Methods for administering cyclic polyribonucleotides

Abstract

A method for administering cyclic polyribonucleotides is provided. [Solution] The present invention generally relates to methods of administering pharmaceutical compositions and formulations of cyclic polyribonucleotides. In one aspect, the present invention features a method of maintaining expression of a protein in a mammal, comprising the steps of: (a) providing to the mammal a first composition comprising a cyclic polyribonucleotide encoding the protein; and (b) 6 hours to 90 days after step (a), providing to the mammal a second composition comprising a cyclic polyribonucleotide encoding the protein, thereby maintaining expression of the protein in the mammal.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS cross reference This application is a continuation of U.S. Provisional Patent Application No. 62 / 863,725, filed June 19, 2019. No. 60 / 699,493, filed on Dec. 1, 2003, the entire contents of which are incorporated herein by reference. is used as a reference. [Background technology]

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

[0003] The present disclosure relates generally to methods of administering cyclic polyribonucleotides. The methods described generally involve expressing proteins in cells or subjects, providing a composition of cyclic polyribonucleotides encoding a protein to a cell or subject; A method for binding a protein in a cell or a subject, comprising the steps of: providing a composition of cyclic polyribonucleotides containing the cyclic polyribonucleotide to a cell or a subject; The method of administration may include providing multiple doses to a cell or a subject. For example, multiple doses may be administered by re-administration or staggered dosing. The method for readministering a cyclic polyribonucleotide composition includes administering two or more compositions. This involves providing the cells or a subject (e.g., a mammal) with the cells, typically over an extended period of time. The staggered administration of polyribonucleotide compositions generally involves administering the polyribonucleotides over a short time interval. The method includes providing two or more compositions.

[0004] In one aspect, the present invention is a method for maintaining expression of a protein in a mammal. (a) administering a first composition comprising a cyclic polyribonucleotide encoding a protein to a mammalian animal; and (b) providing the mammal with a protein-encoding gene 6 hours to 90 days after step (a). providing to the mammal a second composition comprising a cyclic polyribonucleotide, thereby and maintaining expression of the protein in the mammal.

[0005] In some embodiments of these aspects, the cyclic polyribonucleotide is an exogenous synthetic cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide is It lacks a polyA sequence, a replication element, or both.

[0006] In some embodiments of these aspects, the first composition comprises a first circular polyribonucleoside. and the second composition comprises a second cyclic polyribonucleotide, wherein the first The cyclic polyribonucleotide and the second cyclic polyribonucleotide are the same. In one embodiment, the first composition comprises a first cyclic polyribonucleotide and the second composition comprises a comprises a second cyclic polyribonucleotide, wherein the first cyclic polyribonucleotide and the second circular polyribonucleotide are different.

[0007] In some embodiments of these aspects, the step of providing the second composition comprises: and a first level of a protein expressed by the first composition is obtained from the mammal. In some embodiments, the second composition is substantially undetectable in the product. The step of providing the product includes providing the first composition and detecting a target gene expressed by the first composition. This occurs before the first levels of protein are reduced by more than 50% in mammals.

[0008] In one embodiment, the method further comprises: and then administering the composition to a mammal, thereby inducing expression of the protein in the mammal. In one embodiment, the method further comprises maintaining the circular polyribonucleic acid. and providing the mammal with a third composition of the formula after the second composition, thereby providing the mammal with In one embodiment, the method further comprises restoring expression of a protein in a third The step of providing a composition includes providing a second composition and a first and second composition. before the second level of expressed protein is substantially undetectable in the mammal. In one embodiment, the step of providing the third composition is carried out after providing the second composition. and the first and second compositions are expressed in the mammal after administration. In one embodiment, the method is performed before the level of tannins has decreased by more than 50%. The fourth, fifth, sixth, seventh, eighth, ninth, and ninth cyclic polyribonucleotides encoding the protein Or further comprising providing a tenth composition.

[0009] In some embodiments of these aspects, the first composition is a pharmaceutically acceptable carrier or In one embodiment, the first composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the second composition further comprises a drug and does not comprise any carrier. In one embodiment, the second composition further comprises a physiologically acceptable carrier or excipient. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient and does not comprise any carrier. In some embodiments, the third composition further comprises a pharmaceutically acceptable carrier or excipient. wherein the third composition further comprises a pharmaceutically acceptable excipient, and Not at all.

[0010] In some embodiments of these aspects, the protein expressed by the first composition The first level is the highest level of protein 1-2 days after providing the first composition. In one embodiment, the first level of the protein expressed by the first composition is , 40%, 50%, 60% of the highest protein level one day after providing the first composition In one embodiment, the second level of the protein is After providing the second composition, The first composition is administered for 0, 12, 15, 20, 25, 30, 35, or 40 days. At least 30%, 40%, 50%, 60% of the highest protein level after 1 day of feeding ,70%,80%,90%,100%,110%,120%,130%,140%,15 0%, 160%, 170%, 180%, 190%, or 200%. and wherein the third level of protein is administered at least 1, 2, 3 or 4 times after providing the third composition. 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40 days over a period of at least one day after providing the first composition, the highest level of protein 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 12 0%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or In some embodiments, each subsequent composition provided after the first composition is 200%. For each subsequent composition, the subsequent level of protein expressed after each subsequent composition is At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 minutes after serving the product 1 day after providing the first composition for 20, 25, 30, 35, or 40 days. Highest levels of protein at least 30%, 40%, 50%, 60%, 70%, 80% %, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160% , 170%, 180%, 190%, or 200%.

[0011] In some embodiments of these aspects, the average protein content after providing the second composition is the level is at least 40%, 50%, or less than the first level of the protein from the first composition; 60%, 70%, 80%, 90%, 100% or 110% of the average protein level. The test is performed from one day after providing the second composition until the day the protein is substantially undetectable. In some embodiments, after providing each subsequent composition after the first composition, the average level of the protein from the first composition is at least equal to the first level of the protein from the first composition. The percentage is also 40%, 50%, 60%, 70%, 80%, 90%, 100% or 110%. The average protein level was found to be substantially higher than the mean protein level from day 1 after providing each subsequent composition. In one embodiment, the first level of the protein is measured until it is undetectable. However, after providing the first composition, the first composition is administered for at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, or the first composition and the second composition for 14 days, 21 days, 28 days, or 35 days. In one embodiment, the first level of the protein is maintained after providing the composition. After providing the first composition, the first composition and the second composition are administered for 6 hours to 90 days. After providing the composition, the first level of the protein is maintained. After providing the first composition, the circular polyribonucleic acid is administered for 6 hours to 270 days. The first composition, the second composition, and the third composition are provided and then maintained. In some embodiments, the first level of protein is administered between 6 hours and 3 hours after providing the first composition. Substantially undetectable after providing the first composition and the second composition for 5 days. In some embodiments, the first level of protein is determined after providing the first composition. , at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 2 A first composition of cyclic polyribonucleotides, a second composition of cyclic polyribonucleotides, and a third composition of cyclic polyribonucleotides were administered over a period of 8 days or 35 days. In one embodiment, the second composition is provided, and the third composition is maintained. A second level of the protein in the mammal after providing the first composition is measured. at least 1%, 5%, 10% or more than the first level of the protein in the mammal after 20%, 30%, 40%, 50%, or 60% higher. In some embodiments, the third composition After providing the composition, a third level of protein produced in the mammal is obtained from the first composition. After providing the substance, the protein level in the plurality is at least 5%, 10%, or 20%, 30%, 40%, 50%, or 60% higher. The second composition of polynucleotides was provided at 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 Sunday, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 15th, 20th, 25th, 30th, The second level of protein after 40 or 45 days was measured after providing the first composition. At least 1%, 5%, 10%, 20%, 30%, 40%, 5% higher than the first level of protein In one embodiment, the third composition of cyclic polyribonucleotide is 0%, or 60% higher. 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days after providing the item , 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, or 45 days later the third level of protein is less than the first level of protein after providing the first composition. At least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher. In embodiments, the protein is a therapeutic protein, e.g., erythropoietin. In certain embodiments, expression of a protein (e.g., erythropoietin) is Induce a response in the blood (e.g., production of reticulocytes). In some embodiments, the therapeutic protein is an enzyme replacement protein, a replacement protein, Hormones, cytokines, antibodies, proteins for immunotherapy (e.g., cancer), cell reprogramming transdifferentiation factor, transcription factor, chimeric antigen receptor, transposase or nuclease ze, immune effectors (e.g., affecting susceptibility to immune responses / signals), Regulated death effector proteins (e.g., inducers of apoptosis or necrosis), tumor non-lytic inhibitors of tumors (e.g., inhibitors of oncoproteins), epigenetic modifiers, Genetic enzymes, transcription factors, DNA or protein modifying enzymes, DNA intercalators, Efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors, enzyme inhibitors competitive inhibitors of, protein synthesis effectors or inhibitors, nucleases, proteins fragments or domains, ligands or receptors, or CRISPR systems or In some embodiments, the protein is an antigen (e.g., a tumor In one embodiment, the protein is a vaccine antigen. It is a protein for vaccination.

[0012] In a second aspect, the present invention provides a method for maintaining expression of a protein in a cell or a subject. A method comprising: providing a first composition comprising a cyclic polyribonucleotide encoding a protein; providing the cell or subject with the desired protein; thereby maintaining expression of the protein in the cell or subject. The present invention is characterized by a method comprising the steps of:

[0013] In a third aspect, the present invention provides a method for maintaining expression of a protein in a cell or a subject. 6 hours to 90 days after step (a), a circular polyribonucleic acid encoding a protein is obtained. providing to the cell or subject a second composition comprising a protease; thereby providing to the cell or subject a protease; The method includes maintaining expression of the protein in the host.

[0014] In a fourth aspect, the present invention is a method of expressing a protein in a cell or a subject. A first composition comprising a cyclic polyribonucleotide encoding a protein is then introduced into a cell or providing the subject with a first level of the encoded protein, (i) the second level is at least as high as the first level; or (i) i) the second level varies by no more than 20% of the first level; thereby Expression of the encoded protein in the cell or subject is determined by at least a first level of the protein. maintaining the bell.

[0015] In a fifth aspect, the present invention is a method of expressing a protein in a cell or a subject. A second composition comprising a cyclic polyribonucleotide encoding a protein is then added to the cell or providing the cell or subject with a second level of the encoded protein; and (i) the second level is at least as great as the first level, or (i) i) the second level varies by no more than 20% of the first level; thereby Expression of the encoded protein in the cell or subject is determined by at least a first level of the protein. maintaining the bell.

[0016] In a sixth aspect, the present invention provides a first composition of a linear equivalent of a cyclic polyribonucleotide. and comparing the levels of the protein in the cell or subject after providing the compound and the second composition. providing a first composition and a second composition of cyclic polyribonucleotides to a cell or a subject; and expressing a level of a protein in a cell or subject after the protein has been expressed. providing a first composition of cyclic polyribonucleotides encoding a gene to a cell or a subject; The method of claim 1, wherein after providing a cell or a subject with a first composition of cyclic polyribonucleotides, a level of protein; (i) at least a second composition of cyclic polyribonucleotides; or (ii) the level of the protein after providing a second set of cyclic polyribonucleotides. wherein the level of the protein changes by no more than 20% of the level after providing the composition; Thereby, a first composition and a second composition of linear equivalents of cyclic polyribonucleotides the level of the circular polyribonucleic acid in the cell or subject after administration of the After providing the first and second compositions of the peptide, the protein in the cell or subject is The method includes maintaining a quality level of expression.

[0017] In a seventh aspect, the present invention provides a first composition of linear equivalents of cyclic polyribonucleotides. and comparing the levels of the protein in the cell or subject after providing the compound and the second composition. providing a first composition and a second composition of cyclic polyribonucleotides to a cell or a subject; a method for expressing a level of protein in a cell or subject after the synthesis of a cyclic polynucleotide comprising: providing a second composition of ribonucleotides to the cell or subject after the first composition; wherein the cell or subject is (i) at least a second composition of cyclic polyribonucleotides; or (ii) the level of the protein after providing a second set of cyclic polyribonucleotides. wherein the level of the protein changes by no more than 20% of the level after providing the composition; Thereby, a first composition and a second composition of linear equivalents of cyclic polyribonucleotides the level of the circular polyribonucleic acid in the cell or subject after administration of the After providing the first and second compositions of the peptide, the protein in the cell or subject is The method includes maintaining a quality level of expression.

[0018] In some embodiments of these aspects, providing the first composition comprises: to a first cell, and providing the second composition to a second cell in the subject Wherein the first cell and the second cell are the same cell or different cells. In some embodiments, the step of providing the second composition occurs after providing the first composition and A first level of a protein expressed by the first composition is expressed in a cell or a subject. In one embodiment, the second composition is provided before the second composition is qualitatively undetectable. The step includes providing a first composition and detecting a protein expressed by the first composition. In one embodiment, the test is performed before the level of IL-1 is reduced by more than 50% in a cell or subject. In this case, the step of providing the second composition is performed after providing the first composition and by the first composition. a first level of a protein expressed by the This is done before

[0019] In some embodiments of these aspects, the method comprises: The composition is provided to the cell or subject after the second composition, thereby causing and further comprising maintaining expression of the protein in the culture at at least a first level of the protein. In some embodiments, the step of providing the third composition comprises providing the second composition. and (i) a second level of the protein expressed by the first and second compositions is detected in the cells. (ii) before the antibody is substantially undetectable in the cell or subject; or The second level of the protein expressed by the first and second compositions is reduced by more than 50%. In one embodiment, the method comprises the step of: Further comprising providing a fifth, sixth, seventh, eighth, ninth, or tenth composition.

[0020] In some embodiments of these aspects, the second composition comprises a compound expressed by the first composition. at least after the level of the protein in the cell or subject to be treated becomes substantially undetectable. 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks , 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months , 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months , 18 months, 19 months, 20 months, 21 months, or 22 months, provided to the cells or subject. In some embodiments of these aspects, the second composition comprises at least one of the components of the first composition. provided to the cell or subject 4 days later and up to 90 days after the first composition.

[0021] In one embodiment, the first level of protein is measured one day after providing the first composition. In one embodiment, the first level of the protein is the highest level of the protein. However, the highest levels of protein were 40%, 50%, 60%, and 70% of the highest levels one day after providing the first composition. %, 70%, 80%, or 90%.

[0022] In one embodiment, the second level of protein is obtained after providing the second composition. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 3 The highest level of protein was observed after 1 day of providing the first composition over 0 days. Both are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, In one embodiment, the third level of protein is: After providing the third composition, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 The first composition was administered over a period of 2, 15, 20, 25, or 30 days. At least 30%, 40%, 50%, 60%, 70%, 80% of the highest protein levels 90%, 100%, 110%, 120%, or 130%. For each subsequent composition provided after the first composition, The subsequent levels of the protein are increased by at least 1, 2, 3 or 4 times after providing each subsequent composition. , for 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days, At least 30%, 40% of the highest level of protein one day after providing the first composition , 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 13 In one embodiment, the average level of protein after providing the second composition is The rule must be at least 40%, 50%, 60%, 70%, 80%, 90%, 100% of the first level. 00%, or 110%, and the average protein level is The protein is then measured at a later date until the protein is substantially undetectable. and the average level of protein after providing each subsequent composition after the first composition is At least 40%, 50%, 60%, 70%, 80%, 90%, 100% of the level of was 110%, and the average protein levels increased from day 1 after each subsequent composition was provided to The protein is measured until the day when it is substantially undetectable. The first level of protein is maintained for at least 6 hours, 1 day, 2 days after providing the first composition. , for 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days, After providing the first and second compositions of cyclic polyribonucleotides, they are maintained. In one embodiment, the first level of protein is at least At least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days or for 30 days, a first composition of cyclic polyribonucleotides, a second composition, and the third composition are provided, and then maintained. The second level of the protein in the cell or subject is determined after providing the first composition. At least 1%, 5%, 10%, 20%, 30%, 40% higher than the first level of protein In some embodiments, after providing the third composition, the cells or A third level of the protein produced in the subject is measured after providing the first composition. at least 5%, 10%, 20%, 30%, 40% lower than the first level of protein in %, 50%, or 60% higher. The composition was provided for 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days. , 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, or 45 days later the second level of the protein is greater than or equal to the first level of the protein after providing the first composition. At least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than In one embodiment, the third composition of cyclic polyribonucleotides is provided for 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days a third level of protein after 15 days, 20 days, 25 days, or 30 days; at least 1%, 5%, 10%, 20% above the first level of protein after providing 30%, 40%, 50%, or 60% higher.

