator can be a translation enhancer or a translation suppressor. In some embodiments, the polyribonucleotide includes at least one translation modulator adjacent to at least one expression sequence. In some embodiments, the polyribonucleotide includes a translation modulator adjacent to each expression sequence. In some embodiments, the translation modulator is located on one or both sides of each expression sequence, resulting in the separation of the expression product, e.g., peptides and / or polypeptides.

[0266] In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.

[0267] Further examples of modulating elements are provided, for example, in paragraphs

[0154] to

[0161] of International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety.

[0268] Translation start sequence In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of polyribonucleotides) include at least one translation initiation sequence. In some embodiments, the polyribonucleotides include a translation initiation sequence operably ligated to an expression sequence.

[0269] In some embodiments, the polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgano sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence adjacent to the expression sequence, e.g., a Kozak sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, the translation initiation sequence, e.g., a Kozak sequence, is present on one or both sides of each expression sequence, resulting in the separation of the expression product. In some embodiments, the polyribonucleotide includes at least one translation initiation sequence adjacent to the expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is located within a single-stranded region of a cyclic polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs

[0163] to

[0165] of International Publication No. 2019 / 118919, which is incorporated herein by reference in whole.

[0270] A polyribonucleotide may contain two or more start codons, including, 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 downstream of the first start codon.

[0271] In some embodiments, polyribonucleotides may begin with a first start codon, e.g., a codon other than AUG. Translation of polyribonucleotides may begin with alternative translation initiation sequences, e.g., ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG, etc. In some embodiments, translation may begin with an alternative translation initiation sequence under selective conditions, e.g., stress-induced conditions. As an unrestricted example, translation of polyribonucleotides may begin with an alternative translation initiation sequence, e.g., ACG. As another unrestricted example, translation of polyribonucleotides may begin with the alternative translation initiation sequence CTG / CUG. As yet another unrestricted example, translation of polyribonucleotides may begin with the alternative translation initiation sequence GTG / GUG. As another non-limiting example, polyribonucleotides can initiate translation with repeat-associated non-AUG(RAN) sequences, such as short stretches of repetitive RNA, including alternative translation initiation sequences such as CGG, GGGGCC, CAG, and CTG.

[0272] terminating element In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of polyribonucleotides) include at least one termination element. In some embodiments, the polyribonucleotide includes a termination element operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination element.

[0273] In some embodiments, the polyribonucleotide comprises one or more expression sequences, each expression sequence may or may not have a termination element. In some embodiments, the polyribonucleotide comprises one or more expression sequences, and the absence of a termination element allows the polyribonucleotide to be continuously translated. The exclusion of the termination element may result in rolling circle translation or continuous expression of the expression product.

[0274] In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, each expression sequence may or may not have a termination element. In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, and the absence of a termination element allows the cyclic polyribonucleotide to be continuously translated. The absence of a termination element can result in rolling circle translation or continuous expression of an expression product, such as a peptide or polypeptide, due to the absence of ribosome stopping or shedding. In such embodiments, rolling circle translation expresses a continuous expression product through each expression sequence. In some other embodiments, the termination element of an expression sequence may be part of a stagger element. In some embodiments, one or more expression sequences in the cyclic polyribonucleotide contain a termination element. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the cyclic polyribonucleotide is performed. In such cases, the expression product may be shedding from the ribosome when the ribosome encounters a termination element, such as a stop codon, and terminates translation. In some embodiments, translation is terminated while a ribosome, for example, at least one subunit of a ribosome, maintains contact with the cyclic polyribonucleotide.

[0275] In some embodiments, the cyclic polyribonucleotide includes termination elements at the end of one or more expression sequences. In some embodiments, one or more expression sequences include two or more subsequent termination elements. In such embodiments, translation is terminated, and rolling circle translation is terminated. In some embodiments, ribosomes are completely separated from the cyclic polyribonucleotide. In some such embodiments, ribosomes may be required to reassociate with the cyclic polyribonucleotide before the initiation of translation in the generation of subsequent expression sequences (e.g., second, third, fourth, fifth, etc.) in the cyclic polyribonucleotide. The termination elements include an in-frame nucleotide triplet, e.g., UAA, UGA, UAG, which signals the termination of translation. In some embodiments, one or more termination elements in the cyclic polyribonucleotide are frameshifted termination elements, e.g., off-frame or -1 and +1 shifted leading frames (e.g., hidden stops), which can terminate translation. Frameshifted termination elements include triple nucleotides, TAA, TAG, and TGA appearing within the second and third reading frames of the expression sequence. Frameshifted termination elements may be important in preventing mRNA misreading, which is often harmful to cells. In some embodiments, the termination element is a stop codon.

[0276] Further examples of concluding elements are given in paragraphs

[0169] –

[0170] of International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety.

[0277] Non-translated areas In some embodiments, the cyclic polyribonucleotide includes an untranslated region (UTR). The UTR of the genomic region containing the gene is transcribed but not translated. In some embodiments, the UTR is located upstream of the translation initiation sequence of the expression sequence described herein. In some embodiments, the UTR is located downstream of the expression sequence described herein. In some cases, one UTR for the first expression sequence is the same as, contiguous with, or overlaps with another UTR for the second expression sequence. In some embodiments, the intron is a human intron.

[0278] Exemplary untranslated areas are described in paragraphs

[0197] to

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

[0279] In some embodiments, the cyclic polyribonucleotide includes a poly-A sequence. An exemplary poly-A sequence is described in paragraphs

[0202] to

[0205] of International Publication No. 2019 / 118919, which is incorporated herein by reference in whole. In some embodiments, the cyclic polyribonucleotide lacks a poly-A sequence.

[0280] In some embodiments, the cyclic polyribonucleotides include UTRs containing one or more stretches of adenosine and uridine. These AU-rich signatures may increase the turnover rate of the expression product.

[0281] The introduction, removal, or modification of AU-rich elements (AREs) in UTRs may be useful in regulating the stability or immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response) of cyclic polyribonucleotides. When modifying a particular cyclic polyribonucleotide, one or more copies of AREs may be introduced into the cyclic polyribonucleotide, and these copies of AREs may regulate the translation and / or production of the expression product. Similarly, by identifying and removing AREs, or by modifying cyclic polyribonucleotides, intracellular stability can be regulated, and consequently, the translation and production of the resulting protein can be affected.

[0282] Any UTR from any gene may be incorporated into each adjacent region of a circular polyribonucleotide.

[0283] In some embodiments, the cyclic polyribonucleotide lacks a 5'UTR and is capable of protein expression from one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a 3'UTR and is capable of protein expression from one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a poly(A) sequence and is capable of protein expression from one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a termination element and is capable of protein expression from one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site and is capable of protein expression from one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a cap and is capable of protein expression from one or more expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a 5'UTR, 3'UTR, and IRES and is capable of protein expression from one or more expression sequences. In some embodiments, the cyclic polyribonucleotide includes one or more 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.

[0284] In some embodiments, the cyclic polyribonucleotide lacks a 5'UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3'UTR. In some embodiments, the cyclic polyribonucleotide lacks a poly(A) sequence. In some embodiments, the cyclic polyribonucleotide lacks a termination element. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks sensitivity to degradation by exonucleases. In some embodiments, the fact that the cyclic polyribonucleotide lacks degradation sensitivity may mean that the cyclic polyribonucleotide is not degraded by exonucleases, or is degraded to a limited extent in the presence of exonucleases, for example, is equivalent or similar in the absence of exonucleases. In some embodiments, the cyclic polyribonucleotide is not degraded by exonucleases. In some embodiments, the degradation of the cyclic polyribonucleotide is reduced when exposed to exonucleases. In some embodiments, the cyclic polyribonucleotide lacks binding to cap-binding proteins. In some embodiments, the cyclic polyribonucleotide lacks a 5' cap.

[0285] Stagger element In some embodiments, the cyclic polyribonucleotide includes at least one stagger element adjacent to the expression sequence. In some embodiments, the cyclic polyribonucleotide includes a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is located on one or both sides of each expression sequence, resulting in the separation of the expression product, e.g., peptides and / or polypeptides. In some embodiments, the stagger element is a portion of one or more expression sequences. In some embodiments, the cyclic polyribonucleotide includes one or more expression sequences, each of which is separated from subsequent expression sequences by a stagger element in the cyclic polyribonucleotide. In some embodiments, the stagger element prevents the formation of a single polypeptide from (a) two translations of a single expression sequence, or (b) one or more translations of two or more expression sequences. In some embodiments, the stagger element is a sequence detached from one or more expression sequences. In some embodiments, the stagger element includes a portion of one or more expression sequences.

[0286] In some embodiments, the cyclic polyribonucleotide includes a stagger element. To maintain rolling circle translation while avoiding the formation of continuous expression products, such as peptides or polypeptides, the inclusion of a stagger element can induce ribosome pause during translation. In some embodiments, the stagger element is located at the 3' end of at least one expression sequence of one or more sequences. The stagger element can be designed to pause the ribosome during rolling circle translation of the cyclic polyribonucleotide. The stagger element may include, but is not limited to, a 2A-like or CHYSEL (SEQ ID NO: 126) (cis-acting hydrolase element) sequence. In some embodiments, the stagger element encodes a sequence having a C-terminal consensus sequence of 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: 127). In some embodiments, this sequence includes a non-conserved amino acid sequence having a strong α-helix tendency followed by a consensus sequence -D(V / I)EXNPGP (wherein x = any amino acid) (SEQ ID NO: 128). Some non-restrictive examples of stagger elements include GDVESNPGP (sequence number 129), GDIEENPGP (sequence number 130), VEPNPGP (sequence number 131), IETNPGP (sequence number 132), GDIESNPGP (sequence number 133), GDVELNPGP (sequence number 134), GDIETNPGP (sequence number 135), GDVENPGP (sequence number 136), GDVEENPGP (sequence number 137), GDVEQNPGP (sequence number 138), IESNPGP (sequence number 139), GDIELNPGP (sequence number 140), HDIETNGP (sequence number 141), HDVETNPGP (sequence number 142), HDVEMNPGP (sequence number 143), GDMESNPGP (sequence number 144), GDVETNPGP (sequence number 145), GDIEQNPGP (sequence number 146), and DSEFNPGP (sequence number 147).

[0287] In some embodiments, the stagger elements described herein cleave the expression product, for example, between G and P in the consensus sequence described herein. As one non-limiting example, a cyclic polyribonucleotide includes at least one stagger element for cleaving the expression product. In some embodiments, a cyclic polyribonucleotide includes a stagger element adjacent to at least one expression sequence. In some embodiments, a cyclic polyribonucleotide includes a stagger element following each expression sequence. In some embodiments, a cyclic polyribonucleotide includes stagger elements on one or both sides of each expression sequence, resulting in the translation of individual peptides and / or polypeptides from each expression sequence.

[0288] In some embodiments, the stagger element comprises one or more modified or non-native nucleotides that induce ribosome pause during translation. Non-native nucleotides may include peptide nucleic acids (PNA), morpholino and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). These examples are distinguished from naturally occurring DNA or RNA by modifications to the molecular backbone. Modifications may include modifications to sugars, nucleic acid bases, intemucleoside bonds (e.g., to linked phosphate / phosphate diester bonds / phosphate diester backbones), and any combination thereof that can induce ribosome pause during translation. Some of the exemplary modifications provided herein are described elsewhere in this specification.

[0289] In some embodiments, stagger elements exist in other forms in cyclic polyribonucleotides. For example, in some exemplary cyclic polyribonucleotides, the stagger element includes a termination element of a first expression sequence in the cyclic polyribonucleotide and a nucleotide spacer sequence separating the termination element from a first translation initiation sequence of subsequent expression. In some examples, the first stagger element of the first expression sequence is located upstream (5' side) of the first translation initiation sequence of subsequent expression in the cyclic polyribonucleotide. In some cases, the first expression sequence and the subsequent expression sequence are two separated expression sequences in the cyclic polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence may allow for sequential translation of the first expression sequence and the subsequent expression sequence.