[0023] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are After providing, the level of the protein in the cell or subject is maintained for at least 1 hour, 12 hours, or , 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days The dose is maintained for 10, 15, 20, 25, or 30 days. In an embodiment, a first composition and a second composition of cyclic polyribonucleotides are provided. After that, the level of the protein in the cell or subject is determined by the linear isomerization of the cyclic polyribonucleotide. of proteins in a cell or subject after providing a first composition and a second composition of equal quality. At least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the level In one embodiment, the first and second compositions of cyclic polyribonucleotides are After providing, the level of the protein in the cell or subject is determined to be a function of the cyclic polyribonucleotide. At least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days after providing the second composition , 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days The present invention provides a first composition and a second composition of linear equivalents of cyclic polyribonucleotides. at least 5%, 10%, 20%, or more than the level of the protein in the cells or subject after treatment %, 30%, 40%, 50%, or 60% higher.

[0024] In certain embodiments, the protein is a therapeutic protein, e.g., erythropoietin. and / or expression of a protein (e.g., erythropoietin) is In one embodiment, the protein induces a response in the blood (e.g., the production of reticulocytes). The antigen is an antigen (e.g., a tumor antigen, a viral antigen, a bacterial antigen). The protein is a vaccination protein.

[0025] In an eighth aspect, the present invention provides a method for producing cyclic polyribonucleotides in a cell or a subject. The method comprises administering a first composition comprising a cyclic polyribonucleotide to a cell or a subject. providing, wherein the cell or subject is provided with a first composition of cyclic polyribonucleotides; and a second composition of cyclic polyribonucleotides. providing a cell or a subject with a cyclic polyribonucleotide comprising the nucleotide sequence of and (i) the second level of cyclic polyribonucleotides comprises at least or (ii) a second level of cyclic polyribonucleotide is providing the second composition, which then varies by no more than 20% of the first level; to maintain cyclic polyribonucleotides in a cell or subject at at least a first level. The present invention is characterized by a method comprising the steps of:

[0026] In some embodiments of this aspect, the first composition comprises a first cyclic polyribonucleotide. and the second composition comprises a second cyclic polyribonucleotide, wherein: (i) the first the first cyclic polyribonucleotide and the second cyclic polyribonucleotide are the same; or (ii) the first cyclic polyribonucleotide and the second cyclic polyribonucleotide are different In some embodiments of this aspect, the first cyclic polyribonucleotide comprises a first linker. a second circular polyribonucleic acid sequence encoding a first protein and / or a second circular polyribonucleic acid sequence encoding a second protein; The code contains a second binding site and / or encodes a second protein, wherein the first the binding site and the second binding site are the same or different binding sites, and / or The first protein and the second protein may encode the same protein or different proteins. Do it.

[0027] In a ninth aspect, the present invention provides a first composition of a linear equivalent of a cyclic polyribonucleotide. and (b) determining whether the cyclic polyribonucleotide is present in the cell or subject after providing the composition and the second composition. The first and second compositions of cyclic polyribonucleotides are compared to the levels of equivalents. A level of circular polyribonucleic acid in a cell or subject after providing the substance to the cell or subject. 1. A method for producing a cyclic polyribonucleotide, comprising: providing to a subject, wherein the cell or the subject receives the cyclic polylysine after providing the first composition. and a second composition of cyclic polyribonucleotides, providing a cell or subject with a second set of antibodies, wherein the cell or subject (i) (ii) the level of circular polyribonucleotides after providing the composition; the level of the protein after providing the second composition changes by no more than 20%. a step of generating a first set of linear equivalents of a circular polyribonucleotide, and comparing the linear equivalent level in the cell or subject after providing the composition and the second composition. After providing the first and second compositions of cyclic polyribonucleotides, the cells or 1. A method comprising: maintaining a level of cyclic polyribonucleotide in a subject. do.

[0028] In some embodiments, the step of providing the second composition occurs after providing the first composition. and the level of cyclic polyribonucleotides produced by providing the first composition , performed before the antibody is substantially undetectable in the cell or subject.

[0029] In one embodiment, the step of providing a second composition of cyclic polyribonucleotides comprises: After the first composition, and in the cells or subject produced by the first composition, This is done after the level of ribonucleotides becomes substantially undetectable. wherein the second composition is a cyclic polyribonucleotide produced by the first composition. After levels become virtually undetectable, continue for at least 1 minute, 1 hour, 1 day, 2 days, or 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 In one embodiment, the antibody is provided to a cell or subject for 2 months, or 22 months. The second composition is administered at least 14 days after the first composition and not more than 90 days after the first composition. provided to a cell or a subject.

[0030] In one embodiment, the method further comprises: and providing the third composition to the cell or subject, thereby providing the third composition to the cell or subject. further comprising the step of maintaining the level of polyribonucleotides at at least a first level. Optionally, the step of providing a third composition comprises, after providing the second composition, and (i) The circular polyribonucleic acid produced by the first and second compositions in the cell or subject. (ii) before the level of the agonist is substantially undetectable in the cell or subject; or or of the cyclic polyribonucleotides produced by the first and second compositions in the subject. or (iii) before the first and second combinations are present in the cell or subject. The level of cyclic polyribonucleotides produced by the composition is 2 or more in a cell or subject. This is done before a 5% to 75% drop.

[0031] In one embodiment, the method comprises the step of: The method further comprises providing the seventh, eighth, ninth, or tenth composition to a cell or a subject.

[0032] In one embodiment, the first level of cyclic polyribonucleotide comprises a first composition The highest level of cyclic polyribonucleotide is one day after administration. For each subsequent composition provided after the first composition, After providing each subsequent composition, subsequent levels of cyclic polyribonucleotide expressed At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or The maximum amount of cyclic polyribonucleotides was measured one day after providing the first composition over a 30-day period. of levels of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10 In some embodiments, the second composition is 0%, 110%, 120%, or 130%. and after providing the cyclic polyribonucleotides, the average level of the cyclic polyribonucleotides is at least 4 times the first level. 0%, 50%, 60%, 70%, 80%, or 90% cyclic polyribonucleotide The average level of cyclic polyribonucleotides in the blood is substantially higher than that in the blood from day 1 after providing the second composition. In one embodiment, after the first composition, each The average level of cyclic polyribonucleotide after providing the subsequent composition is greater than or equal to the first level. at least 40%, 50%, 60%, 70%, 80%, or 90% cyclic polyribonucleotides The average level of nucleotides increases from 1 day after providing each subsequent composition to 10 days after providing the circular polyribonucleic acid. The measurement is performed until the day when the cyclic polynucleotide is substantially undetectable. The first level of cyclic polyribonucleotides provides a second composition of cyclic polyribonucleotides. After at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days In some embodiments, the second composition is maintained for 10 days, 28 days, or 30 days. After providing the substance, a second level of cyclic polyribonucleotide in the cell or subject is After providing the composition, a first level of cyclic polyribonucleotides in a cell or subject is detected. At least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the In one embodiment, the second composition of cyclic polyribonucleotides is provided within 1 hour. , 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 a second level of cyclic polyribonucleotide after 15 days, 20 days, 25 days, or 30 days; is less than the first level after providing the first composition of cyclic polyribonucleotides. or 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher.

[0033] In one embodiment, the third composition of cyclic polyribonucleotides is provided for 1 hour. 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days a third level of cyclic polyribonucleotide after 15 days, 20 days, 25 days, or 30 days; at least a first level of cyclic polyribonucleotide after providing the first composition 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher.

[0034] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of cyclic polyribonucleotides in the cells or subject after provision is at least 1 Hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, Maintained for 8, 9, 10, 15, 20, 25, or 30 days In one embodiment, the circular polyribonucleic acid produced by the first composition The level of cyclic polyribonucleotides is the highest one day after providing the first composition. In some embodiments, the level is 40%, 50%, 60%, 70%, 80%, or 90%. wherein the level of cyclic polyribonucleotide produced by the second composition is After providing the composition, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 1 The cyclic polylysine was administered one day after the first composition was applied for 5, 20, 25, or 30 days. At least 30%, 40%, 50%, 60%, 70% of the highest level of polynucleotides 80%, 90%, 100%, 110%, 120%, or 130%. and providing the cells with a first composition and a second composition of cyclic polyribonucleotides. or the level of cyclic polyribonucleotides in the subject is determined by measuring the linearity of cyclic polyribonucleotides. and the like. at least 5%, 10%, 20%, 30% above the level of linear equivalents of ribonucleotides; In some embodiments, the cyclic polyribonucleotide is 40%, 50%, or 60% higher. The level of cyclic polyribonucleotide after providing the first composition and the second composition is for at least 1 day, 2 days, 3 days after providing the second composition of polyribonucleotides. , 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, A first set of linear equivalents of cyclic polyribonucleotides is cultured over a period of 25 or 30 days. and the level of linear equivalents of cyclic polyribonucleotides after providing the first composition and the second composition. at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the

[0035] In one embodiment, a third level of cyclic polyribonucleotide is added to the third composition. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 minutes after serving 1 day after providing the first composition for 25 or 30 days. At least 30%, 40%, 50%, 60%, 70%, 80% of the highest level of octide, 90%, 100%, 110%, 120%, or 130%.

[0036] In one embodiment, the first level of cyclic polyribonucleotide is at least 6 hours. 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days or 30 days The third composition of cyclic polyribonucleotides is provided and maintained for a period of time. In some embodiments, the amount of circular polyribonucleic acid in the cell or subject after providing the third composition is The third level of nucleotides is a circular polyribonucleic acid in the plurality after providing the first composition. At least 5%, 10%, 20%, 30%, 40%, 50% lower than the first level of nucleotides %, or 60% higher. In one embodiment, the third composition of cyclic polyribonucleotides We offer 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, Circular polyribonucleic acid after 8, 9, 10, 15, 20, 25, or 30 days The third level of the method comprises providing the first composition followed by the first level of the cyclic polyribonucleotide. At least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% lower than the expensive.

[0037] In certain embodiments, the protein (e.g., erythropoietin) is administered to a subject. In some embodiments, the protein induces an anti-inflammatory response (e.g., reticulocyte production). antigens (e.g., viral antigens, bacterial antigens, tumor antigens).

[0038] In a tenth aspect, the present invention provides a method of binding a target in a cell or a subject, the method comprising: a first composition comprising a cyclic polyribonucleotide comprising a binding site for a target, providing to a subject a target that binds to the binding site at a first level; and A second composition comprising a cyclic polyribonucleotide containing a binding site for the target is administered to the cell or providing to a subject, wherein the target binds to the binding site at a second level, and (i) a second (ii) the second level is at least as high as the first level; or (iii) the second level is at least as high as the the step of varying by no more than 20% of the level of 1; Thereby, binding of the target in the cell or subject is determined to at least a first level of binding. The method includes maintaining the

[0039] In an eleventh aspect, the present invention provides a first set of linear equivalents of a circular polyribonucleotide. and comparing the level of target binding in the cell or subject after providing the composition and the second composition. and providing the first and second compositions of cyclic polyribonucleotides to a cell or a subject. A method for binding a target in a cell or subject after exposure, comprising: (a) a ring containing a binding site; providing a first composition of polyribonucleotides to a cell or a subject, Or the subject is provided with a first composition of cyclic polyribonucleotides and then the level of target binding is assessed. and (b) mixing a second composition of cyclic polyribonucleotides with the first composition. and then providing the cell or subject with (i) at least a circular the level of binding to the target after providing the second composition of polyribonucleotide, or (ii) ) a change of 20% or less in the level after providing the second composition of cyclic polyribonucleotides. the level of binding to the target; thereby determining the linearity of the circular polyribonucleotide; and (c) providing a first composition and a second composition of the same or similar nature to the target in a cell or subject. The first and second compositions of cyclic polyribonucleotides are compared to determine the level of binding. maintaining the level of binding to the target in the cell or subject after providing. It is characterized by:

[0040] In some embodiments, the step of providing the second composition occurs after providing the first composition. and the first level of binding by the first composition is substantially undetectable in the cell or subject. In one embodiment, the step of providing the second composition is performed before the step of providing the first composition. and a first level of binding by the first composition is detected in the cell or subject. In some embodiments, the step of providing a second composition is performed before the concentration of the second composition decreases by more than 50%. After providing the first composition and a first level of binding by the first composition, the cell or antibody is This is done before the disease declines by 25% to 75% in elephants.

[0041] In one embodiment, the method further comprises: and then providing the composition to a cell or subject, thereby inhibiting target binding in the cell or subject. at least the first level of binding. and the step of providing the third composition is performed after providing the second composition and after combining the first and second compositions. a second level of binding of the target in the cell or subject by the substance to a target in the cell or subject; This is done before the virus is physically undetectable.

[0042] In one embodiment, the step of providing the third composition is performed after providing the second composition. and a second level of binding by the first and second compositions in the cell or subject is reduced by more than 50%. This is done before the drop.

[0043] In one embodiment, the method comprises the step of: The method further comprises providing a seventh, eighth, ninth, or tenth composition.

[0044] In one embodiment, the step of providing a second composition of cyclic polyribonucleotides comprises: After the first composition, and the level of binding by the first composition, became substantially undetectable. In one embodiment, the second composition is used to enhance the level of binding by the first composition. The test results are shown for at least 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, and 7 days after the test results become virtually undetectable. Days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months month, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, After 16, 17, 18, 19, 20, 21, or 22 months, cells In one embodiment, the second composition is administered to a subject within 14 days of the first composition. and provided to the cell or subject no more than 90 days after the first composition.

[0045] In one embodiment, the first level of binding is the binding 1 day after providing the first composition. In one embodiment, the first level of binding is 40%, 50%, 60%, 70%, 80% or higher of the highest level of binding after 1 day of administration In one embodiment, the second level of binding is provided by the second composition. After at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, The maximum binding time was 1 day after providing the first composition for 30, 35, 40, or 45 days. High level of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 In some embodiments, the third bond is 00%, 110%, 120%, or 130%. the level of at least 1, 2, 3, 4, 5, 6, 7, 8, The first composition was provided for 9, 10, 12, 15, 20, 25, or 30 days. After 1 day, at least 30%, 40%, 50%, 60%, 70%, and 8 days, the highest level of binding was achieved. 0%, 90%, 100%, 110%, 120%, or 130%. For each subsequent composition provided after the first composition, the results after each subsequent composition are and each subsequent level of the composition is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 3 the first composition for 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days. At least 30%, 40%, 50%, or 60% of the maximum level of binding one day after delivery , 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In an embodiment, the average level of binding after providing the second composition is at least 1 / 2 of the first level. At least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% and the average level of binding is such that from one day after providing the second composition, binding is substantially undetectable. In some embodiments, after the first composition, each subsequent composition The average level of bonding after providing the material is at least 40%, 50%, 60% of the initial level. %, 70%, 80%, 90%, 100%, or 110%, and the average level of binding is The subsequent composition is administered one day after the first administration of the composition, and the subsequent administration of the composition is performed until the day when the binding is substantially undetectable. In one embodiment, the first level of binding occurs after providing the first composition. 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or provides a first composition and a second composition of cyclic polyribonucleotides for a period of 30 days. In one embodiment, the cells or recipients are maintained after providing the second composition. The second level of binding in the subject is determined by measuring the binding in the cell or subject after providing the first composition. At least 1%, 5%, 10%, 20%, 30%, 40%, 50% above the first level of the combination or 60% higher. In one embodiment, the second composition of cyclic polyribonucleotides is Provided 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days A second level of binding after 9, 10, 15, 20, 25, or 30 days is at least 1%, 5%, 10%, 20%, or more than the first level of binding after providing the composition of claim 1; %, 30%, 40%, 50%, or 60% higher.

[0046] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of binding in the cells or subject after provision is maintained for at least 1 hour, 12 hours, 18 hours, or Hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 The effect is maintained for 15 days, 20 days, 25 days, or 30 days. In this embodiment, after providing the first composition and the second composition of cyclic polyribonucleotides, The level of binding in the cell or subject is determined by the first linear equivalent of the cyclic polyribonucleotide. the level of binding in the cell or subject after providing the composition and the second composition is at least In some embodiments, the ion exchange rate is 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher. and a second composition of cyclic polyribonucleotides. the level of binding in the subject is determined after providing the second composition of cyclic polyribonucleotides , at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days , 10 days, 15 days, 20 days, 25 days, or 30 days, a cell or subject after providing a first composition and a second composition of linear equivalents of a nucleotide; at least 5%, 10%, 20%, 30%, 40%, 50%, or is 60% higher.

[0047] In some embodiments, the first level of binding occurs after providing the first composition. 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or administered a first composition of cyclic polyribonucleotides, a second composition of cyclic polyribonucleotides, and After providing the third composition, the method is maintained. a third level of binding in the cell or subject after providing the first composition; At least 5%, 10%, 20%, 30%, 40%, 50%, or 60% lower than the first level In one embodiment, a third composition of cyclic polyribonucleotides is provided. Hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, a third level of binding after 10, 15, 20, 25, or 30 days is greater than or equal to the first composition; at least 1%, 5%, 10%, 20%, 30% above the first level of bonding after providing , 40%, 50%, or 60% higher.

[0048] In some embodiments, the cyclic polyribonucleotide of the first composition and the cyclic polyribonucleotide of the second composition In one embodiment, the cyclic polyribonucleotides of the first composition are the same. The polyribonucleotide and the cyclic polyribonucleotide of the second composition are different.

[0049] In one embodiment, the first composition and the second composition contain approximately equal amounts of cyclic polyribonucleotides. In one embodiment, the first composition contains a greater amount of nucleotides than the second composition. In one embodiment, the first composition comprises a third, A greater amount of circular polyribonucleic acid than the fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition. In one embodiment, the amount of cyclic polyribonucleotide in the second composition is 1%, 5%, 10%, 15%, 20%, or %, or by no more than 25%. The amount of nucleotides may be 1%, 5%, 1% or more of the amount of cyclic polyribonucleotides in the first composition. In some embodiments, the first composition is no more than 0%, 15%, 20%, or 25% less. In one embodiment, the second composition further comprises a pharmaceutically acceptable carrier or excipient. The product further comprises a pharmaceutically acceptable carrier or excipient. The composition further comprises a pharmaceutically acceptable carrier or excipient. The cell is an animal cell (e.g., a mammalian cell, e.g., a human cell). In one embodiment, the cell is a plurality of cells in a subject. and / or the second composition is at a concentration of 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml l, 20ng / ml, 25ng / ml, 30ng / ml, 35ng / ml, 40ng / m l, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / m l, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 50 0ng / ml, 600ng / ml, 1μg / ml, 10μg / ml, 50μg / ml, 1 00μg / ml, 200g / ml, 300μg / ml, 400μg / ml, 500μg / ml, 600μg / ml, 700μg / ml, 800μg / ml, 900μg / ml, 1 mg / ml, 1.5 mg / ml, or 2 mg / ml or less of linear polyribonucleotide molecules In some embodiments, the first composition and / or the second composition comprises and / or at least 30% (w / w) of the total ribonucleotide molecules in the second composition w), 40%(w / w), 50%(w / w), 60%(w / w), 70%(w / w), 8 0%(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), or 99% (w / w) circular polyribonucleic acid In one embodiment, the total ribonucleotides in the first composition and / or the second composition At least 30% (w / w), 40% (w / w), 50% (w / w) of the nucleotide molecules , 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), or 99% (w / w) are circular polyribonucleotide molecules.

[0050] In some embodiments, the subject is an animal (e.g., a mammal). In some embodiments, the subject is a human. antigens, bacterial antigens, and viral antigens).

[0051] In a twelfth aspect, the invention generally relates to a method for producing cyclic polyribonucleotides in a cell. The method includes providing a cell with a first composition comprising a cyclic polyribonucleotide. The method further comprises providing a cell with a first composition, followed by a first sequence of a cyclic polyribonucleotide. and providing a second composition of cyclic polyribonucleotides to the cells. the cell comprises a second level of cyclic polyribonucleotides, The second level of nucleotide is at least as high as the first level after providing the second composition. a step of at least maintaining the temperature at the first level.

[0052] In a thirteenth aspect, the present invention generally relates to a method for producing circular polyribonucleic acid in a mammal. 1. A method for producing a cyclic polyribonucleotide comprising administering a first composition comprising a cyclic polyribonucleotide to a mammal. wherein the mammal is provided with a first composition of cyclic polyribonucleotides. and a second composition of cyclic polyribonucleotides, providing the mammal with a second level of cyclic polyribonucleotides, and a second level of cyclic polyribonucleotide is added to provide a second composition, followed by a second level of cyclic polyribonucleotide. at least as high as the first level; thereby maintaining ribonucleotides at at least a first level. .

[0053] In a fourteenth aspect, the present invention generally provides a method for preparing a linear equivalent of a cyclic polyribonucleotide. of cyclic polyribonucleotides in cells after providing the first composition and the second composition. The first composition and the second composition of cyclic polyribonucleotides are compared to the level of their linear counterparts. A method for producing a certain level of cyclic polyribonucleotides in cells after providing the cells with the composition. 1. A method for producing a cyclic polyribonucleotide comprising the steps of: providing a first composition of cyclic polyribonucleotides to a cell; comprising the level of cyclic polyribonucleotide after the cells are provided with the first composition. and providing a second composition of cyclic polyribonucleotides to the cells, at least includes the level of cyclic polyribonucleotide after providing the second composition , whereby a first composition of linear equivalents of a cyclic polyribonucleotide and a second composition of linear equivalents of a cyclic polyribonucleotide are obtained. the level of circular polyribonucleic acid compared to the linear equivalent in cells after providing the composition Circular polyribonucleic acid in cells after providing the first and second compositions of ribonucleotides. The method includes maintaining the level of leutinone in the patient.

[0054] In a fifteenth aspect, the present invention generally provides a method for preparing a linear equivalent of a cyclic polyribonucleotide. Circular polyribonucleic acid in a mammal after providing the first composition and the second composition. the level of the first and second compositions of cyclic polyribonucleotides compared to their linear equivalents After providing the mammal with the composition, a level of circular polyribonucleic acid is detected in the mammal. 1. A method for producing a cyclic polyribonucleotide comprising administering a first composition of cyclic polyribonucleotides to a mammal. providing, wherein the mammal receives the circular polyribonucleoside after providing the first composition. and providing a second composition of cyclic polyribonucleotides to the mammal. a step of carrying out the step of administering to the mammal at least the circular polyribonucleotides after providing the second composition; at the level of nucleotides; thereby linear synthesis of circular polyribonucleotides and (c) determining the level of linear equivalents in a mammal after providing a first composition and a second composition of the equivalents. After providing the first composition and the second composition of cyclic polyribonucleotides, The present invention relates to a method for producing a cyclic polyribonucleotide comprising the steps of: It is a sign.

[0055] In a sixteenth aspect, the invention generally relates to a method of binding at a target within a cell. providing a first composition comprising a cyclic polyribonucleotide to the cells, the cells contain a first level of binding after providing the first composition; and providing a second composition of polynucleotides to the cells, wherein the cells react with the second polynucleotides of the binding a second level of conjugation, wherein the second level of conjugation comprises at least a first level of conjugation after providing the second composition. 1 level; thereby determining binding in the cells at least at a level comparable to that of the first The method includes maintaining the concentration of the hydroxybenzoate at a level of 0.05%.

[0056] In a seventeenth aspect, the present invention generally relates to a method for binding to a target in a mammal. providing a mammal with a first composition comprising a cyclic polyribonucleotide; wherein the mammal comprises a first level of binding after providing the first composition; and a second composition of cyclic polyribonucleotides to a mammal, The mammal comprises a second level of binding, the second level of binding provided with a second composition. after which the level of binding is at least as great as the first level of binding; thereby maintaining binding at least at a first level in the presence of the hydroxyl group.

[0057] In an eighteenth aspect, the present invention generally provides a method for preparing a linear equivalent of a circular polyribonucleotide. the level of binding in the cells after providing the first composition and the second composition, After providing the cells with the first composition and the second composition of polyribonucleotides, A method of binding a target, comprising providing a first composition of cyclic polyribonucleotides to a cell. measuring the level of binding after the cells have been provided with the first composition; and and a second composition of cyclic polyribonucleotides to the cells, wherein the cells at least the level of binding after providing the second composition; After providing the first and second compositions of linear equivalents of ribonucleotides, the level of binding in the first and second compositions of cyclic polyribonucleotides compared to the level of binding in the cells. and maintaining the level of binding in the cells after providing the composition. do.