[0290] In some embodiments, the first stagger element includes a termination element that separates the expression product of the first expression sequence from the expression product of the subsequent expression sequence, thereby creating separate expression products. In some cases, a cyclic polyribonucleotide containing the first stagger element located upstream of the first translation initiation sequence of the subsequent sequence is translated sequentially, while a corresponding cyclic polyribonucleotide containing the stagger element of the second expression sequence located upstream of the second translation initiation sequence of the expression sequence following the second expression sequence is not translated sequentially. In some cases, there is only one expression sequence in the cyclic polyribonucleotide, and the first expression sequence and the subsequent expression sequence are the same expression sequence. In some exemplary cyclic polyribonucleotides, the stagger element includes a first termination element of the first expression sequence in the cyclic polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from the downstream translation initiation sequence. In some such examples, the first stagger element is located upstream (5' side) of the first translation initiation sequence of the first expression sequence in the cyclic polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence allows for the sequential translation of the first expression sequence and any subsequent expression sequences.

[0291] In some embodiments, the first stagger element separates the expression product of the first expression sequence from the expression product of the next expression sequence, thereby producing separate expression products. In some cases, a cyclic polyribonucleotide containing the first stagger element upstream of the first translation initiation sequence of the first expression sequence in the cyclic polyribonucleotide is translated sequentially, while a corresponding cyclic polyribonucleotide containing the stagger element upstream of the second translation initiation sequence of the second expression sequence in the corresponding cyclic polyribonucleotide is not translated sequentially. In some cases, the distance between the second stagger element and the second translation initiation sequence is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater in the corresponding cyclic polyribonucleotide than the distance between the first stagger element and the first translation initiation in the cyclic polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or greater. In some embodiments, the distance between the second stagger element and the second translation start is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or greater than the distance between the first stagger element and the first translation start. In some embodiments, the cyclic polyribonucleotide comprises two or more expression sequences.

[0292] Examples of stagger elements are given in paragraphs

[0172] to

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

[0293] Non-code array In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of polyribonucleotides) include one or more non-coding sequences, e.g., sequences that do not encode polypeptide expression. In some embodiments, the polyribonucleotides include two, three, four, five, six, seven, eight, nine, ten, or more than ten non-coding sequences. In some embodiments, the polyribonucleotides do not encode polypeptide expression sequences.

[0294] Non-coding sequences can be native or synthetic sequences. In some embodiments, non-coding sequences can modify cellular behavior, such as lymphocyte behavior. In some embodiments, non-coding sequences are antisense to cellular RNA sequences.

[0295] In some embodiments, the polyribonucleotide typically comprises a regulatory nucleic acid that is an RNA or RNA-like structure of about 5 to 500 base pairs, depending on the specific RNA structure (e.g., a 5-30 bps miRNA, a 200-500 bps lncRNA), and may have a nucleic acid base sequence that is identical (complementary) or nearly identical (substantially complementary) to the coding sequence in the target gene expressed in the cell. In embodiments, the cyclic polyribonucleotide comprises a regulatory nucleic acid that encodes an RNA precursor, which may be about 50 to about 1000 bp, that is processable into a smaller miRNA intermediate or mature miRNA, such as a miRNA precursor.

[0296] Long non-coding RNAs (IncRNAs) are defined as non-protein-coding transcripts longer than 100 nucleotides. Many IncRNAs are characterized by tissue specificity. A significant proportion (e.g., about 20% of all IncRNAs in the mammalian genome) of IncRNAs transcribed in reverse to neighboring protein-coding genes may regulate the transcription of neighboring genes. In one embodiment, the polyribonucleotides provided herein comprise the sense strand of an IncRNA. In one embodiment, the polyribonucleotides provided herein comprise the antisense strand of an IncRNA.

[0297] In embodiments, the polyribonucleotide encodes a regulatory nucleic acid that is substantially or completely complementary to all or at least one fragment of an endogenous gene or gene product (e.g., mRNA). In embodiments, the regulatory nucleic acid inhibits the maturation of a newly generated nuclear RNA transcript of a specific gene into mRNA for transcription by complementing the sequence at the boundary between introns and exons, between exons, or adjacent to exons. A regulatory nucleic acid complementary to a specific gene may hybridize with the mRNA for that gene and inhibit its translation. The antisense regulatory nucleic acid may be DNA, RNA, or a derivative or hybrid thereof. In some embodiments, the regulatory nucleic acid includes a protein-binding site that can bind to a protein involved in regulating the expression of an endogenous or exogenous gene.

[0298] In embodiments, the polyribonucleotide encodes a regulatory RNA that hybridizes to the target transcript, the regulatory RNA having a length of approximately 5–30 nucleotides, approximately 10–30 nucleotides, or approximately 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides. In embodiments, the degree of sequence identity of the regulatory RNA with the targeted transcript is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0299] In some embodiments, the polyribonucleotide encodes a microRNA (miRNA) molecule that matches approximately 5 to 25 adjacent nucleotides of the target gene, or encodes a precursor to that miRNA. In some embodiments, the miRNA has a sequence that enables the mRNA to recognize and bind to a specific target mRNA. In some embodiments, the miRNA sequence begins with dinucleotide AA and contains approximately 30-70% (approximately 30-60%, 40-60%, or 45-55%) GC content and does not have a high percentage of identity with any non-target nucleotide sequence in the genome of the subject (e.g., mammal) into which it is to be introduced (as determined by, for example, a standard BLAST search).

[0300] In some embodiments, the polyribonucleotide comprises at least one miRNA (or miRNA precursor), for example, 2, 3, 4, 5, 6, or more miRNAs or miRNA precursors. In some embodiments, the polyribonucleotide comprises a sequence encoding a miRNA (or its precursor) having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100% nucleotide sequence complementarity to the target sequence.

[0301] siRNA and shRNA are analogous to intermediates in the processing pathway of endogenous microRNA (miRNA) genes. In some embodiments, siRNA can function as a miRNA and vice versa. Like microRNA and siRNA, they downregulate target genes using RISC, but unlike siRNA, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein output through translational repression or poly(A) excision and mRNA degradation. Known miRNA binding sites are located within the mRNA 3'UTR; miRNAs target a region that has nearly complete complementarity to nucleotides 2-8 from the 5' end of the miRNA. This region is known as the seed region. Because mature siRNA and miRNA are interchangeable, exogenous siRNA downregulates mRNA that has seed complementarity to siRNA. Lists of known miRNA sequences can be found in databases maintained by research organizations such as the Wellcome Trust Sanger Institute, the Penn Center for Bioinformatics, the Memorial Sloan Kettering Cancer Center, and the European Molecular Biology Laboratory. Known effective siRNA sequences and homologous binding sites are also well described in the relevant literature. RNAi molecules are readily designed and fabricated using techniques known in the art. In addition, computational tools exist that increase the likelihood of finding effective and specific sequence motifs.

[0302] Protein binding sequence In some embodiments, cyclic polyribonucleotides include one or more protein-binding sites that allow proteins, such as ribosomes, to bind to internal sites within an RNA sequence. By designing protein-binding sites, such as ribosome-binding sites, within the cyclic polyribonucleotide, the cyclic polyribonucleotide may evade or reduce detection by the host immune system, modulate degradation, or modulate translation by masking the cyclic polyribonucleotide from components of the host immune system.

[0303] In some embodiments, the cyclic polyribonucleotide evades, for example, an immune response, such as a CTL (cytotoxic T lymphocyte) response, by including at least one immunoprotein binding site. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and assists in masking the cyclic polyribonucleotide as exogenous. In some embodiments, the immunoprotein binding site is a nucleotide sequence that binds to an immunoprotein and assists in concealing the cyclic polyribonucleotide as exogenous or foreign.

[0304] The traditional mechanism of ribosome association to linear RNA involves ribosome binding to the capped 5' end of the RNA. As soon as the ribosome moves from the 5' end to the start codon, the first peptide bond is formed. According to this disclosure, the internal initiation (i.e., cap-independent) of translation of cyclic polyribonucleotides does not require a free or capped end. Rather, the ribosome binds to an uncapped internal site, thereby causing the ribosome to initiate polypeptide elongation at the start codon. In some embodiments, the cyclic polyribonucleotide comprises one or more RNA sequences containing a ribosome binding site, e.g., a start codon.

[0305] The natural 5'UTR possesses features that play a role in translation initiation. It contains a signature similar to the Kozak sequence, which is commonly known to be involved in the process by which ribosomes initiate translation of numerous genes. The Kozak sequence has consensus CCR(A / G)CCAUGG(SEQ ID NO: 125) (where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG)) followed by another "G". The 5'UTR is also known to form secondary structures involved in elongation factor binding.

[0306] 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 the protein.

[0307] In some embodiments, the protein binding site may include, but is not limited to, ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, and 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 Examples of protein binding sites include 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 proteins that bind to RNA.

[0308] Spacer array In some embodiments, the polyribonucleotides described herein include one or more spacer sequences. A spacer refers to any adjacent nucleotide sequence (e.g., one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacers may be present between any of the nucleic acid factors described herein. Spacers may also be present within the nucleic acid factors described herein.

[0309] 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 and second spacer regions may contain a poly-A sequence. The first spacer region, the second spacer region, or the first and second spacer regions may contain a poly-AC sequence. In some embodiments, the first spacer region, the second spacer region, or the first and second spacer regions include a poly-AG array. In some embodiments, the first spacer region, the second spacer region, or the first and second spacer regions include a poly-AT array. In some embodiments, the first spacer region, the second spacer region, or the first and second spacer regions include a random array.

[0310] Spacers may also be present within the nucleic acid regions described herein. For example, a polynucleotide cargo region may contain one or more spacers. Spacers can separate regions within the polynucleotide cargo.

[0311] In some embodiments, the spacer sequence may be, for example, at least 10 nucleotides long, at least 15 nucleotides long, or at least 30 nucleotides long. 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 long. In some embodiments, the spacer sequence is 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides long or less. In some embodiments, the spacer sequence is 20 to 50 nucleotides long. 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.

[0312] The spacer array may be a poly-A array, a poly-AC array, a poly-C array, or a poly-U array.

[0313] In some embodiments, the spacer array may be polyAT, polyAC, polyAG, or a random array.

[0314] By using spacer sequences to separate IRESs from adjacent components, the structure and function of IRESs or adjacent elements can be maintained. Spacers can be specifically modified depending on the IRES. In some embodiments, computer software for RNA folding, such as RNAFold, can be used to guide the design of various elements of the vector, including the spacers.

[0315] In some embodiments, the polyribonucleotide includes a 5' spacer sequence. In some embodiments, the 5' spacer sequence is at least 10 nucleotides long. In another embodiment, the 5' spacer sequence is at least 15 nucleotides long. In yet another embodiment, the 5' spacer sequence is at least 30 nucleotides long. 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 long. In some embodiments, the 5' spacer sequence is 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides long or less. In some embodiments, the 5' spacer sequence is 20 to 50 nucleotides long. 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 long. In one embodiment, the 5' spacer sequence is a poly-A sequence. In another embodiment, the 5' spacer sequence is a poly-AC sequence. In some embodiments, the 5' spacer sequence includes a poly-AG sequence. In some embodiments, the 5' spacer sequence includes a poly-AT sequence. In some embodiments, the 5' spacer sequence includes a random sequence.