[0058] In a nineteenth aspect, the present invention generally provides a method for preparing a linear equivalent of a cyclic polyribonucleotide. and comparing the level of binding in the mammal after providing the first composition and the second composition. providing the mammal with the first composition and the second composition of cyclic polyribonucleotides, A method of binding a target in a mammal, comprising: providing the mammal with the first composition, wherein the mammal is provided with the first composition and then binds the first composition. and providing a second composition of cyclic polyribonucleotides to the mammal. providing the mammal with at least the level of binding after providing the second composition; thereby forming a first composition of linear equivalents of cyclic polyribonucleotides. and the level of binding in the mammal after providing the second composition. and measuring the level of binding in the mammal after providing the first and second compositions of leucine. The method includes maintaining In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for maintaining expression of a protein in a mammal, comprising: (a) providing to the mammal a first composition comprising a cyclic polyribonucleotide encoding the protein; and (b) 6 hours to 90 days after step (a), providing to the mammal a second composition comprising a cyclic polyribonucleotide encoding the protein, thereby maintaining expression of the protein in the mammal. (Item 2) the step of providing the second composition is performed after providing the first composition and (i) before a first level of a protein expressed by the first composition is substantially undetectable in the mammal; or (ii) after the first level of the protein expressed by the first composition becomes substantially undetectable in the mammal; or (iii) The method of item 1, wherein the method is performed before the first level of the protein expressed by the first composition is reduced by more than 50% in the mammal. (Item 3) 3. The method of claim 1 or 2, wherein the cyclic polyribonucleotide (i) is an exogenous synthetic cyclic polyribonucleotide; and / or (ii) lacks a polyA sequence, a replication element, or both. (Item 4) The first composition comprises a first cyclic polyribonucleotide and the second composition comprises a second cyclic polyribonucleotide, wherein: (i) the first cyclic polyribonucleotide and the second cyclic polyribonucleotide are the same; or (ii) The method according to any one of items 1 to 3, wherein the first cyclic polyribonucleotide and the second cyclic polyribonucleotide are different. (Item 5) (i) providing the third composition of cyclic polyribonucleotides to the mammal after the second composition, thereby maintaining expression of the protein in the mammal; or (ii) providing a third composition of cyclic polyribonucleotide to the mammal after the second composition, thereby restoring expression of the protein in the mammal; Optionally, the step of providing the third composition occurs after providing the second composition and before (a) the second level of the protein expressed by the first and second compositions is substantially undetectable in the mammal; or (b) the second level of the protein expressed by the first and second compositions in the mammal is reduced by more than 50%; and / or (iii) providing a fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition of cyclic polyribonucleotides encoding the protein. 5. The method according to any one of items 1 to 4, further comprising: (Item 6) (i) the first level of the protein expressed by the first composition is the highest level of the protein 1 to 2 days after providing the first composition; or (ii) the first level of the protein expressed by the first composition is 40%, 50%, 60%, 70%, 80%, or 90% of the highest level of the protein one day after providing the first composition; and optionally, (iii) the second level of the protein is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of the protein 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing the second composition; and optionally, (iv) a third level of protein is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of said protein 1 day after providing said first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing said third composition; and / or (v) for each subsequent composition provided after the first composition, the subsequent level of the protein expressed after each subsequent composition is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of the protein 1 day after providing the first composition, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing each subsequent composition. (Item 7) 7. The method of any one of items 1 to 6, wherein the average level of the protein after providing the second composition is at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% of the first level of protein from the first composition, wherein the average level of protein is measured (i) from one day after providing the second composition until the day the protein is substantially undetectable; or (ii) after providing each subsequent composition until the day the protein is substantially undetectable. (Item 8) (i) the first level of the protein is maintained after providing the first composition and the second composition for between 6 hours and 90 days after providing the first composition; and / or (ii) the first level of the protein is maintained after providing the first composition, the second composition, and the third composition of the cyclic polyribonucleotide for between 6 hours and 270 days after providing the first composition; and / or (iii) The method of any one of items 1 to 7, wherein the first level of the protein is substantially undetectable after providing the first composition and the second composition for 6 hours to 35 days after providing the first composition. (Item 9) (i) a second level of protein in the mammal after providing the second composition is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than a first level of protein in the mammal after providing the first composition; and / or (ii) after providing the third composition, a third level of protein produced in the mammal is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of protein after providing the first composition; and / or (iii) 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days after providing the second composition of cyclic polyribonucleotide; a second level of protein 15, 20, 25, or 30 days later that is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of protein after providing the first composition; and / or (iv) the third level of protein 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the third composition of cyclic polyribonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of protein after providing the first composition. (Item 10) 10. The method of any one of items 1 to 9, wherein the protein is a therapeutic protein, e.g., erythropoietin; and / or expression of the protein (e.g., erythropoietin) induces a response in the mammal (e.g., production of reticulocytes). (Item 11) 1. A method for maintaining expression of a protein in a cell or a subject, comprising: (a) providing to the cell or subject a first composition comprising a cyclic polyribonucleotide encoding the protein; and (b) 6 hours to 90 days after step (a), providing to the cell or subject a second composition comprising a cyclic polyribonucleotide encoding the protein; thereby maintaining expression of the protein in the cell or subject; optionally, providing the first composition to a first cell in the subject and providing the second composition to a second cell in the subject, wherein the first cell and second cell are the same cell or different cells. (Item 12) 1. A method for expressing a protein in a cell or a subject, comprising: (a) providing to the cell or the subject a first composition comprising a cyclic polyribonucleotide encoding a protein, wherein the cell or the subject expresses a first level of the encoded protein; and (b) providing to the cell or the subject a second composition comprising a cyclic polyribonucleotide encoding a protein, wherein the cell or the subject expresses a second level of the encoded protein; (i) the second level is at least as great as the first level; or (ii) the second level varies by no more than 20% of the first level; thereby maintaining expression of the encoded protein in the cell or the subject at least at the first level of the protein; optionally, providing the first composition to a first cell in the subject and providing the second composition to a second cell in the subject, wherein the first cell and second cell are the same cell or different cells. (Item 13) 1. A method of expressing a level of a protein in a cell or subject after providing said cell or subject with a first composition and a second composition of linear equivalents of cyclic polyribonucleotides, compared to the level of said protein in said cell or subject after providing said first composition and a second composition of linear equivalents of cyclic polyribonucleotides, comprising: (a) providing a first composition of cyclic polyribonucleotides encoding a protein to a cell or subject, wherein the cell or subject contains a level of the protein after providing the first composition of cyclic polyribonucleotides; and (b) providing a second composition of cyclic polyribonucleotides to the cell or subject after the first composition, wherein the cell or subject: (i) at least after providing said second composition of cyclic polyribonucleotides, or (ii) the level of the protein changes by no more than 20% of the level after providing the second composition of the cyclic polyribonucleotide; thereby maintaining expression of the level of the protein in the cell or subject after providing the first and second compositions of cyclic polyribonucleotide compared to the level of the protein in the cell or subject after providing the first and second compositions of linear equivalents of the cyclic polyribonucleotide; and optionally, providing the first composition is to a first cell in the subject and providing the second composition is to a second cell in the subject, wherein the first cell and the second cell are the same cell or different cells. (Item 14) the step of providing the second composition is performed after providing the first composition and (i) before a first level of a protein expressed by the first composition is substantially undetectable in the cell or subject; and / or (ii) before a first level of a protein expressed by the first composition is reduced by more than 50% in the cell or subject; and / or (iii) The method according to item 11 or 12, wherein the method is performed before the first level of the protein expressed by the first composition is reduced by 25% to 75% in the cell or subject. (Item 15) 15. The method of any one of items 12 to 14, further comprising providing the third composition of cyclic polyribonucleotides to the cell or subject after the second composition, thereby maintaining expression of the protein in the cell or subject at at least the first level of protein, optionally wherein providing the third composition occurs after providing the second composition and before (i) the second level of the protein expressed by the first and second compositions is substantially undetectable in the cell or subject, or (ii) the second level of the protein expressed by the first and second compositions in the cell or subject is reduced by more than 50%. (Item 16) 16. The method of any one of items 12 to 15, further comprising providing a fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition of cyclic polyribonucleotides. (Item 17) The second composition comprises: (i) at least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, or 22 months after the level of the protein expressed by the first composition in the cell or subject becomes substantially undetectable; or (ii) the method of any one of items 13, 15, or 16, wherein the first composition is provided to the cell or subject at least 14 days after the first composition and no more than 90 days after the first composition. (Item 18) the first level of the protein (i) the peak level of said protein one day after providing said first composition; and / or (ii) 40%, 50%, 60%, 70%, 80%, or 90% of the highest level of the protein one day after providing the first composition. (Item 19) (i) the second level of the protein is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of the protein 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing the second composition; and / or (ii) the third level of the protein is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of the protein 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing the third composition; and / or (iii) for each subsequent composition provided after the first composition, the subsequent level of the protein expressed after each subsequent composition is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of the protein 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing each subsequent composition; and / or (iv) the average level of the protein after providing the second composition is at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% of the first level, the average level of the protein being measured from 1 day after providing the second composition until the day the protein is substantially undetectable; and / or (v) the average level of the protein after providing each subsequent composition after the first composition is at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% of the first level, the average level of the protein being measured from 1 day after providing each subsequent composition until the day the protein is substantially undetectable; and / or (vi) the first level of the protein is maintained after providing the first composition of cyclic polyribonucleotide and the second composition for at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days after providing the first composition; and / or (vii) the first level of the protein is maintained after providing the first composition, the second composition, and the third composition of the cyclic polyribonucleotide for at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days after providing the first composition; and / or (viii) a second level of the protein in the cell or subject after providing the second composition is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of the protein in the cell or subject after providing the first composition; and / or (ix) after providing the third composition, a third level of the protein produced in the cell or subject is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of the protein in the plurality after providing the first composition; and / or (x) a second level of protein 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, or 45 days after providing the second composition of cyclic polyribonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of protein after providing the first composition; and / or (xi) a third level of protein 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the third composition of cyclic polyribonucleotide is at least 1%, 5%, 10%, 20% or more lower than the first level of protein after providing the first composition. , 30%, 40%, 50%, or 60% higher. (Item 20) (i) the level of the protein in the cell or subject is maintained for at least 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the first composition and the second composition of cyclic polyribonucleotide; and / or (ii) after providing the first and second compositions of the cyclic polyribonucleotide, the level of the protein in the cell or subject is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the level of the protein in the cell or subject after providing the first and second compositions of the linear equivalent of the cyclic polyribonucleotide; and / or (iii) the level of the protein in the cell or subject after providing the first and second compositions of cyclic polyribonucleotides is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the level of the protein in the cell or subject after providing the first and second compositions of the linear equivalent of the cyclic polyribonucleotides for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the second composition of cyclic polyribonucleotides. (Item 21) 21. The method of any one of items 11 to 20, wherein the protein is a therapeutic protein, such as erythropoietin, and / or expression of the protein (e.g., erythropoietin) induces a response in the cell or subject (e.g., production of reticulocytes). (Item 22) 1. A method for producing cyclic polyribonucleotides in a cell or a subject, comprising: (a) providing a first composition comprising the cyclic polyribonucleotide to a cell or a subject, wherein the cell or subject comprises a first level of cyclic polyribonucleotide after providing the first composition; and (b) providing a second composition of cyclic polyribonucleotides to the cell or subject, wherein the cell or subject comprises a second level of cyclic polyribonucleotides; (i) the second level of the cyclic polyribonucleotide is at least as great as the first level; or (ii) the second level of cyclic polyribonucleotide is changed by no more than 20% of the first level after providing the second composition; thereby maintaining cyclic polyribonucleotides in the cell or subject at least at the first level; optionally, the first composition comprises a first cyclic polyribonucleotide and the second composition comprises a second cyclic polyribonucleotide, wherein: (i) the first cyclic polyribonucleotide and the second cyclic polyribonucleotide are the same; or (ii) the first cyclic polyribonucleotide and the second cyclic polyribonucleotide are different; Optionally, the first cyclic polyribonucleotide comprises a first binding site and / or encodes a first protein, and the second cyclic polyribonucleotide comprises a second binding site and / or encodes a second protein, wherein the first binding site and the second binding site are the same or different binding sites, and / or the first protein and the second protein encode the same protein or different proteins. (Item 23) 1. A method of producing a level of cyclic polyribonucleotides in a cell or subject after providing the cell or subject with a first composition and a second composition of linear equivalents of the cyclic polyribonucleotide, compared to a level of the linear equivalent of the cyclic polyribonucleotide in the cell or subject after providing the cell or subject with the first and second compositions of linear equivalents of the cyclic polyribonucleotide, comprising: (a) providing the first composition of cyclic polyribonucleotides to the cell or subject, wherein the cell or subject comprises the level of the cyclic polyribonucleotides after providing the first composition; and (b) providing the second composition of cyclic polyribonucleotides to the cell or subject, wherein the cell or subject comprises (i) at least a level of cyclic polyribonucleotides after providing the second composition, or (ii) a level of the protein after providing the second composition that changes by no more than 20% of the level of the cyclic polyribonucleotides; thereby maintaining the level of the cyclic polyribonucleotide in the cell or subject after providing the first and second compositions of the cyclic polyribonucleotide compared to the level of the linear equivalent in the cell or subject after providing the first and second compositions of the linear equivalent of the cyclic polyribonucleotide. (Item 24) 23. The method of claim 22, wherein the step of providing the second composition occurs after providing the first composition and before the level of cyclic polyribonucleotides produced by providing the first composition is substantially undetectable in the cell or subject. (Item 25) 25. The method of claim 24, wherein the step of providing the second composition of cyclic polyribonucleotide occurs after the first composition and after the level of cyclic polyribonucleotide in the cell or subject produced by the first composition becomes substantially undetectable. (Item 26) The second composition comprises: (i) at least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, or 22 months after the level of cyclic polyribonucleotide produced by the first composition becomes substantially undetectable; or (ii) the method of item 23 or 25, wherein the first composition is provided to the cell or subject at least 14 days after the first composition and no more than 90 days after the first composition. (Item 27) providing a third composition of cyclic polyribonucleotides to the cell or subject after the second composition, thereby maintaining the level of cyclic polyribonucleotides after providing the third composition at at least the first level, and optionally, providing the third composition is performed after providing the second composition and (i) before the level of cyclic polyribonucleotides produced in the cell or subject by the first and second compositions is substantially undetectable in the cell or subject; or (ii) before the level of cyclic polyribonucleotides produced by the first and second compositions in the cell or subject is reduced by more than 50%; or (iii) the level of cyclic polyribonucleotides produced in the cell or subject by the first and second compositions is reduced by 25% to 75% in the cell or subject. 25. The method according to item 22 or 24, carried out before (Item 28) 28. The method of any one of items 22 to 27, further comprising providing the fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition of cyclic polyribonucleotide to the cell or subject. (Item 29) (i) the first level of cyclic polyribonucleotide is the highest level of cyclic polyribonucleotide one day after providing the first composition; and / or (ii) for each subsequent composition provided after the first composition, the subsequent level of cyclic polyribonucleotide expressed after each subsequent composition is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of cyclic polyribonucleotide 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing each subsequent composition; and / or (ii) the average level of the cyclic polyribonucleotide after providing the second composition is at least 40%, 50%, 60%, 70%, 80%, or 90% of the first level, and the average level of the cyclic polyribonucleotide is measured from 1 day after providing the second composition until the day when the cyclic polyribonucleotide is substantially undetectable; and / or (iii) the average level of the cyclic polyribonucleotide after providing each subsequent composition after the first composition is at least 40%, 50%, 60%, 70%, 80%, or 90% of the first level, and the average level of the cyclic polyribonucleotide is measured from 1 day after providing each subsequent composition until the day when the cyclic polyribonucleotide is substantially undetectable; and / or (iv) the first level of cyclic polyribonucleotide is maintained for at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days after providing the second composition of cyclic polyribonucleotide; and / or (v) after providing the second composition, the second level of cyclic polyribonucleotides in the cell or subject is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of cyclic polyribonucleotides in the cell or subject after providing the first composition; and / or (vi) The method of any one of items 22, 24, 27, or 28, wherein the second level of cyclic polyribonucleotide 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the second composition of cyclic polyribonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of cyclic polyribonucleotide after providing the first composition. (Item 30) 30. The method of any one of items 27 to 29, wherein the third level of cyclic polyribonucleotide 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the third composition of cyclic polyribonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of cyclic polyribonucleotide after providing the first composition. (Item 31) (i) the level of cyclic polyribonucleotide in the cell or subject after providing the first composition and the second composition of cyclic polyribonucleotide is maintained for at least 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days. and / or (ii) the level of cyclic polyribonucleotide produced by the first composition is 40%, 50%, 60%, 70%, 80%, or 90% of the highest level of cyclic polyribonucleotide one day after providing the first composition; and / or (iii) the level of cyclic polyribonucleotides produced by the second composition is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of cyclic polyribonucleotides 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing the second composition; and / or (iv) the level of cyclic polyribonucleotides in the cell or subject after providing the first and second compositions of cyclic polyribonucleotides is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the level of linear equivalents of cyclic polyribonucleotides in the cell or subject after providing the first and second compositions of linear equivalents of cyclic polyribonucleotides; and / or (v) the level of cyclic polyribonucleotides after providing the first and second compositions of cyclic polyribonucleotides is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the level of linear equivalents of cyclic polyribonucleotides after providing the first and second compositions of linear equivalents of cyclic polyribonucleotides for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the second composition of cyclic polyribonucleotides. (Item 32) 30. The method of any one of items 27 to 29, wherein the third level of cyclic polyribonucleotide is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of cyclic polyribonucleotide 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing the third composition. (Item 33) (i) the first level of cyclic polyribonucleotide is maintained for at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days after providing the third composition of cyclic polyribonucleotide; and / or (ii) the third level of cyclic polyribonucleotides in the cell or subject after providing the third composition is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of cyclic polyribonucleotides in the plurality after providing the first composition; and / or (iii) The method of any one of items 27 to 29 or 32, wherein the third level of cyclic polyribonucleotide 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the third composition of cyclic polyribonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of cyclic polyribonucleotide after providing the first composition. (Item 34) 34. The method of any one of items 22 to 33, wherein the protein (e.g., erythropoietin) induces a response in the subject (e.g., production of reticulocytes). (Item 35) 1. A method of binding a target in a cell or a subject, comprising: (a) a first composition comprising a cyclic polyribonucleotide comprising a binding site for a target; providing to the cell or subject, wherein the target binds to the binding site at a first level; and (b) providing to the cell or subject a second composition comprising the cyclic polyribonucleotide comprising a binding site for a target, wherein the target binds to the binding site at a second level, and (i) the second level is at least as great as the first level, or (ii) the second level changes by no more than 20% of the first level; thereby maintaining binding of the target in the cell or subject at least at the first level of binding. (Item 36) 1. A method of binding a target in a cell or subject after providing a first composition and a second composition of a cyclic polyribonucleotide to the cell or subject, compared to the level of binding to the target in the cell or subject after providing the first and second compositions of linear equivalents of the cyclic polyribonucleotide, comprising: (a) providing the cell or subject with a first composition of cyclic polyribonucleotides comprising a binding site, wherein the cell or subject comprises a level of binding of the target after providing the first composition of cyclic polyribonucleotides; and (b) providing the second composition of cyclic polyribonucleotide to the cell or subject after the first composition, wherein the cell or subject exhibits (i) at least a level of binding to the target after providing the second composition of cyclic polyribonucleotide, or (ii) a level of binding to the target that changes by no more than 20% of the level after providing the second composition of cyclic polyribonucleotide; thereby maintaining a level of binding to the target in the cell or subject after providing the first and second compositions of cyclic polyribonucleotide compared to a level of binding to the target in the cell or subject after providing the first and second compositions of the linear equivalent of the cyclic polyribonucleotide. (Item 37) 36. The method of claim 35, wherein the step of providing the second composition is performed after providing the first composition and before (i) the first level of binding by the first composition is substantially undetectable in the cell or subject, or (ii) the first level of binding by the first composition is reduced by more than 50% in the cell or subject, or (iii) the first level of binding by the first composition is reduced by 25% to 75% in the cell or subject. (Item 38) 38. The method of claim 35 or 37, further comprising providing a third composition of cyclic polyribonucleotide to the cell or subject after the second composition, thereby maintaining binding of the target in the cell or subject at least the first level of binding, optionally wherein providing the third composition occurs after providing the second composition and before the second level of binding of the target in the cell or subject by the first and second compositions is substantially undetectable in the cell or subject. (Item 39) 39. The method of claim 38, wherein the step of providing the third composition occurs after providing the second composition and before the second level of binding by the first and second compositions in the cell or subject is reduced by more than 50%. (Item 40) 40. The method of any one of items 35 to 39, further comprising providing a fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition of cyclic polyribonucleotides. (Item 41) (i) the step of providing the second composition of cyclic polyribonucleotide occurs after the first composition and after the level of binding by the first composition is substantially undetectable; and / or (ii) the second composition is provided to the cell or subject at least 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, or 22 months after the level of binding by the first composition has become substantially undetectable; and / or (iii) The method of item 36 or 38, wherein the second composition is provided to the cell or subject 14 days after the first composition and no more than 90 days after the first composition. (Item 42) (i) the first level of binding is the highest level of binding one day after providing the first composition; and / or (ii) the first level of binding is 40%, 50%, 60%, 70%, 80%, or 90% of the highest level of binding 1 day after providing the first composition; and / or (iii) the second level of binding is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of binding 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, or 45 days after providing the second composition; and / or (iv) the third level of binding is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of binding 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing the third composition; and / or (v) for each subsequent composition provided after the first composition, the subsequent level of binding after each subsequent composition is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130% of the highest level of binding 1 day after providing the first composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 days after providing each subsequent composition; and / or (vi) the average level of binding after providing the second composition is at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% of the first level, and the average level of binding is measured from 1 day after providing the second composition until the day when the binding is substantially undetectable; and / or (vii) the average level of binding after providing each subsequent composition after the first composition is at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% of the first level, wherein the average level of binding is measured from 1 day after providing each subsequent composition until the day when the binding is substantially undetectable; and / or (viii) the first level of binding is maintained after providing the first composition of cyclic polyribonucleotide and the second composition for at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days after providing the first composition; and / or (ix) the second level of binding in the cell or subject after providing the second composition is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% greater than the first level of binding in the cell or subject after providing the first composition; (x) the second level of binding 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the second composition of cyclic polyribonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of binding after providing the first composition. . (Item 43) (i) the level of binding of the cyclic polyribonucleotide in the cell or subject after providing the first composition and the second composition is maintained for at least 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days; and / or (ii) the level of binding in the cell or subject after providing the first and second compositions of the cyclic polyribonucleotide is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% greater than the level of binding in the cell or subject after providing the first and second compositions of the linear equivalent of the cyclic polyribonucleotide; and / or (iii) the level of binding in the cell or subject after providing the first and second compositions of cyclic polyribonucleotide is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% greater than the level of binding in the cell or subject after providing the first and second compositions of the linear equivalent of the cyclic polyribonucleotide for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the second composition of cyclic polyribonucleotide. (Item 44) (i) the first level of binding is maintained after providing the first, second, and third compositions of cyclic polyribonucleotide for at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days after providing the first composition; and / or (ii) a third level of binding in the cell or subject after providing the third composition is at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% greater than the first level of binding after providing the first composition; (iii) The method of any one of items 38 to 40, or 42, wherein a third level of binding of the cyclic polyribonucleotide 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days after providing the third composition is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the first level of binding after providing the first composition. (Item 45) (i) the cyclic polyribonucleotide of the first composition and the cyclic polyribonucleotide of the second composition are the same; or (ii) The method according to any one of items 1 to 44, wherein the cyclic polyribonucleotides of the first composition and the cyclic polyribonucleotides of the second composition are different. (Item 46) (i) the first composition and the second composition contain approximately the same amount of the cyclic polyribonucleotide; or (ii) the first composition comprises a greater amount of the cyclic polyribonucleotide than the second composition; and / or (iii) the first composition comprises a greater amount of the cyclic polyribonucleotide than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition; and / or (iv) the amount of cyclic polyribonucleotide in the second composition varies by no more than 1%, 5%, 10%, 15%, 20%, or 25% of the amount of cyclic polyribonucleotide in the first composition; (v) the amount of cyclic polyribonucleotide in the second composition is 1%, 5%, 10%, 15%, 20%, or 25% less than the amount of cyclic polyribonucleotide in the first composition; without; and / or (vi) the first composition further comprises a pharmaceutically acceptable carrier or excipient; and / or (vii) the second composition further comprises a pharmaceutically acceptable carrier or excipient; and / or (viii) the third composition further comprises a pharmaceutically acceptable carrier or excipient; and / or (x) the cell is an animal cell (e.g., a mammalian cell, e.g., a human cell); and / or (xi) the cell is a plurality of cells in a subject; (xii) the first composition and / or the second composition is 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500ng / ml, 600ng / ml, 1µg / ml, 10µg / ml, 50µg / ml, 100µg / ml, 200g / ml, 300µg / ml, 400µg / ml, 500µg / ml, 600µg / ml, 700µg / ml, 800µg / ml, 900µg / ml, 1mg / ml, 1.5mg / ml, or 2mg / ml or less of linear polyribonucleotide molecules; (xiii) the first composition and / or the second composition comprises 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), or 99% (w / w) of circular polyribonucleotide molecules relative to the total ribonucleotide molecules in the first composition and / or the second composition; and / or (xiv) The method of any one of items 1 to 45, wherein 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), or 99% (w / w) of the total ribonucleotide molecules in the first composition and / or the second composition are circular polyribonucleotide molecules. (Item 47) 47. The method of any one of items 11 to 46, wherein the subject is an animal (e.g., a mammal). (Item 48) 48. The method according to any one of items 11 to 47, wherein the subject is a human. (Item 49) 23. The method of any one of items 1, 11, 12 and 22, wherein the protein is an antigen (e.g., a tumor antigen, a bacterial antigen, a viral antigen).