[0316] In some embodiments, the polyribonucleotide includes a 3' spacer sequence. In some embodiments, the 3' spacer sequence is at least 10 nucleotides long. In another embodiment, the 3' spacer sequence is at least 15 nucleotides long. In yet another embodiment, the 3' spacer sequence is at least 30 nucleotides long. 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 long. In some embodiments, the 3' spacer sequence is 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides or less long. In some embodiments, the 3' spacer sequence is 20 to 50 nucleotides long. 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 long. In one embodiment, the 3' spacer sequence is a poly-A sequence. In another embodiment, the 5' spacer sequence is a poly-AC sequence. In some embodiments, the 5' spacer sequence includes a poly-AG sequence. In some embodiments, the 5' spacer sequence includes a poly-AT sequence. In some embodiments, the 5' spacer sequence includes a random sequence.

[0317] In one embodiment, the polyribonucleotide includes a 5' spacer sequence but does not include a 3' spacer sequence. In another embodiment, the polyribonucleotide includes a 3' spacer sequence but does not include a 5' spacer sequence. In yet another embodiment, the polyribonucleotide does not include either a 5' or 3' spacer sequence. In yet another embodiment, the polyribonucleotide does not include an IRES sequence. In a further embodiment, the polyribonucleotide does not include an IRES sequence, a 5' spacer sequence, or a 3' spacer sequence.

[0318] In some cases, the spacer sequence consists of at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, and at least about 80 ribonucleotides. The creotide contains 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 1,000 ribonucleotides.

[0319] bioreactor In some embodiments, any method for purifying polyribonucleotides (e.g., cyclic polyribonucleotides) as described herein may be carried out in a bioreactor. A bioreactor refers to any container in which a chemical or biological process involving a biochemically active substance of an organism or a biochemically derived organism is carried out. The bioreactor may be adapted to a cell-free method for producing or purifying cyclic RNA as described herein. Containers for the bioreactor may include culture flasks, dishes, or bags that are disposable, autoclavable, or sterilizable. The bioreactor may be made of glass, polymer-based, or manufactured from other materials.

[0320] Examples of bioreactors include, but are not limited to, agitated tank (e.g., well-mixed) bioreactors and plate (e.g., plug-flow) bioreactors, air-transport bioreactors, membrane agitated tanks, spin-filter agitated tanks, vibrating mixers, fluidized bed reactors, and membrane bioreactors. The mode of operation for a bioreactor may be batch or continuous process. A bioreactor is continuous if a flow of reagents and products is continuously supplied and removed from the system. A batch bioreactor may have a continuous recirculation flow but may not have a continuous supply of reagents or product recovery.

[0321] Some of the methods described herein are intended for the large-scale production of polyribonucleotides. In the case of large-scale production methods, the methods can be carried out in volumes of 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 carried out with volumes of 5L-10L, 5L-15L, 5L-20L, 5L-25L, 5L-30L, 5L-35L, 5L-40L, 5L-45L, 5L-50L, 10L-15L, 10L-20L, 10L-25L, 20L-30L, 10L-35L, 10L-40L, 10L-45L, 10L-50L, 15L-20L, 15L-25L, 15L-30L, 15L-35L, 15L-40L, 15L-45L, or 15L-50L.

[0322] In some embodiments, the bioreactor may produce at least 1 g of RNA. In some embodiments, the bioreactor may produce 1 to 200 g of RNA (e.g., 1 to 10 g, 1 to 20 g, 1 to 50 g, 10 to 50 g, 10 to 100 g, 50 to 100 g, 50 to 200 g of RNA). In some embodiments, the amount produced is measured per liter (e.g., 1 to 200 g per liter), per batch or reaction (e.g., 1 to 200 g per batch or reaction), or per unit time (e.g., 1 to 200 g per hour or per day).

[0323] In some embodiments, two or more bioreactors can be used in succession to enhance production capacity (for example, one, two, three, four, five, six, seven, eight, or nine bioreactors can be used in succession).

[0324] How to use In some embodiments, polyribonucleotides (e.g., cyclic polyribonucleotides) prepared as described herein are used as effectors in therapy or agriculture.

[0325] For example, polyribonucleotides purified by the methods described herein may be administered to a subject (e.g., in pharmaceutical, animal, or agricultural compositions). In some embodiments, the subject is a vertebrate (e.g., mammal, bird, fish, reptile, or 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., monkey, ape), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horse, donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or rabbit (e.g., rabbit). In the embodiments, the subjects are birds such as members of avian taxa such as Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In the embodiments, the subjects are invertebrates such as arthropods (e.g., insects, spiders, crustaceans), nematodes, annelids, helminths, or mollusks. In the embodiments, the subjects are invertebrates that are agricultural pests of invertebrates or parasites of invertebrate or vertebrate hosts. In the embodiments, the subjects are plants such as angiosperms (which may be dicotyledonous or monocotyledonous) or gymnosperms (e.g., conifers, cycads, Gnetophytes, ginkgo), ferns, horsetails, clubmosses, or mosses. In the embodiments, the subjects are eukaryotic algae (unicellular or multicellular). In the embodiments, the subjects are agricultural or horticulturally important plants such as furrow crops, fruit-bearing plants and trees, vegetables, trees, and ornamental plants, such as ornamental flowers, shrubs, trees, ground cover, and turf.

[0326] In some embodiments, this disclosure provides methods for modifying a subject by providing 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 RNA molecule as described herein), and polynucleotides are provided for a eukaryotic subject. In some embodiments, the composition or preparation is or comprises a eukaryotic or prokaryotic cell containing a nucleic acid as described herein.

[0327] In some embodiments, this disclosure provides methods for treating symptoms in subjects requiring the use of compositions or preparations described herein. In some embodiments, the compositions or preparations are or comprise nucleic acid molecules (e.g., DNA molecules or polyribonucleotides as described herein), and the polynucleotides are provided for eukaryotic subjects. In some embodiments, the compositions or preparations are or comprise eukaryotic or prokaryotic cells containing nucleic acids as described herein.

[0328] In some embodiments, the Disclosure provides a method for providing polyribonucleotides (e.g., cyclic polyribonucleotides) by providing them to eukaryotic or prokaryotic cells containing the polynucleotides described herein.

[0329] preparation In some embodiments of this disclosure, the polyribonucleotides described herein (e.g., cyclic polyribonucleotides) can be formulated in compositions, for example, compositions for delivery to cells, plants, invertebrates, non-human vertebrates, or human subjects, for example, agricultural, animal, or pharmaceutical compositions. In some embodiments, the polyribonucleotides are formulated in pharmaceutical compositions. In some embodiments, the composition comprises the polyribonucleotides and a diluent, carrier, adjuvant, or a combination thereof. In certain embodiments, the composition comprises the polyribonucleotides described herein and a carrier or a diluent without a carrier. In some embodiments, a composition comprising the polyribonucleotides and a diluent without a carrier is used for naked delivery of the polyribonucleotides (e.g., cyclic polyribonucleotides) to a subject.

[0330] The pharmaceutical composition may optionally contain one or more additional active substances, such as therapeutic and / or prophylactic active substances. The pharmaceutical composition may optionally contain an inert substance that serves as a medium or culture medium for the compositions described herein (e.g., cyclic polyribonucleotides, for example, compositions containing any one of the inert substances approved by the U.S. Food and Drug Administration (FDA) and listed in the Inert Ingredients Database). The pharmaceutical compositions 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, dispersions, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulators, disintegrants, binders, buffers (e.g., phosphate-buffered saline (PBS)), smoothing agents, oils, and mixtures thereof.

[0331] The descriptions of pharmaceutical compositions provided herein primarily concern pharmaceutical compositions suitable for administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animals, such as non-human animals, such as non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or carry out such modifications simply by performing ordinary (if possible) experiments. Targets to which the administration of pharmaceutical compositions is intended include, but are not limited to, humans and / or other primates; mammals, including commercially important mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and birds, including commercially important birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0332] Preparations of the pharmaceutical compositions described herein may be prepared by any known or subsequently developed method in the field of pharmacology. Generally, such preliminary methods include associating the active ingredient with excipients and / or one or more other accessory components, and then, if necessary and / or desirable, dividing, forming and / or packaging the product.

[0333] In some embodiments, the reference standards for the amount of linear polyribonucleotide molecules present in the preparation are 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, and 500 ng / mL. The concentrations are 600 ng / mL, 650 mg / mL, 700 mg / mL, 700 ng / mL, 750 mg / mL, 800 ng / mL, 850 ng / mL, 900 ng / mL, 950 ng / mL, 1 μg / mL, 10 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, or 2 mg / mL or less.

[0334] In some embodiments, the reference standard for the amount of cyclic polyribonucleotide molecules present in the 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), of the total ribonucleotide molecules in the pharmaceutical preparation. The numerators are 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).

[0335] In some embodiments, the reference standard for the amount of 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), and 50%(w / w) or less of linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical preparation.

[0336] In some embodiments, the reference standard for the amount of nic polyribonucleotide molecules present in the preparation is 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), or 15% (w / w) or less of the total ribonucleotide molecules in the pharmaceutical preparation.

[0337] In some embodiments, the reference standard for the amount of nic and linear polyribonucleotide molecule combinations present in the preparation is a combination of nic and linear polyribonucleotide molecules that constitutes 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), or 50% (w / w) of the total ribonucleotide molecules in the pharmaceutical preparation. In some embodiments, the pharmaceutical preparation is an intermediate pharmaceutical preparation of the final cyclic polyribonucleotide formulation. In some embodiments, the pharmaceutical preparation is a raw material or active pharmaceutical ingredient (API). In some embodiments, the pharmaceutical preparation is a formulation for administration to a subject.

[0338] In some embodiments, the cyclic polyribonucleotide preparation is further processed (before, during, or after linear RNA reduction) to remove DNA, protein contaminants (e.g., cellular proteins such as host cell proteins or process impurity proteins), endotoxins, mononucleotide molecules, and / or process-related impurities.

[0339] salt In some cases, the compositions or pharmaceutical compositions provided herein contain one or more salts. Physiological salts, such as sodium salts, may be included in the compositions provided herein to control osmotic pressure. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride. In some cases, the compositions are formulated with one or more pharmaceutically acceptable salts. One or more pharmaceutically acceptable salts may include, for example, those of inorganic ions such as sodium, potassium, calcium, and magnesium ions. Such salts may 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 may exist in either linear or cyclic forms.

[0340] Buffering agent / pH The compositions or pharmaceutical compositions provided herein may contain one or more buffers, such as Tris buffers; borate buffers; succinate buffers; histidine buffers (e.g., having an aluminum hydroxide adjuvant); or citrate buffers. In some cases, the buffer is contained in a range of 5 to 20 mM.

[0341] The compositions or pharmaceutical compositions provided herein may 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 may have a pH of about 7. Polyribonucleotides may exist in either a linear or cyclic form.

[0342] Cleaning agents / surfactants The compositions or pharmaceutical compositions provided herein, depending on the intended route of administration, include one or more detergents and / or surfactants, for example, polyoxymethylene sorbitan surfactants (commonly referred to as "Tween®"), for example, polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) sold under the trade name DOWFAX®, for example, linear EO / PO block copolymers; octoxynols with varying numbers of repeating ethoxy(oxy-1,2-ethanediyl) groups, for example, octoxynol-9 (Triton® X-100, or t-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, e.g., phosphatidylcholine (lecithin); nonylphenol ethoxylates, e.g., Tergitol® NP series; polyoxyethylene aliphatic ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (known as Brij® surfactants), e.g., triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as "SPAN"), e.g., sorbitan trioleate (Span 85) and sorbitan monolaurate, octoxynol (such as octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide ("CTAB"), or sodium deoxycholate may be included. 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. Nonionic surfactants may be used in this specification. Surfactants can be classified by their "HLB" (hydrophilic / lipophilic balance). In some examples, surfactants have an HLB of at least 10, at least 15, and / or at least 16. Polyribonucleotides may be included in linear or cyclic forms.

[0343] Diluent In some embodiments, the compositions of the Disclosure comprise a polyribonucleotide and a diluent. In some embodiments, the compositions of the Disclosure comprise a linear polyribonucleotide and a diluent.

[0344] Diluents may be non-carrier excipients. Non-carrier excipients act as vehicles or media for the composition, such as cyclic polyribonucleotides described herein. Non-carrier excipients act as vehicles or media for the composition, such as linear polyribonucleotides described herein. Non-limiting examples of non-carrier excipients include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidases, dispersants, granulators, disintegrants, binders, buffers (e.g., phosphate-buffered saline (PBS)), lubricants, oils, and mixtures thereof. Non-carrier excipients may be any of the non-active ingredients listed in the Inactive Ingredient Database approved by the United States Food and Drug Administration (FDA) that do not exhibit cell permeability. Non-carrier excipients can be, for example, any inactive component suitable for administration to non-human animals suitable for veterinary use. Modifications of compositions suitable for human administration to make them suitable for administration to various animals are well understood, and a veterinary pharmacologist with ordinary skills can design and / or make such modifications, if any, with only ordinary experiments.

[0345] In one embodiment, polyribonucleotides (e.g., cyclic polyribonucleotides) are delivered as a naked delivery formulation, such as one containing a diluent. The naked delivery formulation delivers polyribonucleotides to cells without a carrier, and without modification or partial or complete encapsulation, capped polyribonucleotides, or complexes thereof.

[0346] Naked delivery formulations are carrier-free formulations in which the polyribonucleotide (e.g., cyclic polyribonucleotide) does not have a covalent modification that binds to a portion that assists delivery to cells, or is not partially or completely encapsulated. In some embodiments, a polyribonucleotide that does not have a covalent modification that binds to a portion that assists delivery to cells is a polyribonucleotide that is not covalently bound to a protein, small molecule, particle, polymer, or biopolymer. A polyribonucleotide that does not have a covalent modification that binds to a portion that assists delivery to cells does not contain a modified phosphate group. For example, a polyribonucleotide that does not have a co-covalent modification that binds to a portion that assists delivery to cells does not contain phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotryesters.

[0347] In one embodiment, the naked delivery formulation does not contain any or all of the following: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. In one embodiment, the naked delivery formulation includes phytoglycogen octenyl succinate, phytoglycogen β-dextrin, anhydrous-modified phytoglycogen β-dextrin, lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-β-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimer, 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 It does not contain -(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), 3B-[N-(N,N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterolHCl), diheptadecylamide glycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxypropa-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.

[0348] In some embodiments, the naked delivery formulation includes a non-carrier excipient. In some embodiments, the non-carrier excipient includes an inert component that does not exhibit cell permeability. In some embodiments, the non-carrier excipient includes a buffer, such as PBS. In some embodiments, the non-carrier excipient is a solvent, a non-aqueous solvent, a diluent, a suspension aid, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersant, a granulator, a disintegrant, a binder, a buffer, a lubricant, or an oil.

[0349] In some embodiments, the naked delivery formulation includes a diluent. The diluent may be a liquid or solid diluent. In some embodiments, the diluent is an RNA solubilizer, a buffer, or an isotonic agent. Examples of RNA solubilizers 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-acetamide)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-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 isotonic agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.

[0350] Carrier In some embodiments, the composition of the Disclosure comprises a cyclic polyribonucleotide and a carrier. In some embodiments, the composition of the Disclosure comprises a linear polyribonucleotide and a carrier.

[0351] In certain embodiments, the composition comprises a cyclic polyribonucleotide described herein in a vesicle or other membrane-based carrier. In certain embodiments, the composition comprises a linear polyribonucleotide described herein in a vesicle or other membrane-based carrier.

[0352] In other embodiments, the composition comprises cyclic polyribonucleotides in or via cells, vesicles, or other membrane-based carriers. In other embodiments, the composition comprises linear polyribonucleotides in or via cells, vesicles, or other membrane-based carriers. In one embodiment, the composition comprises cyclic polyribonucleotides in liposomes or other similar vesicles. In one embodiment, the composition comprises linear polyribonucleotides in liposomes or other similar vesicles. Liposomes are spherical vesicular structures consisting of a monolayer or multilayer 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, capable of delivering both hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes, and transporting their load across biological membranes and the blood-brain barrier (BBB) ​​(see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679).

[0353] Vesicles can be prepared from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilayer vesicle lipids are known in the art (see, for example, U.S. Patent No. 6,693,086, the teaching relating to the preparation of multilayer vesicle lipids is incorporated herein by reference). Vesicle formation can occur spontaneously when lipid membranes are mixed with aqueous solutions, but it can also be facilitated by applying force in the form of shaking using homogenizers, sonicators, or extruders (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for an overview). The extruded lipids can be prepared by extruding them through a reduced-size filter, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings relating to the preparation of the extruded lipids are incorporated herein by reference.

[0354] In certain embodiments, the compositions of the Disclosure include polyribonucleotides and lipid nanoparticles as described herein, for example, lipid nanoparticles. In certain embodiments, the compositions of the 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 important components 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), which are a novel type of carrier combining liposomes and polymers, may also be used. These nanoparticles have complementary advantages to PNPs and liposomes. PLNs consist of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Therefore, the two components increase drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs. For an overview, see, for example, Li et al. 2017, Nanomaterials 7, 122; doi:10.3390 / nano7060122.

[0355] Further non-limiting examples of carriers include carbohydrate carriers (e.g., anhydrous-modified phytoglycogen or glycogen-type materials), protein carriers (e.g., proteins covalently bonded to polyribonucleotides or proteins covalently bonded 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 anhydrous-modified phytoglycogen β-dextrin. Non-limiting examples of cationic carriers include lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-β-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-Hyd Examples include roxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propaneaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterolHC1), diheptadecylamide glycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristiloxypropa-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). Non-specific examples of protein carriers include human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin.

[0356] 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.

[0357] Ex vivo differentiated erythrocytes may also be used as carriers for the compositions or preparations described herein. Ex vivo differentiated erythrocytes may also be used as carriers for the linear polyribonucleotide compositions or preparations described herein. For example, International Publication No. 2015 / 073587; International Publication No. 2017 / 123646; International Publication No. 2017 / 123644; International Publication No. 2018 / 102740; International Publication No. 2016 / 183482; International Publication No. 2015 / 153102; International Publication No. 2018 / 151829; International Publication No. 2018 / 009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28):10131-10136; U.S. Patent No. 9,644,180; Huang et al. 2017. Nature Communications 8:423; Shi et al. 2014. Proc Natl Acad Sci See USA.111(28):10131-10136.

[0358] For example, fusosome compositions, such as those described in International Publication No. 2018 / 208728, can also be used as carriers for delivering the polyribonucleotide molecules described herein.

[0359] Virosomes and virus-like particles (VLPs) can also be used as carriers for delivering the polyribonucleotide molecules described herein to target cells.

[0360] For example, plant nanovesicles and plant messenger packs (PMPs), such as those described in International Patent Publication No. 2011 / 097480, International Publication No. 2013 / 070324, International Publication No. 2017 / 004526, or International Publication No. 2020041784, can also be used as carriers for delivering the compositions or preparations described herein. Plant nanovesicles and plant messenger packs (PMPs) can also be used as carriers for delivering the linear polyribonucleotide compositions or preparations described herein.

[0361] 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. Patent 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, J. et al., Adv Drug Deliv Rev. 2014 Jun; 72: 82-93. In one embodiment, the microbubbles are albumin-coated perfluorocarbon microbubbles.

[0362] Silk fibroin can also be used as a carrier for delivering the compositions and formulations described herein. See, for example, Boopathy, A. et al., PNAS. 116.33 (2019): 16473-1678; and He, H. ct al., ACS BIOMATER. SCI. ENG. 4.5 (2018): 1708-1715.

[0363] The polyribonucleotide-containing carriers described herein may comprise a plurality of particles. The particles may have a median article size of 30 to 700 nanometers (e.g., 30 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 100 to 500, 50 to 500, or 200 to 700 nanometers). The particle size can be optimized to favor the deposition of the polyribonucleotide-containing payload into cells. Different particle sizes may be favorable for the deposition of polyribonucleotides into specific cell types. For example, a particle size may be optimized for the deposition of polyribonucleotides into antigen-presenting cells. A particle size may be optimized for the deposition of polyribonucleotides into dendritic cells. Furthermore, a particle size may be optimized for the deposition of polyribonucleotides into influx region lymph node cells.

[0364] Lipid nanoparticles In some embodiments, the compositions of the present disclosure include cyclic polyribonucleotides and lipid nanoparticles (LNPs). In some embodiments, the lipid nanoparticles include one or more ionic lipids, such as noncationic lipids (e.g., neutral, anionic, or amphoteric lipids); one or more conjugate lipids (such as PEG conjugate lipids or lipids conjugated to polymers as described in Table 5 of International Publication No. 2019217941, which is incorporated herein by reference); and one or more sterols (e.g., cholesterol).

[0365] Lipids that can be used in nanoparticle formation (e.g., lipid nanoparticles) include, for example, those listed in Table 4 of International Publication No. 2019217941, which is incorporated by reference—for example, lipid-containing nanoparticles may include one or more lipids from Table 4 of International Publication No. 2019217941. Lipid nanoparticles may also include further elements, such as polymers, for example, polymers listed in Table 5 of International Publication No. 2019217941, which is incorporated by reference.

[0366] In one embodiment, the conjugated lipid, if present, is PEG-diacylglycerol (DAG) (e.g., l-(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(tetradecane)). It may include one or more of the following: noyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), PEG dialkoxypropyl carbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those listed in Table 2 of International Publication No. 2019051289 (as referenced by reference), as well as combinations thereof.

[0367] In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more cholesterol or cholesterol derivatives, such as those described in International Publication No. 2009 / 127060 or U.S. Patent Application Publication No. 2010 / 0130588, as incorporated herein by reference. Further exemplary sterols include plant sterols, such as those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, as incorporated herein by reference.

[0368] In one embodiment, the lipid particles include ionizable lipids, noncationic lipids, conjugate lipids that inhibit particle aggregation, and sterols. The amounts of these components can be independently varied to achieve the desired properties. For example, in one embodiment, the lipid nanoparticles include about 20 mol% to about 90 mol% of the total lipids in the ionizable lipids (in other embodiments, this may be 20-70% (mol), 30-60% (mol), or 40-50% (mol); about 50 mol% to about 90 mol%), about 5 mol% to about 30 mol% of the total lipids in the lipid nanoparticles in the noncationic lipids, about 0.5 mol% to about 20 mol% of the total lipids in the conjugate lipids, and about 20 mol% to about 50 mol% of the total lipids in the sterols. The ratio of total lipids to nucleic acids can be varied as needed. For example, the ratio of total lipids to nucleic acids (mass or weight) may be about 10:1 to about 30:1.

[0369] In some embodiments, the lipid-to-nucleic acid ratio (mass / mass ratio; w / w ratio) may 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 lipids and nucleic acids may 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 higher. Generally, the total lipid content of a lipid nanoparticle formulation may range from about 5 mg / ml to about 30 mg / mL.