[0059] definition While the present invention will be described with respect to particular embodiments and with reference to certain drawings, the present invention The present invention is not limited thereto, but is limited only by the claims. Terms generally should be understood in their ordinary sense unless otherwise indicated.

[0060] As used herein, "circular RNA (circRNA)" or "circular polyribonucleotides" refers to RNA that is The terms "nucleotidyl polynucleotide" and "circular RNA" are synonyms. and structures that have no free ends (i.e., no free 3' and / or 5' ends). Polyribonucleotide molecules having a structure, e.g., cyclic or polyribonucleotides, which are linked together via covalent or non-covalent bonds. means a polyribonucleotide molecule that forms an endless structure.

[0061] As used herein, the term "aptamer sequence" refers to an aptamer sequence that is specific to a target molecule. Aptamers are non-natural or synthetic oligonucleotides that bind to Aptamers are typically identified by secondary structure rather than sequence homology. It binds to its target via

[0062] As used herein, the term "encryptogen" refers to the ability of immune cells to Reduce, avoid and / or prevent detection and / or Nucleic acid sequences or structures of polyribonucleotides that are useful for reducing the induction of an immune response against It is made of

[0063] As used herein, the term "expressed sequence" refers to the expression of a product, e.g., a peptide. or a nucleic acid sequence encoding a peptide or polypeptide, or a regulatory nucleic acid. An exemplary expression sequence encoding a peptide includes multiple nucleotide triplets, which are Each code for an amino acid is called a "codon."

[0064] As used herein, the term "exogenous" refers to a biological molecule (such as a circular RNA) ), when used in connection with a biomolecule, refers to a biological molecule that has been inserted by human intervention into a host genome, cell, or organism. means introduced into cells, e.g., by recombinant DNA techniques and / or by introducing biomolecules into cells. Circular RNA added to an existing genome, cell, tissue, or subject using methods for incorporating , existing nucleic acid sequences, cells, tissues or subjects, and nucleic acid sequences, cells, tissues carrying biomolecules or exogenous to any offspring of the subject.

[0065] As used herein, the term "immunity protein binding site" refers to an immune protein. In one embodiment, the immunoglobulin-binding protein is a nucleotide sequence that binds to the immunoglobulin. The site serves to mask the cyclic polyribonucleotide as being exogenous, e.g. For example, the immune protein binding site is a site where the cyclic polyribonucleotide is bound by the immune protein. It is bound by a protein (e.g., a competitive inhibitor) that prevents it from being recognized and bound. This reduces or avoids the immune response to cyclic polyribonucleotides.

[0066] As used herein, the term "immunoprotein" refers to, for example, an immunogen, For example, any protein or protein associated with an immune response to cyclic polyribonucleotides, etc. is a peptide. Non-limiting examples of immune proteins include T cell receptors (TCRs), Antibodies (immunoglobulins), major histocompatibility complex (MHC) proteins, complement proteins , and RNA-binding proteins.

[0067] As used herein, the term "modified ribonucleotide" refers to a naturally occurring unmodified ribonucleotide. Nucleotides adenosine (A), uridine (U), guanine (G), cytidine (C), etc. any ribonucleotide having one or more chemical modifications relative to the chemical composition of unmodified natural ribonucleotides, In certain embodiments, modified ribonucleotides are Chemical modifications of ribonucleotides can involve modifications to the sugar, nucleobase, or internucleoside linkages of ribonucleotides. to one or more functional groups of the bond (e.g., phosphate / phosphodiester It is a modification to the ester bond / phosphodiester backbone.

[0068] As used herein, the term "quasi-helical structure" refers to a circular polyribonucleic acid The higher-order structure of a cyclic polyribonucleotide, wherein at least a portion of the cyclic polyribonucleotide is a helical structure. It folds into a structure.

[0069] As used herein, the phrase "quasi-double-stranded secondary structure" refers to a circular polyribonucleotide. A higher-order structure of nucleotides, wherein at least a portion of the cyclic polyribonucleotide is Forms an internal duplex.

[0070] As used herein, the term "regulatory element" refers to a circular polyribonucleic acid These are portions such as nucleic acid sequences that regulate the expression of an expression sequence within the gene.

[0071] As used herein, the term "repetitive nucleotide sequence" refers to a sequence of DNA or or repetitive nucleic acid sequences within stretches of RNA or throughout the genome. In some embodiments, the repetitive nucleotide sequence comprises a poly-CA or poly-TG (UG) sequence. In the form, the repeated nucleotide sequence is repeated in the Alu family of introns. Contains arrays.

[0072] As used herein, the term "replication element" refers to a molecule that is useful for replication or is a sequence and / or motif that initiates transcription of a circular polyribonucleotide.

[0073] As used herein, the term "stagger element" refers to a staggered element that is present on the ribosome during translation. In one embodiment, the staggered portion is a nucleotide sequence that induces pausing. The element consists of a non-conserved sequence of amino acids with a strong α-helical propensity, followed by the consensus sequence -D (V / I)ExNPG P, where x=any amino acid. wherein the stagger element is glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or The chemical moieties may include any combination thereof.

[0074] As used herein, the term "substantially resistant" refers to a condition in which the At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, It may refer to having 95%, 96%, 97%, 98% or 99% resistance.

[0075] As used herein, the term "stoichiometric translation" refers to the synthesis of a circular polyribonucleic acid. This means substantially equal production of expression products translated from the nucleic acids. For example, two expressions In a cyclic polyribonucleotide with a sequence, the stoichiometric translation of the cyclic polyribonucleotide This means that the expression products of the two expression sequences can be in substantially equal amounts, e.g., The difference in quantity (e.g., molar difference) between the present sequences is about 0, or 1%, 2%, 3%, 4%, 5%, This means it can be less than 6%, 7%, 8%, 9%, 10%, 15%, or 20% obtain.

[0076] As used herein, the term "translation initiation sequence" refers to the sequence of a circular polyribonucleic acid sequence. It is a nucleic acid sequence that initiates translation of an expression sequence in a nucleotide.

[0077] As used herein, the term "termination element" refers to a terminal element of a circular polyribonucleic acid. A portion of a gene, such as a nucleic acid sequence, that stops the translation of an expression sequence in a gene.

[0078] As used herein, the term "translation efficiency" refers to the rate at which ribonucleotide transcript production In one embodiment, the expression level refers to the rate or amount of protein or peptide production from a given organism. Translation efficiency can be measured, for example, using a given translation system, e.g., an in vitro system such as rabbit reticulocyte lysate. in a translation system, or in an in vivo translation system, such as a eukaryotic or prokaryotic cell, e.g. For example, in a given period of time, the number of transcripts encoding a protein or peptide is It can be expressed as the amount of protein or peptide produced.

[0079] As used herein, the term "circularization efficiency" refers to the efficiency of the resulting circular polyribonucleotides. It is a measure of a nucleotide relative to its starting material.

[0080] As used herein, the term "immunogenic" refers to the ability to induce an immune response to a substance. In some embodiments, the immune system of an organism or certain immune cells may be induced to When exposed to an immunogenic agent, an immune response can be induced. In certain embodiments, the absence or lack of an immune response above a detectable threshold to the disease. When the immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic substance, an immune response is initiated. In certain embodiments, the non-immunogenic cyclic polynucleotides provided herein are not detectable. Ribonucleotides are those that produce an immunogenicity above a predetermined threshold as measured by an immunogenicity assay. For example, immunogenicity assays are used to detect the presence of cyclic polyribonucleotides. When measuring the innate immune response (e.g., measuring inflammatory markers) of the The non-immunogenic polyribonucleotides used in this study inhibit the innate immune response at levels below a certain threshold. The predetermined threshold may be, for example, the number of antibodies produced by an innate immune response relative to a control. The level of the markers made is 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x 10 times or less.

[0081] As used herein, the term "substantially undetectable" refers to the relative detection Techniques (e.g., chromatography (columns, filter paper, gels, HPLC, UHPLC, IC) , SEC, etc.), electrophoresis (UREA PAGE, tip-based, polyacrylamide gel electrophoresis, etc.), detection techniques, such as RNA, capillary, c-IEF, and fluorescence-based detection techniques cyclic polyribonucleotides or cyclic polyribonucleotides at levels lower than those which can be detected It may refer to the level of a protein expressed.

[0082] As used herein, the term "linear equivalent" refers to a circular polyribonucleic acid. The nucleotide sequence is the same as or similar to the nucleotide sequence of ... , 80%, 75%, or any percentage of sequence similarity therebetween), and Polyribonucleotide molecules (and fragments thereof) with two free ends (i.e. , the non-cyclized form of a circularized polyribonucleotide (and fragments thereof). In embodiments, the linear equivalent (e.g., pre-circularized form) is a cyclic polyribonucleotide. A nucleotide sequence that is the same or similar to (e.g., 100%, 95%, 90%, 85%, 8 0%, 75%, or any percentage sequence similarity therebetween) and the same or similar A polyribonucleotide molecule (and its fragments) having the nucleic acid modification and having two free ends. fragments) (i.e., the non-cyclized forms of the circularized polyribonucleotides (and their fragments) In some embodiments, the linear equivalent is a cyclic polyribonucleotide. The same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%) %, 75%, or any percentage of sequence similarity therebetween) and different nucleic acid modifications or a polyribonucleotide molecule having no nucleic acid modification and having two free ends. Polyribonucleotides (and fragments thereof) (i.e., non-cyclized forms of cyclized polyribonucleotides ( and fragments thereof). In some embodiments, the linear equivalent is a polyribonucleic acid. A fragment of a nucleotide molecule is a linear equivalent that is shorter than the linear equivalent polyribonucleotide molecule. A linear equivalent is any portion of a polyribonucleotide molecule. In certain embodiments, a linear equivalent In one embodiment, the linear equivalent further comprises a 5' cap. In one embodiment, the linear equivalent further comprises a 3'UTR. In some embodiments, the linear equivalent further comprises a 5'UTR.

[0083] As used herein, the term "conjugated moiety" refers to a compound used in a conjugation method. refers to a modified nucleotide that contains a functional group for the purpose of

[0084] As used herein, the term "carrier" refers to a cyclic polyribonucleotide. by covalent modification, partially or completely by encapsulating agents, or a combination thereof facilitates transport or delivery of compositions (e.g., cyclic polyribonucleotides) into cells Carriers refer to compounds, compositions, reagents, or molecules. Non-limiting examples of carriers include carbohydrate carriers. bodies (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles ( For example, nanoparticles encapsulating or covalently attached to cyclic polyribonucleotides. ), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, proteins Protein carriers (e.g., proteins covalently bound to cyclic polyribonucleotides), or cationic Examples include ionic carriers (e.g., cationic lipopolymers or transfection reagents). It can be obtained.

[0085] As used herein, "naked delivery" refers to The term "carrier-free" refers to a compound that is carrier-free and has covalent modifications to moieties that aid in delivery to cells. Naked delivery formulations refer to formulations for delivery to cells that do not contain any transfection agent. an injection reagent, a cationic carrier, a carbohydrate carrier, a nanoparticle carrier, or a protein carrier For example, naked delivery formulations of cyclic polyribonucleotides do not contain covalent modifications. The formulation contains a cyclic polyribonucleotide that does not contain a carrier.

[0086] The term "diluent" refers to a composition described herein (e.g., a circular polyribonucleic acid The term "dilution" refers to a vehicle containing an inert solvent in which a composition containing an oxalate can be diluted or dissolved. The diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof. The diluent may be a liquid or a solid. Non-limiting examples of liquid diluents include water or other Solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, carbonate Chill, Ethyl Acetate, Benzyl Alcohol, Benzyl Benzoate, Propylene Glycol, 1 ,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, Sorghum oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran Fatty acid esters of hydroxyfurfuryl alcohol, polyethylene glycol and sorbitan, and and 1,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate. Sodium, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, Calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline Cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, Examples include dry starch, corn starch, or powdered sugar.