[0370] Some non-limiting examples of lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of the compositions described herein, for example, nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) include the following: [ka] In one embodiment, an LNP comprising formula (i) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0371] [ka] In one embodiment, an LNP comprising formula (ii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0372] [ka] In one embodiment, an LNP comprising formula (iii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0373] [ka] In one embodiment, an LNP comprising formula (v) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0374] [ka] In one embodiment, an LNP comprising formula (vi) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0375] [ka] In one embodiment, an LNP comprising formula (viii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0376] [ka] In one embodiment, an LNP comprising formula (ix) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0377] [ka] During the ceremony, X 1 However, O, NR 1 , or direct bond, X 2 However, C2-5 alkylenes, X 3 However, C (=O) or direct bond, R 1 However, H or Me, and R 3 However, it is C1-3 alkyl, R 2 However, it is C1-3 alkyl, or R 2 However, the nitrogen atom and X to which it is bonded 2 Together with 1 to 3 carbon atoms, they form a 4-membered, 5-membered, or 6-membered ring, or X 1 However, NR 1 And R 1 and R 2 However, together with the nitrogen atom to which they are bonded, they form a 5-membered or 6-membered ring, or R 2 However, R 3 And together with the nitrogen atom to which they are bonded, they form a 5-membered, 6-membered, or 7-membered ring, Y 1 However, it is a C2-12 alkylene, Y 2 but, [ka] Selected from, n is between 0 and 3, and R 4However, it is C1-15 alkyl, Z 1 However, it is a C1-6 alkylene or a direct bond. Z 2 but [ka] (In either orientation) or not present, however Z 1 This is a direct bond, Z 2 If it does not exist; R 5 However, it is C5-9 alkyl or C6-10 alkoxy, R 6 However, W is a C5-9 alkyl or C6-10 alkoxy, and W is a methylene group or a direct bond, R 7 However, H or Me, or a salt thereof, provided that R 3 and R 2 However, it is a C2 alkyl group, X 1 However, O and X 2 However, it is a linear C3 alkylene, X 3 However, C (=0), and Y 1 However, it is a linear Ce alkylene, (Y 2 )nR 4 but, [ka] And R 4 However, it is a linear C5 alkyl, Z 1 However, it is C2 alkylene, Z 2 However, it does not exist, W is methylene, and R 7 However, if H, then R 5 and R 6 However, it is not a Cx alkoxy.

[0378] In one embodiment, an LNP comprising formula (xii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. [ka] In one embodiment, an LNP comprising formula (xi) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0379] [ka] In one embodiment, the LNP includes the compound of formula (xiii) and the compound of formula (xiv).

[0380] [ka] In one embodiment, an LNP comprising formula (xv) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0381] [ka] In one embodiment, an LNP comprising a formulation of formula (xvi) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0382] [ka]

[0383] In one embodiment, the lipid compounds used to form lipid nanoparticles for the delivery of the compositions described herein, for example, the nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)), are produced by one of the following reactions: [ka]

[0384] In some embodiments, LNPs comprising formula (xxi) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. In some embodiments, the LNP of formula (xxi) is an LNP described in International Publication No. 2021113777 (e.g., lipids of formula (1), such as the lipids in Table 1 of International Publication No. 2021113777). [ka] During the ceremony, Each n is an integer between 2 and 15, independently of the other two integers; L1 and L3 are each independently of the other integers. * or -C(O)O- * And in the formula, * " refers to the junction with 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 carbon atoms are 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 alkylsulfonalkyl.20 Alkyl or C9-C 20 It is an alkenyl; and R2 is [ka] It is selected from the group consisting of the following.

[0385] In some embodiments, LNPs comprising formula (xxii) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. In some embodiments, the LNP of formula (xxii) is an LNP described in International Publication No. 2021113777 (e.g., lipids of formula (2), such as the lipids in Table 2 of International Publication No. 2021113777). [ka] During the ceremony, Each n is an integer between 1 and 15, independently; R1 and R2 are independent of each other. [ka] It is selected from the group consisting of the following. R3 is [ka] It is selected from the group consisting of the following.

[0386] In some embodiments, LNPs comprising formula (xxiii) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. In some embodiments, the LNP of formula (xxiii) is an LNP described in International Publication No. 2021113777 (e.g., lipids of formula (3), such as the lipids in Table 3 of International Publication No. 2021113777). [ka] During the ceremony, X is -O-, -S-, or -OC(O)- * Selected from, in the formula, * This refers to the junction with R1; R1 is [ka] Selected from the group consisting of, R2 is [ka] It is selected from the group consisting of the following.

[0387] In some embodiments, the compositions described herein (e.g., nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) or proteins) are provided in LNPs containing ionizable lipids. In some embodiments, the ionizable lipid is, for example, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102) as described in Example 1 of U.S. Patent No. 9,867,888 (the entirety of which is incorporated herein by reference). In one embodiment, the ionizable lipid is, for example, 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate (LP01), as synthesized in Example 13 of International Publication No. 2015 / 095340 (the entirety of which is incorporated herein by reference). In another embodiment, the ionizable lipid is, for example, di((Z)-nona-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), as synthesized in Example 7, 8, or 9 of U.S. Patent Application Publication No. 2012 / 0027803 (the entirety of which is incorporated herein by reference). In one embodiment, the ionizable lipid is, for example, 1,1'-((2-(4-(2-((2-((bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azandiyl)bis(dodecane-2-ol)(C12-200), as synthesized in Examples 14 and 16 of International Publication No. 2010 / 053572 (the whole of which is incorporated herein by reference).In one embodiment, 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]phenanthrene-3-yl3-(1H-imidazole-4-yl)propanoate, for example, structure (I) from International Publication No. 2020 / 106946 (the whole of which is incorporated herein by reference).

[0388] In some embodiments, the ionizable lipid may be a cationic lipid, an ionizable cationic lipid, for example, a cationic lipid that can exist in a positively charged or neutral form depending on the pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is, for example, a lipid that can be positively charged under physiological conditions. An example cationic lipid contains one or more positively charged amine groups. In some embodiments, the lipid particles include cationic lipids in a formulation with one or more of the following: neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugate lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. The example cationic lipids disclosed herein may have an effective pKa greater than 6.0. In embodiments, the lipid nanoparticles may include a second cationic lipid having a different effective pKa (e.g., higher than the first effective pKa) from the first cationic lipid. Lipid nanoparticles may comprise 40–60 mol percent of cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and therapeutic agents, such as nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) encapsulated within or bound to the lipid nanoparticles. In some embodiments, nucleic acids are compounded simultaneously with cationic lipids. Nucleic acids may be adsorbed onto the surface of LNPs, such as LNPs containing cationic lipids. In some embodiments, nucleic acids may be encapsulated within LNPs, such as LNPs containing cationic lipids. In some embodiments, lipid nanoparticles may comprise, for example, a target moiety coated with a targeting agent. In some embodiments, the LNP formulation is biodegradable.In one embodiment, lipid nanoparticles comprising one or more lipids described herein, for example, 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 RNA molecules.

[0389] Examples of 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. 2019051289, which is incorporated herein by reference. Further examples of lipids include, but are not limited to, one or more of the following formulas: X of U.S. Patent Application Publication No. 2016 / 0311759; I of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224; I, II, or III of U.S. Patent Application Publication No. 20160151284; I, IA, II, or IIA of U.S. Patent Application Publication No. 20170210967; U.S. Patent Application Publication No. 20150140070 Ic of the specification; A of the specification of U.S. Patent Application Publication No. 2013 / 0178541; I of the specification of U.S. Patent Application Publication No. 2013 / 0303587 or U.S. Patent Application Publication No. 2013 / 0123338; ​​I of the specification of U.S. Patent Application Publication No. 2015 / 0141678; II, III, IV, or V of the specification of U.S. Patent Application Publication No. 2015 / 0239926; I of the specification of U.S. Patent Application Publication No. 2017 / 0119904; Brochure of International Publication No. 2017 / 117528 I or II; A of the specification of U.S. Patent Application Publication 2012 / 0149894; A of the specification of U.S. Patent Application Publication 2015 / 0057373; A of the brochure of International Publication 2013 / 116126; A of the specification of U.S. Patent Application Publication 2013 / 0090372; A of the specification of U.S. Patent Application Publication 2013 / 0274523; A of the specification of U.S. Patent Application Publication 2013 / 0274504; A of the specification of U.S. Patent Application Publication 2013 / 0053572; International Publication 2013 / 0 Pamphlet A of 16058; Pamphlet A of International Publication 2012 / 162210; Specification I of U.S. Patent Application Publication 2008 / 042973; Specification I, II, III, or IV of U.S. Patent Application Publication 2012 / 01287670; Specification I or II of U.S. Patent Application Publication 2014 / 0200257; Specification I, II, or III of U.S. Patent Application Publication 2015 / 0203446; Specification I or III of U.S. Patent Application Publication 2015 / 0005363;Sections I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of U.S. Patent Publication No. 2014 / 0308304; Sections I, II, III, or IV of U.S. Patent Publication No. 2013 / 0338210; Section A of International Publication No. 2009 / 132131; Section I or XXXV of U.S. Patent Publication No. 2012 / 0027796; Section XIV or XVII of U.S. Patent Publication No. 2012 / 0058144; U.S. Patent Publication No. 2 Specification 013 / 0323269; Specification I of U.S. Patent Application Publication 2011 / 0117125; Specification I, II, or III of U.S. Patent Application Publication 2011 / 0256175; Specification I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of U.S. Patent Application Publication 2012 / 0202871; Specification I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of U.S. Patent Application Publication 2011 / 0076335; Specification 2006 / 008378 I or II of the detailed specification; I of the specification of U.S. Patent Application Publication No. 2013 / 0123338; ​​I or XAYZ of the specification of U.S. Patent Application Publication No. 2015 / 0064242; XVI, XVII, or XVIII of the specification of U.S. Patent Application Publication No. 2013 / 0022649; I, II, or III of the specification of U.S. Patent Application Publication No. 2013 / 0116307; I, II, or III of the specification of U.S. Patent Application Publication No. 2013 / 0116307; I or II of the specification of U.S. Patent Application Publication No. 2010 / 0062967; the detailed specification of U.S. Patent Application Publication No. 2013 / 0189351 I-X of the document; I of the specification of U.S. Patent Application Publication 2014 / 0039032; V of the specification of U.S. Patent Application Publication 2018 / 0028664; I of the specification of U.S. Patent Application Publication 2016 / 0317458; I of the specification of U.S. Patent Application Publication 2013 / 0195920; 5, 6, or 10 of U.S. Patent Application Publication 10,221,127; III-3 of Brochure 2018 / 081480; I-5 or I-8 of Brochure 2020 / 081938; 18 or 25 of the specification of U.S. Patent Application Publication 9,867,888;A of the specification in U.S. Patent Application Publication 2019 / 0136231; II of the brochure in International Publication 2020 / 219876; 1 of the specification in U.S. Patent Application Publication 2012 / 0027803; OF-02 of the specification in U.S. Patent Application Publication 2019 / 0240349; 23 of the specification in U.S. Patent Application Publication 10,086,013; cKK-E12 / A6 of Miao et al (2020); C12-200 of the brochure in International Publication 2010 / 053572; 7C1 of Dahlman et al (2017); Whitehead et al Examples include al 304-O13 or 503-O13; TS-P4C2 of U.S. Patent No. 9,708,628; I of International Publication No. 2020 / 106946; and (1), (2), (3), or (4) of International Publication No. 2021 / 113777. Exemplary lipids include any one of the lipids listed in Tables 1 to 16 of International Publication No. 2021 / 113777.

[0390] In one embodiment, the ionizable lipid is, for example, MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), as described in Example 9 of International Publication No. 2019051289A9 (the entirety of which is incorporated herein by reference). In another embodiment, the ionizable lipid is, for example, lipid ATX-002, as described in Example 10 of International Publication No. 2019051289A9 (the entirety of which is incorporated herein by reference). In one embodiment, the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3-nonyldocosa-l3,l6-diene-l-amine (compound 32), as described in Example 11 of International Publication No. 2019051289A9 (the entirety of which is incorporated herein by reference). In another embodiment, the ionizable lipid is compound 6 or compound 22, as described in Example 12 of International Publication No. 2019051289A9 (the entirety of which is incorporated herein by reference).

[0391] Examples of noncationic lipids include, but are not limited to, distearoyl-sn-glycerol-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidyl 18-1-trans ethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethylPE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethylPE), l8-l-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diylcoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidyl phosphate Examples include phatidylglycerol (POPG), dierydoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, 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 a C10-C24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Further exemplary lipids are, in certain embodiments, but are not limited to, those described in Kim et al. (2020)dx.doi.org / 10.1021 / acs.nanolett.0c01386, which are incorporated herein by reference. Such lipids include, in some embodiments, plant lipids that have been shown to improve hepatic transfection with mRNA (e.g., DGTS).

[0392] Other examples of noncationic lipids suitable for use in lipid nanoparticles include, but are not limited to, nonphospholipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl-aryl sulfate polyethyl oxy-fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin. Other noncationic lipids are described in International Publication No. 2017 / 099823 or U.S. Patent Application Publication No. 2018 / 0028664 (the entire contents of which are incorporated herein by reference).

[0393] In some embodiments, the noncationic lipid is oleic acid or a compound of formula I, II, or IV of U.S. Patent Application Publication 2018 / 0028664 (which is incorporated herein by reference in its entirety). The noncationic lipid may account for, for example, 0 to 30% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the noncationic lipid content is 5 to 20% (mol) or 10 to 15% (mol) of the total lipids present in the lipid nanoparticles. In embodiments, the molar ratio of ionizable lipids to neutral lipids 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).

[0394] In one embodiment, the lipid nanoparticles do not contain any phospholipids.

[0395] In one embodiment, lipid nanoparticles may further contain components such as sterols to provide membrane integrity. One exemplary sterol that may be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs, e.g., 5α-cholestanol, 53-coprostanol, cholesteryl-(2 , Examples include (-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs, such as 5a-cholestane, cholestane, 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 International Publication No. 2009 / 127060 and U.S. Patent Application Publication No. 2010 / 0130588 (each, respectively, is incorporated herein by reference in whole).

[0396] In some embodiments, components that provide membrane integrity, such as sterols, may account for 0-50% (mol) of the total lipids present in the lipid nanoparticles (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%). In some embodiments, such components account for 20-50% (mol) or 30-40% (mol) of the total lipid content of the lipid nanoparticles.

[0397] In some embodiments, lipid nanoparticles may include polyethylene glycol (PEG) or conjugated lipid molecules. Generally, these are used to inhibit aggregation of lipid nanoparticles 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, for example, a (methoxypolyethylene glycol) conjugated lipid.

[0398] Examples of PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (e.g., l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), and PEG succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradecanoyloxy)propyl-l Examples include -0-(w-methoxy(polyethoxy)ethyl)butanediate (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, for example, U.S. Patent No. 5,885,613, U.S. Patent No. 6,287,591, and U.S. Patent Application Publication No. 2003 / 0077829. The following are the full contents of the following publications: U.S. Patent Application Publication 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 International Publication No. 099823 (all of which are full contents of which are by reference). As described herein (incorporated herein). 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 as of U.S. Patent Publication No. 2018 / 0028664 (the entire content of which is incorporated herein by reference). In some embodiments, the PEG-lipid is of formula II as of U.S. Patent Publication No. 20150376115 or U.S. Patent Publication No. 2016 / 0376224 (both of which the entire content of which is incorporated herein by reference).In one embodiment, the PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid may be PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (l-[8'-(cholesta-5-ene-3[β]-oxy)carboxamide-3',6'-dioxaoctanyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB( It may be one or more of 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, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid is... [ka] Includes structures selected from.

[0399] In some embodiments, lipids conjugated with molecules other than PEG may also be used instead of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (GPL) conjugates may be used instead of or in addition to PEG-lipids.

[0400] Exemplary conjugate lipids, namely PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in PCT and LIS patent applications listed in Table 2 of International Publication No. 2019051289A9 (all of which are incorporated herein by reference in their entirety).

[0401] In some embodiments, PEG or conjugate lipids may account for 0-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the PEG or conjugate lipid content is 0.5-10% or 2-5% (mol) of the total lipids present in the lipid nanoparticles. The molar ratios of ionizable lipids, noncationic lipids, sterols, and PEG / conjugate lipids may be varied as needed. For example, lipid particles may contain 30-70% ionizable lipids, 0-60% cholesterol, 0-30% noncationic lipids, and 1-10% conjugate lipids per mole or total weight of the composition. Preferably, the composition contains 30-40% ionizable lipids, 40-50% cholesterol, and 10-20% noncationic lipids per mole or total weight of the composition. In another embodiment, the composition comprises 50-75% ionizable lipids per mole or total weight of the composition, 20-40% cholesterol per mole or total weight of the composition, 5-10% noncationic lipids per mole or total weight of the composition, and 1-10% conjugate lipids per mole or total weight of the composition. The composition may also contain 60-70% ionizable lipids per mole or total weight of the composition, 25-35% cholesterol per mole or total weight of the composition, and 5-10% noncationic lipids per mole or total weight of the composition. The composition may also contain up to 90% ionizable lipids per mole or total weight of the composition and 2-15% noncationic lipids per mole or total weight of the composition.The formulation may also contain, for example, 8-30% ionizable lipids per mole or total weight of the composition, 5-30% noncationic lipids per mole or total weight of the composition, and 0-20% cholesterol per mole or total weight of the composition; 4-25% ionizable lipids per mole or total weight of the composition, 4-25% noncationic lipids per mole or total weight of the composition, 2-25% cholesterol per mole or total weight of the composition, 10-35% conjugate lipids per mole or total weight of the composition, and 5% cholesterol per mole or total weight of the composition; or the composition may contain The lipid nanoparticle formulation may contain 2-30% ionizable lipids per mole or total weight of the composition, 2-30% noncationic lipids per mole or total weight of the composition, 1-15% cholesterol per mole or total weight of the composition, 2-35% conjugate lipids 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 lipids and 2-10% noncationic lipids per mole or total weight of the composition, or 100% cationic lipids per mole or total weight of the composition. In one embodiment, the lipid particle formulation contains ionizable lipids, phospholipids, cholesterol and PEGylated lipids in a molar ratio of 50:10:38.5:1.5. In another embodiment, the lipid particle formulation contains ionizable lipids, cholesterol and PEGylated lipids in a molar ratio of 60:38.5:1.5.

[0402] In one embodiment, the lipid particles include ionizable lipids, noncationic lipids (e.g., phospholipids), sterols (e.g., cholesterol), and PEGylated lipids, where the molar ratio of lipids is in the range of 20 to 70 mole percent for ionizable lipids, with a target of 40 to 60; the molar percentage of noncationic lipids is in the range of 0 to 30, with a target of 0 to 15; the molar percentage of sterols is in the range of 20 to 70, with a target of 30 to 50; and the molar percentage of PEGylated lipids is in the range of 1 to 6, with a target of 2 to 5.

[0403] In one embodiment, the lipid particles contain ionizable lipids / noncationic lipids / sterols / conjugate lipids in a molar ratio of 50:10:38.5:1.5.

[0404] In one embodiment, the present disclosure provides a lipid nanoparticle formulation comprising phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[0405] In some embodiments, one or more further compounds may also be included. These compounds may be administered separately, or the further 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 nucleic acids or at least a second nucleic acid different from a first nucleic acid. The other further 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, peptide mimetic drugs, nucleic acids, nucleic acid analogs and derivatives, extracts made from biomaterials, or any combination thereof.

[0406] In some embodiments, the LNP comprises a biodegradable, ionizable lipid. In some embodiments, the LNP comprises (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. For example, see International Publication No. 2019 / 067992, International Publication No. 2017 / 173054, International Publication No. 2015 / 095340, and International Publication No. 2014 / 136086, and the lipids in the references provided therein. In some embodiments, the terms cationic and ionizable with respect to LNP lipids are synonymous, for example, ionizable lipids are cationic depending on pH.

[0407] In one embodiment, the average LNP diameter of the LNP formulation may be in the range of several tens to several hundred nm, measured, for example, by dynamic light scattering (DLS). In another embodiment, the average LNP diameter of the LNP formulation may be in the range of approximately 40 nm to approximately 150 nm, for example, approximately 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 some embodiments, the average LNP diameter of the LNP formulation may be approximately 50 nm to 100 nm, approximately 50 nm to 90 nm, approximately 50 nm to 80 nm, approximately 50 nm to 70 nm, approximately 50 nm to 60 nm, approximately 60 nm to 100 nm, approximately 60 nm to 90 nm, approximately 60 nm to 80 nm, approximately 60 nm to 70 nm, approximately 70 nm to 100 nm, approximately 70 nm to 90 nm, approximately 70 nm to 80 nm, approximately 80 nm to 100 nm, approximately 80 nm to 90 nm, or approximately 90 nm to 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be approximately 70 nm to 100 nm. In certain embodiments, the average LNP diameter of the LNP formulation may be approximately 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be approximately 100 nm. In one embodiment, the average LNP diameter of the LNP formulation is approximately 1 mm to 500 mm, 5 mm to 200 mm, 10 mm to 100 mm, 20 mm to 80 mm, 25 mm to 60 mm, 30 mm to 55 mm, 35 mm to 50 mm, or 38 mm to 42 mm.

[0408] LNPs can be relatively homogeneous in some cases. The polydispersity index can be used to indicate the homogeneity of LNPs, for example, the particle size distribution of lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. LNPs may have a polydispersity index of about 0 to about 0.25, for example, 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 an LNP may be about 0.10 to about 0.20.

[0409] The zeta potential of LNPs can be used to indicate the interfacial dynamic potential of a composition. In some embodiments, the zeta potential may represent the surface charge of the LNPs. Lipid nanoparticles with relatively low positive or negative charges are generally preferred because more highly charged species may unnecessarily interact with cells, tissues, and other elements in the body. In one embodiment, the zeta potential of the LNP may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0410] The efficiency of protein and / or nucleic acid encapsulation represents the amount of protein and / or nucleic acid encapsulated by LNPs or otherwise bound to LNPs after preparation, compared to the initial amount provided. High encapsulation efficiency (e.g., nearly 100%) is desirable. 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 degrading 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. In the case of lipid nanoparticles described herein, the encapsulation efficiency of proteins and / or nucleic acids may be at least 50%, for example 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%.

[0411] LNP may optionally comprise one or more coatings. In some embodiments, LNP may be formulated into capsules, films, or tablets having the coatings. Capsules, films, or tablets comprising the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0412] Additional exemplary lipids, formulations, methods, and characterizations of LNP are taught in International Publication No. 2020 / 061457 and International Publication No. 2021 / 113777 (each of which is incorporated herein by reference in whole). Further exemplary lipids, formulations, methods, and characterizations of LNP are taught in Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021).doi.org / 10.1038 / s41578-021-00358-0 (incorporated herein by reference in whole) (see, for example, the exemplary lipids and lipid derivatives in Figure 2 of Hou et al.).

[0413] In some 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) (the entire work is incorporated herein by reference).

[0414] CRISPR-Cas systems, such as LNP formulations optimized for the delivery of Cas9-gRNA RNP, gRNA, and Cas9 mRNA, are described in International Publication No. 2019067992 and International Publication No. 2019067910 (both incorporated by reference) and are useful for the delivery of cyclic polyribonucleotides and linear polyribonucleotides as described herein.

[0415] Further specific LNP formulations useful for the delivery of nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) are described in U.S. Patent No. 8,158601 and U.S. Patent No. 8,168775 (both incorporated by reference), which include formulations used in patisirane marketed under the name ONPATTRO.

[0416] 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 cationic lipids, neutral lipids, cholesterol, and PEG lipids; (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) formulated in an LNP is a vaccine.