[0087] As used herein, the terms "response" or "level of response" refer to a response to a stimulus. Any measurable change or any level of measurable change that results from exposure, e.g. A measurable change can be a phenotypic shift or change (e.g., cellular phenotype, physical phenotype, etc.). phenotype) or any characteristic that provides information that a stimulus is working, e.g., Changes in cell morphology, increased or decreased production of certain cell types, increased or decreased muscle mass after exposure to stimuli, Further examples include proteins containing stimulatory binding sites (e.g., cyclic polynucleotides, erythropoietin expressed from cyclic polyribonucleotides) or cyclic polyribonucleotides the response or level of response is determined by measuring the level of a protein or circular polyribonucleotide containing the binding site in the subject. A measurable shift in phenotype after exposure to nucleotides (e.g., reticulocyte count in a subject) (increased production or level of

[0088] Incorporation by Reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. The patent or patent application is specifically and individually indicated to be incorporated by reference. and the same is incorporated herein by reference to the same extent.

[0089] The following detailed description of the embodiments of the present invention is better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the embodiments illustrated herein are shown in the drawings. However, the present invention does not depend on the precise arrangements and instrumentalities of the embodiments shown in the drawings. It should be understood that this is not limiting. [Brief explanation of the drawings]

[0090] [Figure 1] After injection into mice, circular RNA was detected at higher levels than linear RNA in the liver of mice at 3, 4, and 7 days after injection. [Figure 2] Figures 2A and 2B show that after injection of circular or linear RNA expressing Gaussia luciferase into mice, Gaussia luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23 days after administration of circular RNA, whereas the activity was detected in plasma only at 1 and 2 days after administration of modified linear RNA. [Figure 3] Following injection of RNA, circular RNA, but not linear RNA, was detected in the liver and spleen at 16 days after administration of RNA. [Figure 4] Figure 1 shows that after RNA injection, linear RNA, but not circular RNA, was immunogenic as assessed by RIG-I, MDA-5, IFN-B, and OAS. [Figure 5] 1 shows experimental data demonstrating increased persistence of Gaussia luciferase expression in mice following re-administration of cyclic polyribonucleotides ("endless") compared to their linear polyribonucleotide equivalents ("linear"). [Figure 6]1 shows experimental data demonstrating increased persistence of Gaussia luciferase expression in mice following staggered administration of cyclic polyribonucleotides ("endless triple dose") compared to linear polyribonucleotide equivalents ("linear triple dose"), or a single dose of cyclic polyribonucleotides ("endless"), or a single dose of linear polyribonucleotide equivalents ("linear"); [Figure 7] 1 shows experimental data demonstrating increased persistence of Gaussia luciferase expression in mice following a single dose of circular polyribonucleotide ("endless RNA") compared to a single dose of linear polyribonucleotide equivalent ("linear RNA"), staggered administration of linear polyribonucleotide equivalent ("three doses of linear RNA") compared to a single dose ("linear RNA"), or staggered administration of circular polyribonucleotide ("three doses of endless RNA") compared to a single dose ("endless RNA"). [Figure 8] Intravenously administered cyclic polyribonucleotides, with (TransIT) and without (unformulated) carrier, demonstrated long-term protein expression in vivo, with plasma protein activity levels at multiple days post-infusion. [Figure 9] This shows that intramuscularly administered cyclic polyribonucleotides, without a carrier, expressed protein in vivo for an extended period of time, with levels of protein activity in plasma at multiple days post-infusion. [Figure 10] This shows that intravenously administered cyclic polyribonucleotides express protein in vivo for an extended period of time, with levels of protein activity in plasma at multiple days post-infusion, and can be readministered at least five times. [Figure 11] We show that cyclic polyribonucleotides expressed proteins in vivo for extended periods of time, with increased levels of protein activity in plasma after multiple serial infusions. [Figure 12]Increased numbers of reticulocytes were detected in whole blood at 3, 5, 7, 14, 21, and 28 days after administration of the first dose of unformulated RNA, indicating that reticulocyte counts subsequently returned to the normal range of 3-5% in the mouse population. [Figure 13] Increased numbers of reticulocytes were detected in whole blood at 3, 5, 7, 14, 21, and 28 days after administration of the first dose of TransIT-formulated RNA, indicating that reticulocyte counts subsequently returned to the normal range of 3-5% in the mouse population. [Figure 14] 1 shows that an increase in reticulocyte counts was detected for circular RNA administration and mRNA administration when unformulated compared to vehicle-only controls. [Figure 15] 1 shows that an increase in reticulocyte counts was detected for circular RNA and mRNA administration when formulated with TransIT compared to vehicle-only controls. [Figure 16] FIG. 1 shows a schematic diagram of an exemplary in vitro generation process of circular RNA containing a start codon, an ORF (open reading frame) encoding GFP, a stagger element (2A), an encryptogen, and an IRES (internal ribosome entry site). [Figure 17] FIG. 1 shows a schematic diagram of an exemplary in vivo generation process of circular RNA. [Figure 18] An exemplary circular RNA design is shown, including a start codon, an ORF encoding GFP, a stagger element (2A), and an encryptogen. [Figure 19] 19A and 19B are schematic diagrams showing the in vivo stoichiometric protein expression of two different circular RNAs. [Figure 20] 1 is a graph showing qRT-PCR analysis of immune-related genes from 293T cells transfected with circular or linear RNA. [Figure 21] FIG. 1 is a schematic diagram showing in vivo protein expression in a mouse model from exemplary circular RNAs. [Figure 22] FIG. 1 is a schematic diagram showing the in vivo biodistribution of exemplary circular RNAs in a mouse model. [Figure 23] FIG. 1 is a schematic diagram showing in vivo protein expression in a mouse model from an exemplary circular RNA with an encryptogen (intron). [Figure 24] 1 is a denaturing PAGE gel image showing an exemplary circular RNA after an exemplary purification process. [Figure 25] 1 is a Western blot image showing expression of Flag protein (approximately 15 kDa) by an exemplary circular RNA lacking an IRES, cap, 5' and 3' UTRs. DETAILED DESCRIPTION OF THE INVENTION

[0091] The present invention relates generally to methods for administering cyclic polyribonucleotides. The indicated methods of administration generally involve administering at least two compositions of cyclic polyribonucleotides. After providing the protein, the level of the protein in the cell is expressed or the level of the cyclic polyribonucleotide is wherein the cyclic polyribonucleotide encodes a protein. The methods of administration disclosed herein also generally involve the administration of cyclic polyribonucleotides. After providing at least two compositions, the compounds bind to a target within the cell, wherein the ring In some embodiments, the circular polyribonucleotide encodes a protein. Ribonucleotides are exogenous synthetic cyclic polyribonucleotides.

[0092] In one aspect, the present invention provides a method for expressing a protein in a cell, comprising: providing to the cell a first composition comprising a cyclic polyribonucleotide encoding a gene; wherein the cell expresses a first level of the protein; and providing a second composition comprising a nucleotide to the cells, wherein the cells are capable of expressing the second nucleotide of the protein. wherein the second level is at least as great as the first level; This allows the expression of the protein in the cell to be at least at the first level of the protein. In one embodiment, the method further comprises the step of expressing a protein in a cell. The method comprises administering to a cell a first composition comprising a cyclic polyribonucleotide encoding a protein. providing a cell to express a first level of the protein; providing a second composition comprising a polyribonucleotide to the cells, wherein the cells: expressing a second level of the protein, the second level varying by no more than 20% of the first level; thereby increasing the expression of the protein in the cell by at least In one embodiment, the method comprises maintaining the circular polyribonucleic acid (CPN) level in the cell at a first level of 0.05 to 0.15. The method for producing a cyclic polyribonucleotide comprises providing a first composition to a cell, the first composition comprising a cyclic polyribonucleotide. a step of inducing a first cyclic polyribonucleotide into the cells after providing the first composition; and providing the cells with a second composition of cyclic polyribonucleotides. wherein the cells comprise a second level of cyclic polyribonucleotides and the second level of ribonucleotides is at least as great as the first level; This maintains cyclic polyribonucleotides in the cells at at least a first level. In one embodiment, the method for producing a cyclic polyribonucleotide in a cell comprises the step of: providing a first composition comprising a cyclic polyribonucleotide to a cell, After providing the first composition, the cell is then subjected to a step of cleaving the first composition containing a first level of cyclic polyribonucleotide. and providing a second composition of cyclic polyribonucleotides to the cells, the cell comprises a second level of cyclic polyribonucleotide, and the first level of cyclic polyribonucleotide the level of the second composition changes by no more than 20% of the level of the first composition after providing the second composition. thereby increasing the amount of cyclic polyribonucleotides in the cells to at least a first level. In some embodiments, the method includes maintaining the cells in a circumferentially oriented state. The method comprises providing a cell with a first composition comprising a cyclic polyribonucleotide. and wherein the cells contain a first level of cyclic polyribonucleotides after providing the first composition. and providing a second composition of cyclic polyribonucleotides to the cells. and wherein the cell comprises a second level of the circular polyribonucleotide, The second level of the compound changes by no more than 20% of the first level after providing the second composition. thereby converting the cyclic polyribonucleotide in the cell into at least a first In one embodiment, the step of providing the second composition comprises: After providing the first composition and determining a first level of a protein expressed by the first composition, The assay is performed before the test is substantially undetectable in the cells.

[0093] In one embodiment, the present invention provides a first composition of linear equivalents of cyclic polyribonucleotides. and the level of the cyclic polynucleotide compared to the level of the protein in the cells after providing the second composition. After providing the cells with the first and second compositions of ribonucleotides, the target gene in the cells is expressed as A method for expressing the level of a protein, comprising: providing a first composition of nucleotides to the cells, wherein the cells are capable of synthesizing the circular polyribonucleic acid; providing a first composition of peptides, and then measuring the protein level; and providing a second composition of cyclic polyribonucleotides to the cell after providing the second composition of cyclic polyribonucleotides; the cells are provided with at least the second composition of cyclic polyribonucleotides, a step of forming a linear equivalent of a cyclic polyribonucleotide, the step of and comparing the levels of the protein in the cells after providing the first composition and the second composition. After providing the first and second compositions of cyclic polyribonucleotides, In one embodiment, the method comprises maintaining the level of expression of a protein comprising: After providing a first composition and a second composition of linear equivalents of cyclic polyribonucleotides, a first composition of cyclic polyribonucleotide relative to the level of a protein in the cell; and a method for expressing the level of a protein in a cell after providing the cell with the second composition. providing a first composition of cyclic polyribonucleotides encoding a protein to the cell; a step of inducing a cell to undergo transcription after providing the cell with a first composition of cyclic polyribonucleotides; and after providing the first composition to the cells, providing a second composition of cyclic polyribonucleotides, and a level of the protein that changes by no more than 20% of the level after providing the second composition. , whereby a first composition of linear equivalents of a cyclic polyribonucleotide and a second composition of linear equivalents of a cyclic polyribonucleotide are obtained. the level of the circular polyribonucleic acid protein in the cells after providing the composition of After providing the first and second compositions of protease, the level of the protein in the cells is In one embodiment, the linear synthase of the circular polyribonucleotide is maintained. Circular polyribonucleoside production in cells after providing a first composition and a second composition of equal quality. a first composition of cyclic polyribonucleotides and a second composition of cyclic polyribonucleotides, After providing the two compositions to cells, a certain level of cyclic polyribonucleotides is produced in the cells. The method for producing the cyclic polyribonucleotides comprises providing a first composition of cyclic polyribonucleotides to the cells. and wherein the cells are provided with the first composition and then subjected to a step of increasing the level of cyclic polyribonucleotides. and providing a second composition of cyclic polyribonucleotides to the cells, The cells contain at least the level of cyclic polyribonucleotide after providing the second composition. thereby producing a first composition of linear equivalents of cyclic polyribonucleotides and The level of circular polyribonucleic acid in cells after administration of the two compositions was compared to the level of linear equivalents. Circular polyribonucleic acid in cells after providing a first composition and a second composition of nucleotides. In one embodiment, the method comprises maintaining the level of a circular polyribonucleic acid. The circular polynucleotides in the cells after providing a first composition of linear equivalents of the nucleotides and a second composition of linear equivalents of the nucleotides. The first set of cyclic polyribonucleotides is compared to the level of their linear equivalents. After providing the cells with the composition and the second composition, a certain level of circular polyribonucleic acid is produced in the cells. The method for producing a cyclic polyribonucleotide comprises providing a first composition of cyclic polyribonucleotides to a cell. The method further comprises: providing the cells with the first composition, and then increasing the level of cyclic polyribonucleotides in the cells. and providing a second composition of cyclic polyribonucleotides to the cells. Thus, the cells have a 20% level of cyclic polyribonucleotides after providing the first composition. the level of the protein after providing the second composition varies only by: providing a first composition and a second composition of linear equivalents of cyclic polyribonucleotides, The first level of cyclic polyribonucleotides compared to the level of their linear counterparts in cells after exposure to The level of cyclic polyribonucleotides in cells after providing the first composition and the second composition. In one embodiment, the second cyclic polyribonucleotide is maintained at 100° C. The step of providing the composition includes providing the first composition and the cell expressed by the first composition. This is done after the level of the protein in the test sample becomes substantially undetectable. The cyclic polyribonucleotides used in the described methods contain one or more expression sequences. In certain embodiments, at least one of the expressed sequences encodes a protein. The protein can be an intracellular protein, a membrane protein, or a secreted protein. The protein can be a therapeutic protein. In certain embodiments, the therapeutic protein may have activity, for example, antioxidant activity, binding, cargo receptor activity, catalytic activity, Molecular carrier activity, molecular function regulator, molecular transducer activity, nutrient storage activity, protein It has tag, structural molecule activity, toxin activity, transcriptional regulatory activity, translational regulatory activity, or transport activity.

[0094] The methods described herein may be therapeutic or veterinary methods for treating a subject The methods described herein can be used to treat diseases in multiple cells. In certain embodiments, the methods described herein involve the production of non-functional, less functional, or non-functional Used to treat diseases resulting from a protein or gene product that is fully expressed In certain embodiments, the methods described herein are directed to the treatment of genetic disorders (e.g., mutations, , substitution, deletion, expansion, or recombination), cancer, neurodegenerative diseases , cardiovascular disease, pulmonary disease, kidney disease, liver disease, genetic disease, vascular disease, eye disease, musculoskeletal disease, Lymphatic system disorders, hearing and inner ear disorders, metabolic disorders, inflammatory disorders, autoimmune disorders, or immunological disorders It is used to treat infections.

[0095] Method of administration administering to the cells at least two doses or compositions of cyclic polyribonucleotides; To produce a certain level of cyclic polyribonucleotides or to increase a certain level of protein in a cell Methods of administration of cyclic polyribonucleotides to express the desired properties are disclosed herein. After providing (e.g., administering) at least two doses or compositions to a subject, a certain level of or in a subject (e.g., a mammal, e.g., a human). Methods of administration to express levels of the protein in the composition are disclosed herein. The composition may comprise a cyclic polyribonucleotide encoding the composition protein. The method of administration is generally long-term administration. The re-administration of the cyclic polyribonucleotide composition may be in two or more doses. The method may involve staggered administration of the compositions at short time intervals. The composition further comprises a pharmaceutically acceptable carrier or excipient. Proteins from can be expressed intracellularly.