[0417] Typical doses of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) LNPs may contain approximately 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 polyribonucleotides, linear polyribonucleotides) antigen composition described herein is 30 to 200 mcg, for example, 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg.

[0418] kit In some embodiments, the Disclosure provides a kit. In some embodiments, the kit comprises (a) a cyclic polyribonucleotide or pharmaceutical composition as described herein, and optionally, (b) informational material. In some embodiments, the cyclic polyribonucleotide or pharmaceutical composition may be part of a prescribed dosing plan. The informational material may be descriptive, instructional, marketing, or other material relating to the methods described herein and / or the use of the pharmaceutical composition or cyclic polyribonucleotide for the methods described herein. The pharmaceutical composition or cyclic polyribonucleotide may comprise material for a single dose (e.g., a single dosage form) or material for multiple doses (e.g., a “multiple doses” kit).

[0419] The informational materials in the kit are not limited in form. In one embodiment, the informational materials may include information about the production of a pharmaceutical composition, a pharmaceutical raw material, or a pharmaceutical preparation, and information about the molecular weight, concentration, expiration date, batch, or production site of the pharmaceutical composition, pharmaceutical raw material, or pharmaceutical preparation. In one embodiment, the informational materials relate to a method for administering a dosage form of a pharmaceutical composition. In one embodiment, the informational materials relate to a method for administering a dosage form of a cyclic polyribonucleotide.

[0420] In addition to the pharmaceutical compositions and dosage forms of cyclic polyribonucleotides described herein, the kit may also contain other components, such as solvents or buffers, stabilizers, preservatives, flavoring agents (e.g., bitter antagonists or sweeteners), fragrances, pigments or colorants for coloring or tinting one or more components in the kit, or other cosmetic ingredients, and / or a second agent for treating the conditions or disorders described herein. Alternatively, other components may be included in the kit, but they cannot be included in a composition or container different from the pharmaceutical compositions or cyclic polyribonucleotides described herein. In such embodiments, the kit may also include instructions for mixing the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein with other components, or for using the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein together with other components.

[0421] In some embodiments, the kit components are stored under inert conditions (e.g., under another inert gas such as nitrogen or argon). In some embodiments, the kit components are stored under anhydrous conditions (e.g., in the presence of a desiccant). In some embodiments, the components are stored in a light-blocking container such as an amber vial.

[0422] The dosage forms of the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein may be provided in any form, such as liquid, dry, or lyophilized. It is preferable that the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein are substantially pure and / or sterile. When the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein are provided in solution, the solution is preferably an aqueous solution, and preferably a sterile aqueous solution. When the pharmaceutical compositions or nucleic acid molecules (e.g., cyclic polyribonucleotides) described herein are provided in dry form, reconstitution is generally by adding a suitable solvent. The solvent, such as sterile water or buffer solution, may optionally be provided in the kit.

[0423] The kit may comprise one or more containers for compositions containing the dosage forms described herein. In some embodiments, the kit comprises separate containers, dividers, or compartments for the compositions and informational materials. For example, the pharmaceutical composition or cyclic polyribonucleotide may be contained in a bottle, vial, or syringe, and the informational materials may be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained in a single, undivided container. For example, the pharmaceutical composition or dosage form of a nucleic acid molecule (e.g., cyclic polyribonucleotide) described herein is contained in a bottle, vial, or syringe with the informational materials attached in the form of a label. In some embodiments, the kit comprises multiple (e.g., packs) of individual containers, each containing one or more unit dosage forms of the pharmaceutical composition or cyclic polyribonucleotide described herein. For example, the kit comprises multiple syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of the dosage form described herein.

[0424] The kit container may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-shielding.

[0425] The kit optionally includes a device suitable for use with the dosage form, such as a syringe, pipette, forceps, measuring spoon, swab (e.g., cotton swab or wooden swab), or any such device.

[0426] The kit of the present invention may include various strengths of dosage forms to provide doses suitable for one or more of the commencement-phase, induction-phase, or maintenance-phase regimens described herein. Alternatively, the kit may include scored tablets that allow the user to administer divided doses as needed. [Examples]

[0427] The following examples are provided to those skilled in the art to illustrate how the compositions and methods described herein may be used, prepared, and evaluated, and are intended to be purely illustrative of the disclosure and not intended to limit the scope of what the inventors consider to be the invention.

[0428] Example 1: Linear RNA pulldown method for enriching circular RNA This example describes a method for removing linear RNA byproducts from circular RNA generated by self-splicing.

[0429] When circular RNA is generated by self-splicing, several major linear byproducts may be present in the in vitro transcription (IVT) mixture: unspliced ​​linear RNA, partially spliced ​​linear RNA, fully spliced ​​but unligated linear RNA, and spliced ​​introns (Figure 3).

[0430] In one embodiment, the linear polyribonucleotide is designed to contain an aptamer near its 5' end (Figure 1). In another embodiment, the linear polyribonucleotide is designed to contain an aptamer at its 3' end. The linear polyribonucleotide is cyclic, thereby producing a cyclic polyribonucleotide that does not contain an aptamer. The reagent, conjugated to particles, is added to the mixture. The reagent binds to the aptamer on the linear polyribonucleotide, while the cyclic polyribonucleotide is not bound by the reagent, thereby separating the aptamer-containing linear polyribonucleotide from the aptamer-deficient cyclic polyribonucleotide.

[0431] In one embodiment, a linear polyribonucleotide is designed to contain a cyclic sequence (e.g., an intron fragment) near its 5' end (Figure 2). The aptamer-containing polyribonucleotide also contains a region that hybridizes to the cyclic sequence. The linear polyribonucleotide is cyclic, thereby producing a cyclic polyribonucleotide that does not contain the cyclic sequence that hybridizes to the aptamer. The reagent, conjugated to particles, is added to the mixture. The reagent binds to the aptamer hybridized to the linear polyribonucleotide, while the cyclic polyribonucleotide is not bound by the reagent, thereby separating the aptamer-containing linear polyribonucleotide from the aptamer-deficient cyclic polyribonucleotide.

[0432] Example 2: Lambda peptides can capture linear RNA byproducts containing BoxB aptamers and enrich circular RNA. This example describes enrichment of circular RNA by capturing linear byproducts via BoxB aptamer-lambdapeptide interactions. In this example, the linear RNA has a BoxB aptamer at the 5' end of a 3' half-intron that is spliced ​​out during self-splicing.

[0433] In this embodiment, the construct is designed to have 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 construct also has an extended sequence at the 5' end that includes a 15-nucleotide BoxB aptamer (5'-GCCCUGAAGAAGGGC-3' (SEQ ID NO: 148) or 5'-GCCCUGAAAAAGGGC-3' (SEQ ID NO: 149)).

[0434] For a linear pulldown to remove linear byproducts containing BoxB aptamers, a lambda peptide is designed to bind to the BoxB aptamer. The peptide also contains biotin linked by triethylene glycol (TEG).

[0435] Linear RNA is synthesized by in vitro transcription using T7 RNA polymerase from a DNA template in the presence of 7.5 mM NTP. The template DNA is removed by treatment with deoxyribonuclease for 20 minutes. The synthesized linear RNA is purified using an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurs during transcription; no additional reactions are required.

[0436] Self-spliced ​​RNA (200 picomoles) is mixed with 400 picomoles of biotinylated peptide in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) (final RNA concentration is 400 nM, peptide concentration is 800 nM). RNA without the BoxB aptamer is used as a negative control. The RNA-peptide mixture is incubated at room temperature (RT) for 30 minutes, then mixed with 100 μL of streptavidin-SEPHAROSE® beads (Sigma). The mixture is incubated on a rotor mix at RT for 1 hour, and the unbound fraction is collected by spinning down the SEPHAROSE® beads by centrifugation. The beads are washed three times with 1x binding buffer. RNA bound to the beads is eluted by heating the beads at 75°C for 10 minutes in the presence of 1x binding buffer. The beads were heated at 95°C for 5 minutes in the presence of 95% formamide to elute the RNA still bound to the beads. The concentration of unbound, eluted RNA was measured by a Qubit assay, and 200 ng of RNA was separated by urea-polyacrylamide gel electrophoresis (urea PAGE), stained using gel staining, and visualized using an image processing system. The RNA bound to the beads was linear RNA containing a BoxB aptamer, indicating that the lambdapeptide specifically captures linear RNA containing a BoxB aptamer.

[0437] Example 3. Tetracycline can capture linear RNA byproducts containing tetracycline aptamers and enrich circular RNA. This example describes enrichment of circular RNA by capturing linear byproducts via tetracycline-tetracycline aptamer interactions. In this example, the linear RNA has a tetracycline aptamer at the 5' end of a 3' half-intron that is spliced ​​out during self-splicing.

[0438] In this embodiment, the construct is designed to have 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 has an extended sequence at the 5' end containing a 60-nucleotide tetracycline aptamer.

[0439] For linear pulldown to remove linear byproducts containing the tetracycline aptamer (5'-GGCCUAAAACAUACCAGAUUUCGAUCUGGAGAGGUGAAGAAUUCGACCACCUAGGCCGGU-3' (SEQ ID NO: 150)), agarose beads conjugated with tetracycline are used.

[0440] Linear RNA is synthesized by in vitro transcription using T7 RNA polymerase from a DNA template in the presence of 7.5 mM NTP. The template DNA is removed by treatment with deoxyribonuclease for 20 minutes. The synthesized linear RNA is purified using an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurs during transcription; no additional reactions are required.

[0441] Self-spliced ​​RNA (200 picomoles) is mixed with tetracycline conjugated on 200 μL of agarose beads in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) (final RNA concentration is 400 nM). RNA without a tetracycline aptamer is used as a negative control. The mixture is incubated on RotorMix at RT for 2 hours, and the unbound fraction is collected by spinning down the agarose beads by centrifugation. The beads are washed three times with 1x binding buffer. RNA bound to the beads is eluted by heating the resin at 75°C for 10 minutes in the presence of 1x binding buffer. RNA still bound to the beads is eluted by heating the beads at 95°C for 5 minutes in the presence of 95% formamide. The concentration of unbound eluted RNA was measured by a Qubit assay, 200 ng of RNA was separated by urea PAGE, stained using gel staining, and visualized using an image processing system. The RNA bound to the beads was linear RNA containing a tetracycline aptamer, demonstrating that tetracycline specifically captures linear RNA containing a tetracycline aptamer.

[0442] Example 4. BoxB aptamer is conjugated to linear RNA byproducts to enrich circular RNA. This example describes the enrichment of circular RNA by binding a BoxB aptamer to a linear RNA byproduct and capturing the linear RNA via a BoxB aptamer-lambdapeptide interaction. In this example, the BoxB aptamer containing the oligomer has an extended 23-nucleotide sequence complementary to the 5' end of the 3' half-intron sequence.

[0443] In this embodiment, the construct is designed to have a 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing an ORF, exon fragment 1 (E1), and a 5' half of the catalytic intron. The oligomer is designed to have a 15-nucleotide BoxB sequence (5'-GCCCUGAAGAAGGGC-3' (SEQ ID NO: 151) or 5'-GCCCUGAAAAAGGGC-3' (SEQ ID NO: 152)) and an extended 23-nucleotide sequence complementary to the 5' end of the 3' half-intron sequence.

[0444] For a linear pulldown to remove linear byproducts bound to the BoxB aptamer, a lambda peptide is designed to bind to the BoxB aptamer. The peptide contains biotin linked by a TEG.

[0445] Linear RNA is synthesized by in vitro transcription using T7 RNA polymerase from a DNA template in the presence of 7.5 mM NTP. The template DNA is removed by treatment with deoxyribonuclease for 20 minutes. The synthesized linear RNA is purified using an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurs during transcription; no additional reactions are required.