[0096] In one embodiment, the method comprises administering at least a first composition and a second composition to a cell. or providing (e.g., administering) the compound to a subject (e.g., a mammal, e.g., a human). In one embodiment, the method includes adding a third composition, a fourth composition, a fifth composition, a third composition, a fourth composition, a fifth composition, a fifth composition, a sixth ... Composition 6, composition 7, composition 8, composition 9, composition 10, or any other In some embodiments, the method further comprises providing (e.g., administering) the above. The composition is provided for the duration of the cell's lifespan. The product is provided (eg, administered) while the cell or subject benefits from the composition. In certain embodiments, multiple compositions are provided in a staggered administration regimen (e.g., administration provided), where any composition is administered after the previous composition has been provided (e.g., administered). the level of protein or cyclic polyribonucleotide from the previous composition in the plurality of substantially undetectable in a cell or subject (e.g., mammal) prior to being provided ( For example, a first composition and a second composition may be administered in a staggered regimen. To achieve this, after the first composition is provided (e.g., administered) and The levels of protein or cyclic polyribonucleotide from the composition are measured in a cell or subject (e.g., The second composition is provided before the antibody is substantially undetectable in a mammal (e.g., a mammal). In some embodiments, multiple compositions are provided in a re-administration regimen. wherein a plurality of proteins or cyclic polyribonucleotides from the preceding composition are after the level of After the previous composition has been provided (e.g., administered), any composition may be provided (e.g., administered). For example, a re-administration regimen may be used to generate a first composition and a second composition. To achieve this, the first composition is provided (e.g., administered) and then administered to a cell or subject. The level of protein or cyclic polyribonucleotide from the first composition in the cell or After the antibody is substantially undetectable in the subject, a second composition is provided (e.g., administered (will be done).

[0097] In one embodiment, the first composition in the staggered or re-administration regimen comprises: In one embodiment, the staggered regimen includes a first amount of a cyclic polyribonucleotide. The second composition in the re-administration regimen comprises a second amount of cyclic polyribonucleotide. In one embodiment, the third composition in the staggered or re-administration regimen, Composition 4, Composition 5, Composition 6, Composition 7, Composition 8, Composition 9, Ten or more compositions may comprise a third, fourth, fifth, sixth, or In some embodiments, the cyclic poly(amino acid) may comprise a 7th, 8th, 9th, 10th, or more amount. The second amount of cyclic polyribonucleotide is the same as the first amount of cyclic polyribonucleotide. In an embodiment, the third amount of cyclic polyribonucleotide is In one embodiment, the fourth, fourth, and sixth amounts of the cyclic polyribonucleotide are the same as the first amount of the cyclic polyribonucleotide. 5, 6, 7, 8, 9, 10, or more cyclic polyribonucleotides In one embodiment, the second amount of cyclic polyribonucleotide is the same as the first amount of cyclic polyribonucleotide. is less than the first amount of cyclic polyribonucleotide. The third amount of ribonucleotides is less than the first amount of cyclic polyribonucleotides. In an embodiment, the fourth, fifth, sixth, seventh, eighth, ninth, The tenth or more amount is less than the first amount of cyclic polyribonucleotide. In some embodiments, the second amount of cyclic polyribonucleotide is In one embodiment, the third amount of cyclic polyribonucleotide is greater than 1. In one embodiment, the first amount of polyribonucleotide is greater than the first amount of circular polyribonucleotide. The fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amounts of the nucleotide are cyclic poly(lysine). In some embodiments, the amount of cyclic polynucleotides in the second composition is greater than the first amount of cyclic polynucleotides in the second composition. The amount of cyclic polyribonucleotides in the first composition is 1%, 5%, 1%, or 2% of the amount of cyclic polyribonucleotides in the first composition. The second set varies by no more than 0%, 15%, 20%, or 25%. the amount of cyclic polyribonucleotide in the first composition is greater than the amount of cyclic polyribonucleotide in the second composition In some embodiments, the ion exchange rate is 1%, 5%, 10%, 15%, 20%, or 25% less than the ion exchange rate. and the amount of cyclic polyribonucleotide in the second composition is greater than the amount of cyclic polyribonucleotide in the first composition. In one embodiment, the amount of the ring of the second composition is 0.1 to 1000 times greater than the amount of the hydroxybenzoate. The amount of cyclic polyribonucleotides in the first composition is 0.5 to 10 times less than the amount of cyclic polyribonucleotides in the first composition. 1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold more. The circular polyribonucleic acid of the subsequent composition (e.g., the composition administered after the first composition) The amount of cyclic polyribonucleotide in the first composition is 0.1 times, 1 time, 5 times, 1 time, or 1 time greater than the amount of cyclic polyribonucleotide in the first composition. In some embodiments, the cyclic polysaccharide of the second composition is 0, 100, or 1000 times more. The amount of ribonucleotides is 0.1 to 1 times the amount of cyclic polyribonucleotides in the first composition. In one embodiment, the cyclic polyribonucleotides of the second composition are 1000 times less. The amount is 0.1 times, 1 time, 5 times, or 10 times the amount of cyclic polyribonucleotide in the first composition. , 100-fold, or 1000-fold less. In some embodiments, the subsequent composition (e.g., The amount of cyclic polyribonucleotide in the composition administered after the first composition is 0.1 times, 1 times, 5 times, 10 times, 100 times, or 1 times the amount of cyclic polyribonucleotide In some embodiments, the amount of a subsequent composition (e.g., a quantity of circular polyribonucleotides) The amount of cyclic polyribonucleotide in the first composition of nucleotides (after the first composition of nucleotides) is The amount of cyclic polyribonucleotide is 0.1 to 1000 times more or less than the amount of cyclic polyribonucleotide. In some embodiments, a subsequent composition (e.g., a first composition of a quantity of cyclic polyribonucleotides) the amount of cyclic polyribonucleotide in the first composition (after 0.1, 1, 5, 10, 100, or 1000 times more or less than the amount. For example, the first composition contains 1x cyclic polyribonucleotide and the second composition contains 1x cyclic polyribonucleotide. The third composition contains 5 times as much cyclic polyribonucleotide as the first composition, and the third composition contains 5 times as much cyclic polyribonucleotide as the first composition. In one embodiment, the composition contains 0.2 times as much cyclic polyribonucleotide as the composition. The second composition contains at least 5 times the amount of cyclic polyribonucleotides as compared to the amount of cyclic polyribonucleotides in the first composition. The polyribonucleotides include:

[0098] In one embodiment, the first composition contains a greater amount of circular polyribonucleic acid than the second composition. In one embodiment, the first composition comprises a third, fourth, fifth, sixth, or The seventh, eighth, ninth, or tenth composition contains a greater amount of cyclic polyribonucleotide.

[0099] In certain embodiments, the first composition further comprises a pharmaceutically acceptable carrier or excipient. In certain embodiments, the second composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the third composition, the fourth composition, the fifth composition, the sixth composition, Composition 7, composition 8, composition 9, composition 10, or more , further comprising a pharmaceutically acceptable carrier or excipient.

[0100] In certain embodiments, the first composition further comprises a pharmaceutically acceptable excipient, and optionally In one embodiment, the second composition does not contain any pharmaceutically acceptable carrier. In one embodiment, the third composition further comprises a vehicle and does not comprise any carrier. Composition 4, Composition 5, Composition 6, Composition 7, Composition 8, Composition 9 The tenth, tenth, or more compositions further comprise a pharmaceutically acceptable excipient, and any It does not include a carrier.

[0101] In some embodiments, the compositions described herein (e.g., a first composition, a second composition, a The composition, the third composition, etc.) is delivered to a subject (e.g., a mammal). The compositions described herein (e.g., the first composition, the second composition, the third composition, etc.) The method of delivery involves administering to a subject in need thereof a composition described herein (e.g., a first a first composition, a second composition, a third composition, etc.) to a subject in need thereof parenterally. In another example, the composition (e.g., the first composition, the second composition, the third composition) is added to the The method of delivering a composition of formula (e.g., ... In some embodiments, the compositions described herein (e.g., a first composition, a second composition, In some embodiments, the composition described herein (e.g., the first composition, the second composition, the third composition, etc.) comprises a carrier. The compositions (e.g., first composition, second composition, third composition, etc.) may contain a diluent. In certain embodiments, parenteral administration is intravenous, intramuscular, or , intraocularly, or topically.

[0102] In some embodiments, the compositions (e.g., a first composition, a second composition, a third composition, In some embodiments, the compositions (e.g., the first composition, the second composition, etc.) are administered orally. In one embodiment, the compositions (composition 2, composition 3, etc.) are administered intranasally. The compositions (e.g., the first composition, the second composition, the third composition, etc.) are administered by inhalation. In some embodiments, the compositions (e.g., a first composition, a second composition, a third composition, In some embodiments, the composition is administered intraocularly. In some embodiments, the compositions (e.g., a first composition, a second composition, a third composition, In some embodiments, the composition (e.g., the first composition) is administered rectally. In some embodiments, the first composition, the second composition, the third composition, etc., is administered by injection. In this case, the compositions (e.g., the first composition, the second composition, the third composition, etc.) are administered by infusion. Administration can be systemic or local. In certain embodiments, The compositions (e.g., the first composition, the second composition, the third composition, etc.) are administered parenterally. In some embodiments, the compositions (e.g., a first composition, a second composition, a third composition, (compositions, etc.) can be administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, or intralymphatically. The drug is administered intralymphatically, subcutaneously, or intramuscularly. In the embodiment, the composition (e.g., the first composition, the second composition, the third composition, etc.) comprises: Administration is by intraocular, intracochlear (inner ear) or intratracheal administration. In some embodiments, any of the methods of delivery described herein are carried out using a carrier. In some embodiments, any of the delivery methods described herein are performed without a carrier. .

[0103] The cyclic polyribonucleotide compositions described herein induce a response in a subject. In certain embodiments, the method of inducing a response in a subject comprises: To induce the In certain embodiments, the method further comprises providing (e.g., administering) a composition comprising the nucleotide. In the method for inducing a response, a circular polyribonucleic acid encoding erythropoietin is providing (e.g., administering) a cyclic polynucleotide to a subject, wherein the cyclic polynucleotide in the subject Expression of erythropoietin from ribonucleotides induces the production of reticulocytes in the subject. In one embodiment, the method for inducing a response level in a subject comprises: (a) inducing a response level in a subject; and a first composition comprising a cyclic polyribonucleotide as described herein that induces and 14 to 90 days after step (a), administering (e.g., administering) a compound as described herein. and providing (e.g., administering) to the subject a second composition comprising the cyclic polyribonucleotide. whereby a response level in the subject after providing the first composition and the second composition In one embodiment, the cyclic polyribonucleotide composition comprises: A therapeutic protein encoding a therapeutic agent for inducing a response or level of response in a subject after administration. In one embodiment, the cyclic polyribonucleotide encoding erythropoietin The composition is provided to a subject to induce the production of reticulocytes in the subject. In one embodiment, a method for inducing reticulocytes in a subject includes: (a) administering erythropoietin to a subject; A first composition comprising a cyclic polyribonucleotide to be administered to a subject is provided (e.g., administered) to the subject. and (b) administering to the subject a gene encoding erythropoietin 6 hours to 90 days after step (a). and providing (e.g., administering) to the subject a second composition comprising a cyclic polyribonucleotide. In some embodiments, the method includes administering the method to a subject, thereby inducing reticulocyte production in the subject. In the method, the second composition is provided (e.g., 6 hours to 30 days after step (a)). In one embodiment, the method includes a step of administering step (a) for 14 to 90 days. The method further comprises providing (e.g., administering) a second composition days later. to provide a circular polyribonucleotide containing a binding site or encoding a protein. The response level from the linear equivalent of polyribonucleotides is higher than the response level from the linear equivalent of polyribonucleotides. In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and and comparing the response level in the subject after providing the composition of claim 2 with the circular polyribonucleic acid. and inducing a response level in the subject after providing the subject with the first composition and the second composition of the method. The method comprises the step of: providing (e.g., administering) a first composition to a subject, wherein the subject is a step of detecting a response level after the first composition of ribonucleotides is provided; and A second composition of cyclic polyribonucleotides encoding the protein is administered to the subject after the first composition. The step of providing (e.g., administering) a method for treating a subject, comprising administering to the subject at least a portion of a circular polyribonucleic acid molecule comprising administering to the subject a method for treating a subject ... a step of providing a second composition of the cyclic polynucleotide containing the cyclic polynucleotide; A first composition and a second composition of linear equivalents of ribonucleotides were provided (e.g., the response level in the subject after the first dose of cyclic polyribonucleotide was administered (administered). Response in a subject after one composition and a second composition have been provided (e.g., administered) For example, maintaining the level of the first linear equivalent of the cyclic polyribonucleotide. and comparing the level of reticulocyte production in the subject after providing the composition and the second composition. providing the subject with the first composition and the second composition of cyclic polyribonucleotides, and then administering to the subject The method for inducing the level of reticulocyte production in a subject is to use a cyclic erythropoietin-encoding gene. providing (e.g., administering) a first composition of polyribonucleotides to a subject; Thus, the subject is provided with (e.g., administered) a first composition of cyclic polyribonucleotides. and a step of encoding erythropoietin, comprising the level of reticulocyte production after the step of A second composition of cyclic polyribonucleotides is provided to the subject after the first composition (e.g., For example, administering) a step of administering to the subject at least a second portion of the cyclic polyribonucleotide. comprising the level of reticulocyte production after the composition is provided (e.g., administered). thereby forming a first composition and a second composition of linear equivalents of cyclic polyribonucleotides; and comparing the level of reticulocyte production in the subject after the substance has been provided (e.g., administered). Thus, a first composition and a second composition of cyclic polyribonucleotides are provided (e.g., The method includes maintaining the level of reticulocyte production in the subject after the administration of the compound (e.g., administered).

[0104] In certain embodiments, the present invention provides a method for administering (e.g., staggered administration or re-administration) The cyclic polyribonucleotide compositions described herein (e.g., a first composition, a second composition, a composition, the third composition, etc.) are 1ng / ml, 5ng / ml, 10ng / ml, 15ng g / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35ng / ml, 40n g / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90n g / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml , 500ng / ml, 600ng / ml, 1μg / ml, 10μg / ml, 50μg / m l, 100μg / ml, 200g / ml, 300μg / ml, 400μg / ml, 500 μg / ml, 600μg / ml, 700μg / ml, 800μg / ml, 900μg / m Linear polyribonucleic acid at 1 mg / ml, 1.5 mg / ml, or 2 mg / ml or less In certain embodiments, the cyclic polyribonucleotides described herein include The composition (e.g., the first composition, the second composition, the third composition, etc.) is a cyclic polyribonucleotide. Total ribonucleic acid in a composition of nucleotides (e.g., a pharmaceutical composition described herein) At least 30% (w / w), 40% (w / w), 50% (w / w) of the tide molecule , 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), or In one embodiment, the present invention comprises 99% (w / w) circular polyribonucleotide molecules. At least 30% (w / w) of the total ribonucleotide molecules in the compositions described herein, 0%(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%( 98% (w / w), or 99% (w / w) of the cyclic polyribonucleotide molecules is.

[0105] Staggered administration After providing the plurality of cells with at least two compositions of cyclic polyribonucleotides, produce certain levels of circular polyribonucleotides or express certain levels of protein or a method of staggered administration to generate a level of binding to multiple intracellular targets. The present invention relates to a method for administering to a subject (e.g., a mammal) a small amount of a cyclic polyribonucleotide. After providing (e.g., administering) at least two compositions, a certain level of cyclic polyribonucleic acid is obtained. to produce a nucleotide, express a certain level of a protein, or to induce a cell death in a subject (e.g., mammalian cell). Staggered administration to generate a level of binding to the target in mammals, e.g., humans In one embodiment, the method of preparing a cyclic polyribonucleotide is disclosed herein. At least two of the compositions are the same composition. At least two compositions of nucleotides are different compositions. The same composition may be a circular polyribonucleic acid molecule encoding the same protein or containing the same binding site. In one embodiment, the different compositions encode different proteins. The present invention also includes circular polyribonucleotides containing different or different binding sites, or combinations thereof.

[0106] In one embodiment, the expression of a protein in a mammal (e.g., a human) is maintained. The method comprises (a) providing a circular polyribonucleic acid molecule encoding a protein to be provided (e.g., administered) to a mammalian cell; providing a mammal (e.g., a human) with a first composition comprising a nucleotide; and ) 6 hours to 90 days after step (a), a cyclic polyribonucleotide encoding a protein is and providing (e.g., administering) to the mammal a second composition comprising The method includes maintaining expression of the protein in the host.