[0446] Self-spliced ​​RNA (200 picomoles) is mixed with 400 picomoles of oligomers containing complementary sequences for the BoxB aptamer and introns in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) (final RNA concentration is 400 nM, oligomer concentration is 800 nM). The RNA-oligomer mixture is incubated at RT for 30 minutes, and then 800 picomoles of biotinylated peptide are added to the RNA-oligomer mixture. An oligomer without the BoxB aptamer is used as a negative control. The RNA-peptide mixture is incubated at RT for 30 minutes, and then mixed with 100 μL of streptavidin-SEPHAROSE® (Sigma). The mixture is incubated on a rotor mix at RT for 1 hour, and the unbound fraction is collected by spinning down the SEPHAROSE® beads by centrifugation. The beads were washed three times with 1x binding buffer. RNA bound to the beads was eluted by heating the resin at 75°C for 10 minutes in the presence of 1x binding buffer. RNA still bound to the beads was eluted by heating the beads at 95°C for 5 minutes in the presence of 95% formamide. The concentration of unbound eluted RNA was measured by a Qubit assay, and 200 ng of RNA was separated by urea PAGE, stained using gel staining, and visualized using an imaging system. The RNA bound to the beads was linear RNA bound to BoxB aptamers, indicating that the lambdapeptide specifically captures linear RNA bound to BoxB aptamers.

[0447] Example 5. Circular RNA is enriched by conjugating a tetracycline aptamer to a linear RNA byproduct. This example describes the enrichment of circular RNA by binding a tetracycline aptamer to a linear RNA byproduct and capturing the linear RNA via a tetracycline aptamer-tetracycline interaction. In this example, the oligomer-containing tetracycline aptamer has an extended 23-nucleotide sequence complementary to the 5' end of the 3' half-intron sequence.

[0448] In this embodiment, the construct is designed to have a 3' half of the catalytic intron, exon fragment 2 (E2), a polyribonucleotide cargo containing an ORF, exon fragment 1 (E1), and a 5' half of the catalytic intron. The oligomer is designed to have a 60-nucleotide tetracycline aptamer sequence (5'-GGCCUAAAACAUACCAGAUUUCGAUCUGGAGAGGUGAAGAAUUCGACCACCUAGGCCGGU-3' (SEQ ID NO: 153)) and an extended sequence of 23 nucleotides complementary to the 5' end of the 3' half-intron sequence.

[0449] For linear pulldown to remove linear byproducts bound to tetracycline aptamers, agarose beads conjugated with tetracycline are used.

[0450] Linear RNA is synthesized by in vitro transcription using T7 RNA polymerase from a DNA template in the presence of 7.5 mM NTP. The template DNA is removed by treatment with deoxyribonuclease for 20 minutes. The synthesized linear RNA is purified using an RNA cleanup kit (New England Biolabs, T2050). Self-splicing occurs during transcription; no additional reactions are required.

[0451] Self-spliced ​​RNA (200 picomoles) is mixed with 400 picomoles of oligomers containing complementary sequences for tetracycline aptamers and introns in the presence of 1x binding buffer (150 mM NaCl, 15 mM sodium citrate, 0.5 mM EDTA) (final RNA concentration is 400 nM, oligomer concentration is 800 nM). A tetracycline aptamer-free RNA oligomer is used as a negative control. The RNA-oligomer mixture is incubated at RT for 30 minutes, then 200 μL of tetracycline conjugate agarose beads are added to the RNA-oligomer mixture and incubated on a rotor mix at RT for 2 hours. The unbound fraction is collected by spinning down the agarose beads by centrifugation. The beads are washed three times with 1x binding buffer. RNA bound to the beads is eluted by heating the resin at 75°C for 10 minutes in the presence of 1x binding buffer. The beads were heated at 95°C for 5 minutes in the presence of 95% formamide to elute the RNA still bound to the beads. The concentration of unbound eluted RNA was measured by a Qubit assay, and 200 ng of RNA was separated by urea PAGE, stained using gel staining, and visualized using an imaging system. The RNA bound to the beads was linear RNA bound to a tetracycline aptamer, indicating that tetracycline specifically captures linear RNA bound to a tetracycline aptamer.

[0452] Other Embodiments While the present invention is described in relation to its specific embodiments, it is understood that it is subject to further modification and that this application is intended to encompass any variations, uses, or adaptations of the invention, including departures from the invention that generally fall within the scope of known or customary practices in the art to which the invention relates, in accordance with the principles of the invention, and that these are applicable to the essential features described above and are within the scope of the claims. Other embodiments are included within the scope of the claims.

Claims

1. A method for separating a linear polyribonucleotide containing an aptamer from a plurality of polyribonucleotides, including a mixture of linear and cyclic polyribonucleotides, (a) Prepare a sample comprising a plurality of polyribonucleotides, wherein a subset thereof comprises the linear polyribonucleotide containing the aptamer; (b) Contacting the sample with a reagent that binds to the aptamer; (c) Separating the linear polyribonucleotide containing the aptamer bound to the reagent from the plurality of polyribonucleotides; Methods that include...

2. The method according to claim 1, wherein the linear polyribonucleotide containing the aptamer is transcribed from a deoxyribonucleotide encoding the linear polyribonucleotide containing the aptamer.

3. The method according to claim 1, further comprising the step of generating the linear polyribonucleotide containing the aptamer by binding the aptamer to the linear polyribonucleotide.

4. A method for separating linear polyribonucleotides from a plurality of polyribonucleotides, including a mixture of linear polyribonucleotides and cyclic polyribonucleotides, (a) Prepare a sample comprising the plurality of polyribonucleotides, wherein a subset thereof includes the linear polyribonucleotide; (b) Attaching the aptamer to the linear polyribonucleotide; (c) Contacting the sample with a reagent that binds to the aptamer; (d) Separating the linear polyribonucleotide containing the aptamer bound to the reagent from the plurality of polyribonucleotides, Methods that include...

5. The method according to claim 3 or 4, wherein attaching the aptamer to the linear polyribonucleotide includes covalently attaching the aptamer to the 3' or 5' end of the linear polyribonucleotide.

6. The method according to claim 3 or 4, wherein attaching the aptamer to the linear polyribonucleotide comprises hybridizing the aptamer to a region of the linear polyribonucleotide.

7. The method according to any one of claims 1 to 6, wherein the cyclic polyribonucleotide lacks the aptamer.

8. The method according to any one of claims 1 to 7, wherein the separation step includes recovering a portion of the sample that is not bound by the reagent.

9. The method according to claim 7 or 8, wherein the portion of the sample not bound by the reagent comprises the cyclic polyribonucleotide.

10. The method according to any one of claims 1 to 9, wherein the reagent is a polypeptide, a small molecule, a lipid, a carbohydrate, RNA, or a metal.

11. The method according to claim 10, wherein the reagent is a polypeptide.

12. The method according to claim 11, wherein the polypeptide is selected from Table 1.

13. The method according to claim 12, wherein the aptamer comprises a nucleic acid sequence selected from any one of sequence numbers 1 to 66.

14. The method according to claim 11, wherein the polypeptide is protein A, streptavidin, lambdapeptide, or MS2 bacteriophage coat protein.

15. The method according to claim 10, wherein the reagent is a small molecule.

16. The method according to claim 15, wherein the small molecule is selected from Table 2.

17. The method according to claim 16, wherein the aptamer comprises a nucleic acid sequence selected from any one of sequence numbers 67 to 119.

18. The method according to claim 10, wherein the small molecule is biotin or tetracycline.

19. The method according to claim 18, wherein the reagent is RNA.

20. The method according to claim 19, wherein the RNA is selected from Table 3.

21. The method according to claim 20, wherein the aptamer comprises a nucleic acid sequence selected from SEQ ID NO: 120 or 121.

22. The method according to claim 10, wherein the reagent is a metal.

23. The method according to claim 22, wherein the metal is selected from Table 4.

24. The method according to claim 23, wherein the aptamer comprises a nucleic acid sequence selected from any one of sequence numbers 122 to 124.

25. The separation step includes immobilizing the reagent. The method according to any one of claims 1 to 24.

26. The method according to claim 25, wherein the reagent is conjugated to particles.

27. The method according to claim 26, wherein the particles include magnetic beads.

28. The method according to claim 26, wherein the reagent is conjugated to a resin containing a plurality of particles.

29. The method according to claim 28, wherein the resin comprises crosslinked poly[styrene-divinylbenzene], agarose, or SEPHAROSE®.

30. The method according to claim 28 or 29, wherein the column comprises the resin.

31. The method according to claim 30, comprising contacting the sample with the column and recovering an eluate containing a portion of the sample that is not bound to the reagent from the plurality of polyribonucleotides in the sample.

32. The method according to any one of claims 1 to 31, further comprising preparing a linear precursor polyribonucleotide before step (a), and cyclizing the linear precursor to produce the cyclic polyribonucleotide.

33. The method according to claim 32, wherein the linear precursor comprises a 5' self-splicing intron fragment and a 3' self-splicing intron fragment, and the cyclic polyribonucleotide is produced by self-splicing of the linear precursor.

34. The method according to claim 33, wherein the 5' self-splicing intron fragment and the 3' self-splicing intron fragment are each a group I or group II self-splicing intron fragment.

35. The method according to claim 32, wherein the cyclization of the cyclic polyribonucleotide is brought about by sprint ligation of the linear precursor.

36. The method according to any one of claims 1 to 35, wherein the cyclic polyribonucleotide comprises ORF.

37. The method according to claim 36, wherein the ORF encodes a polypeptide.

38. The method according to claim 36 or 37, wherein the level of expression of the purified cyclic polyribonucleotide from the ORF is increased by at least 10% compared to the level of expression from the ORF before separation.

39. The method according to any one of claims 36 to 38, wherein the cyclic polyribonucleotide includes an internal ribosome entry site (IRES).

40. The method according to claim 39, wherein the ORF is operably connected to the IRES.

41. The method according to any one of claims 1 to 40, wherein the separation step further comprises washing the polyribonucleotide containing the aptamer bound to the reagent once or more times.

42. The method according to any one of claims 1 to 41, wherein the separation step further comprises eluting the polyribonucleotide containing the aptamer from the reagent.

43. The method according to any one of claims 1 to 42, comprising preparing a plurality of reagents, each of which is bound to a different aptamer region.

44. The method according to any one of claims 1 to 43, comprising preparing the reagent in a molar ratio of 10:1 to 1:10 with respect to the polyribonucleotide containing the aptamer region.

45. The method according to any one of claims 1 to 44, comprising separating at least 500 μg of the cyclic polyribonucleotide.

46. The method according to claim 45, wherein 500 μg to 1000 mg of the cyclic polyribonucleotide is separated.

47. A group of polyribonucleotides produced by the method described in any one of claims 1 to 46.

48. A group of polyribonucleotides according to claim 47, comprising a cyclic polyribonucleotide lacking an aptamer, wherein the cyclic polyribonucleotide constitutes at least 40% (mol / mol) of the total polyribonucleotides in the composition.

49. The polyribonucleotide population according to claim 47 or 48, comprising less than 40% (mol / mol) of the total polyribonucleotides in the composition.

50. The polyribonucleotide population according to claim 49, comprising 30%, 20%, 10%, 5%, or less than 1% (mol / mol) of the total polyribonucleotides in the composition.

51. The polyribonucleotide population according to any one of claims 47 to 50, wherein the total weight of polyribonucleotides in the polyribonucleotide population is at least 500 μg.

52. The polyribonucleotide population according to claim 51, wherein the total weight of polyribonucleotides in the polyribonucleotide population is 500 μg to 1000 mg.

53. A pharmaceutical composition comprising a group of polyribonucleotides according to any one of claims 47 to 52 and a diluent, carrier, or excipient.

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