[0107] In one embodiment, a method for maintaining expression of an antigen in a mammal (e.g., a human) (a) administering a first composition comprising a cyclic polyribonucleotide encoding an antigen to a mammal and (b) 6 hours to 90 days after step (a), providing (e.g., administering) the antigen to the subject. providing to the mammal a second composition comprising a cyclic polyribonucleotide encoding the (e.g., administering) thereby maintaining expression of the protein in the mammal. .

[0108] In certain embodiments, the cyclic polyribonucleotide is an exogenous synthetic cyclic polyribonucleotide. In one embodiment, the cyclic polyribonucleotide is a polyribonucleotide having a polyA sequence, a polynucleotide having ... lacking the manufacturing element, or both.

[0109] In some embodiments, the step of providing (e.g., administering) the second composition comprises the steps of: After a), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 7 0, 75, 80, 85, or 90 days, or any time in between. In some embodiments, the step of providing (e.g., administering) the second composition is performed in step (a) 6. In some embodiments, a second composition is provided (e.g., administration The step of (a) is performed 6 hours to 30 days after the step (a). The step of providing (e.g., administering) the composition may be performed within 6 hours to 30 days of step (a) plus the cyclic polysaccharide. The half-life of the protein encoded by the ribonucleotide is approximately equal to the half-life of the protein encoded by the ribonucleotide. In the method, the step of providing (e.g., administering) the second composition is performed within 14 days of step (a). After ~30 days plus the half-life of the protein encoded by cyclic polyribonucleotide In some embodiments, the step of providing (e.g., administering) the second composition comprises: Step (a) for 14 to 65 days plus the tag encoded by the cyclic polyribonucleotide In some embodiments, a second composition is provided (e.g., The step (a) of administering the cyclic polyribonucleotide to a subject is carried out for 21 to 41 days in addition to the step (a). Thus, after the half-life of the encoded protein.

[0110] In one embodiment, the first composition comprises a first cyclic polyribonucleotide, The second composition comprises a second circular polyribonucleotide, wherein the first circular polyribonucleotide The nucleotide and the second cyclic polyribonucleotide are the same. The first composition comprises a first cyclic polyribonucleotide and the second composition comprises a second cyclic polyribonucleotide. a first circular polyribonucleotide and a second circular polyribonucleotide, In one embodiment, the first composition comprises a first protein. The second composition comprises a first cyclic polyribonucleotide encoding a second protein. a second circular polyribonucleotide encoding a protein, wherein the first protein In one embodiment, the first composition and the second protein are the same protein. comprises a first circular polyribonucleotide encoding a first protein and a second composition The article includes a second circular polyribonucleotide encoding a second protein, wherein The first protein and the second protein are different proteins. wherein the first composition comprises a first cyclic polyribonucleotide comprising a first binding site; The second composition comprises a second cyclic polyribonucleotide comprising a second binding site, wherein In one embodiment, the first binding site and the second binding site are the same binding site. The first composition comprises a first cyclic polyribonucleotide comprising a first binding site and a second The composition includes a second cyclic polyribonucleotide comprising a second binding site, wherein the first The first binding site and the second binding site are different binding sites. The composition comprises a first circular polyribonucleotide encoding a protein and a binding site. The second circular polyribonucleotide comprises:

[0111] In some embodiments, the step of providing the second composition occurs after providing the first composition. and a first level of the protein expressed by the first composition is detected in a mammal (e.g., a human). In one embodiment, the second set of The step of providing (e.g., administering) the composition may be performed after the first composition is provided (e.g., administered). and a first level of the protein expressed by the first composition is detected in a mammal. In some embodiments, the method for providing the second composition is performed before the concentration of the first composition decreases by more than 50%. The step of providing (e.g., administering) the first composition may be performed after the first composition is provided (e.g., administered). and a first level of the protein expressed by the first composition is substantially In one embodiment, the second composition is provided ( The step of providing (e.g., administering) the first composition is performed after the first composition is provided (e.g., administered) and a first level of a protein expressed by the composition of claim 1 being 25% to 30% higher in a mammal; In one embodiment, the method provides (e.g., administers) a 75% or greater reduction in the amount of erythrocyte permeability (e.g., ... administering a third composition of cyclic polyribonucleotides to the mammal after the second composition; Thereby, maintaining expression of the protein in the mammal. In some embodiments, providing (e.g., administering) the third composition is performed by administering the second composition to the subject. After being provided (e.g., administered) and the proteins expressed by the first and second compositions The second level of protein is then substantially undetectable in the mammal. In some embodiments, providing (e.g., administering) the third composition comprises administering the second composition After the product is provided (e.g., administered) and the first and second compositions are This occurs before the second level of expressed protein is reduced by 25% to 75%. do.

[0112] In one embodiment, the cyclic polylysine is administered to a subject (e.g., a mammal, e.g., a human). The method for producing cyclic polyribonucleotides comprises: providing (e.g., administering) a therapeutic agent to a subject (e.g., a mammal), a mammal) receives the circular polyribonucleic acid after the first composition is provided (e.g., administered) a first level of cyclic polyribonucleotide; and a second composition of cyclic polyribonucleotide. providing (e.g., administering) the compound to a subject (e.g., a mammal), a mammal (e.g., a mammal) comprising a second level of circular polyribonucleotides, the second level of leutidine is at least as great as the first level; and chromatographically determining whether the cyclic polyribonucleotide in the subject (e.g., mammal) is a cyclic polyribonucleotide comprising at least a first In one embodiment, the subject (e.g., a mammal, e.g., The method for producing cyclic polyribonucleotides in a mammal (e.g., a human) includes providing (e.g., administering) a first composition comprising the compound to a subject (e.g., a mammal); a subject (e.g., a mammal) to which a first composition has been provided (e.g., administered) a step of forming a first level of circular polyribonucleotide after the first level of circular polyribonucleotide has been formed; and A second composition of nucleotides is provided (e.g., administered) to a subject (e.g., a mammal). ) a step in which a subject (e.g., a mammal) is subjected to a second level of cyclic polyribonucleotides. a second composition comprising a cyclic polyribonucleotide, and a second level of cyclic polyribonucleotide is provided (e.g., (e.g., administered) changes by 20% or less of the first level; thereby A cyclic polyribonucleotide in a subject (e.g., a mammal) is administered at least at a first level. In one embodiment, the subject (e.g., a mammal, e.g., a human) The method for producing cyclic polyribonucleotides in providing (e.g., administering) to a subject (e.g., a mammal) a first composition comprising wherein the subject (e.g., mammal) is provided with (e.g., administered with) a first composition. ) followed by a first level of circular polyribonucleotide; and providing (e.g., administering) a second composition of the ointment to a subject (e.g., a mammal); wherein the subject (e.g., mammal) receives a second level of cyclic polyribonucleotide. and a second level of cyclic polyribonucleotide is provided in a second composition (e.g., , administered) at the first level of 1%, 5%, 15%, 20%, 25%, 30%, 35% %, 40%, or 50% or less; thereby maintaining the cyclic polyribonucleotides in the product at at least a first level. nothing.

[0113] In one embodiment, the first level of protein is determined after the first composition is provided: and maintaining the first composition and the second composition for a period of 6 hours to 90 days. In one embodiment, the first level of protein is determined after the first composition is provided: A first composition of cyclic polyribonucleotide, a second composition of cyclic polyribonucleotide, and a third composition of cyclic polyribonucleotide are administered over a period of 6 hours to 270 days. In one embodiment, the protein is maintained after providing the product and the third composition. The first level is administered to the first group for 6 hours to 35 days after the first composition is provided. The composition and the second composition are substantially undetectable after being provided.

[0114] Additionally, the second composition may be administered to a subject (e.g., a mammal) after providing the first composition. The level of cyclic polyribonucleotide from the first composition in the subject (e.g., mammal) In some embodiments, the second The step of providing the composition of the present invention is carried out after the first composition is provided and after the first composition is provided. a first level of cyclic polyribonucleotide to be administered in a subject (e.g., a mammal) In some embodiments, the step of providing a second composition is performed before the concentration of the second composition decreases by more than 50%. After the first composition is provided and the circular polyribonucleic acid produced by the first composition is The first level of the oxidase is reduced by 25% to 75% in a subject (e.g., a mammal). In one embodiment, the step of providing the second composition is performed before the step of providing the first composition. and a first composition of cyclic polyribonucleotides produced by the first composition. The level may be 1%, 5%, 15%, 20%, 25%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 16%, 17%, 18%, 21%, 22%, 23%, 24%, 25%, 36%, 38%, 39%, 40%, This is done before it drops by more than 0%, 35%, 40%, or 50%.

[0115] In certain embodiments, a cyclic polyribonucleotide is synthesized in a mammal (e.g., a human). The method for producing the cyclic polyribonucleotide comprises providing a first composition to a mammal comprising the cyclic polyribonucleotide. administering (e.g., administering) the first composition to the mammal to which it was provided (e.g., a step of administering a first level of cyclic polyribonucleotide to a subject after administration of the cyclic polyribonucleotide; providing (e.g., administering) a second composition of ribonucleotides to the mammal; wherein the mammal comprises a second level of cyclic polyribonucleotide, the second level of nucleotide is at least as great as the first level; Thus, maintaining cyclic polyribonucleotides in mammals at at least a first level. In one embodiment, the method comprises the step of: The method for producing nucleotides comprises administering a first composition comprising cyclic polyribonucleotides to a mammal. providing (e.g., administering) to an animal, wherein the mammal is provided with the first composition; a step of forming a first level of circular polyribonucleotide after the first level of circular polyribonucleotide has been formed; and providing (e.g., administering) a second composition of methicillin to a mammal, the mammal comprises a second level of cyclic polyribonucleotides, the second level is obtained after the second composition is provided (e.g., administered) and by 20% or less of the circular polyribonucleic acid in mammals; In one embodiment, the method further comprises maintaining the amount of oxalate in the mammal at at least a first level. Methods for producing cyclic polyribonucleotides in animals (e.g., humans) include the production of cyclic polyribonucleotides. providing (e.g., administering) to a mammal a first composition comprising a polynucleotide; and wherein the mammal receives the cyclic polylysine after the first composition is provided (e.g., administered). a first level of cyclic polyribonucleotides; and a second level of cyclic polyribonucleotides. providing (e.g., administering) the composition to a mammal, wherein the mammal is a second level of ribonucleotides, wherein the second level of cyclic polyribonucleotides comprises: 1%, 5%, 15%, or 20% of the first level after the second composition is provided (e.g., administered). %, 20%, 25%, 30%, 35%, 40%, or 50% or less; This allows the mammal to produce cyclic polyribonucleotides at least to a first level. The method includes a step of maintaining the

[0116] Additionally, the second composition may be administered after the first composition has been provided and after the first group has been administered in the mammal. The level of cyclic polyribonucleotides from the composition is substantially undetectable in mammals. In some embodiments, a second composition may be provided (e.g., before administration). The step of providing (e.g., administering) the first composition is performed after the first composition is provided (e.g., administered) and after the first composition is administered. The first level of cyclic polyribonucleotides produced by In some embodiments, a second composition is provided (e.g., The step of administering includes administering the first composition after the first composition has been administered and administering the first composition to the patient. First, levels of cyclic polyribonucleotides are reduced by 25% to 75% in mammals. In some embodiments, the step of providing (e.g., administering) the second composition The process begins after the first composition is provided (e.g., administered) and after the first composition is produced. The first level of cyclic polyribonucleotides produced in mammals is 1%, 5%, 1% This occurs before a drop of more than 5%, 20%, 25%, 30%, 35%, 40%, or 50% occurs.

[0117] In certain embodiments, the protein is expressed in a subject (e.g., a mammal, e.g., a human). The method for expressing the protein comprises providing a first composition comprising a cyclic polyribonucleotide encoding the protein. providing (e.g., administering) an agent to a subject (e.g., a mammal), expressing a first level of the encoded protein in a host (e.g., a mammal); and providing a second composition comprising a cyclic polyribonucleotide to a subject (e.g., a mammal); (e.g., administering) a gene to a subject (e.g., a mammal) that expresses the encoded protein. and expressing a second level of the protein, the second level being at least as great as the first level. thereby inhibiting expression of the encoded protein in a subject (e.g., a mammal). The method includes maintaining expression at least at a first level of the encoded protein. In embodiments, the protein is expressed in a subject (e.g., a mammal, e.g., a human). The method comprises: providing a first composition comprising a cyclic polyribonucleotide encoding a protein; providing (e.g., administering) a compound to a subject (e.g., a mammal), expressing a first level of the encoded protein; and A second composition comprising a ribonucleotide is provided to a subject (e.g., a mammal) (e.g., administering the encoded protein to a subject (e.g., a mammal) expressing a second level, the second level being at least as great as the first level. thereby producing a similar level of the encoded protein compared to the first level of the protein; The method includes maintaining expression of the encoded protein in the organism (e.g., mammal). In one embodiment, the protein is expressed in a subject (e.g., a mammal, e.g., a human). The method of expression comprises administering a first composition comprising a cyclic polyribonucleotide encoding a protein. providing (e.g., administering) to a subject (e.g., a mammal) expressing a first level of the protein; and A second composition comprising the nucleotide is provided (e.g., administered) to a subject (e.g., a mammal). wherein the subject (e.g., mammal) expresses a second level of the protein. , the second level varies by no more than 20% of the first level; thereby, the target Expression of a protein in a mammal (e.g., a mammal) can be achieved by at least detecting a first level of the protein. In some embodiments, the subject (e.g., a mammal, e.g., a human) A method for expressing a protein in a vector comprising the steps of: A first composition comprising the peptide is provided (e.g., administered) to a subject (e.g., a mammal). a step of: a subject (e.g., a mammal) expressing a first level of a protein; and administering a second composition comprising the cyclic polyribonucleotide to a subject (e.g., a mammal). providing (e.g., administering) a protein and the second level is 1%, 5%, 15%, 20% of the first level. , by 25%, 30%, 35%, 40%, or 50% or less; The expression of the protein in a subject (e.g., a mammal) is determined by at least one of the first and second sequences of the protein. Furthermore, the second composition may be maintained at a level equal to or greater than the level at which the first composition was provided. after (e.g., administered to) and the level of protein produced by the first composition can be provided (e.g., In some embodiments, the second composition may be administered in the same manner as the first composition. after (e.g., administered) and a first level of a protein expressed by the first composition. The drug is provided (e.g., administered) before the drug is reduced by more than 50% in a subject (e.g., a mammal). In some embodiments, the second composition is administered in the same manner as the first composition was administered (e.g., and a first level of a protein expressed by the first composition is provided (e.g., before it is reduced by 25% to 75% in a subject (e.g., a mammal) In some embodiments, a second composition is provided (e.g., administered). The process may be carried out after the first composition is provided (e.g., administered) and The first level of expressed protein is 1%, 5%, or , 15%, 20%, 25%, 30%, 35%, 40%, or 50% decrease do.

[0118] In one embodiment, a method for expressing a protein in a mammal (e.g., a human) a first composition comprising a cyclic polyribonucleotide encoding a protein, the ... providing (e.g., administering) to a mammal a first level of a protein; and providing a second composition comprising the cyclic polyribonucleotide to the mammal. providing (e.g., administering) a second level of the protein to the mammal, wherein the second level is at least as high as the first level; whereby Maintaining expression of a protein in a mammal at least at a first level of the protein In some embodiments, the method includes the steps of: expressing a protein in a mammal (e.g., a human); The method comprises administering to a mammal a first composition comprising a cyclic polyribonucleotide encoding a protein. providing (e.g., administering) to a mammal a first portion of the protein; and administering a second composition comprising cyclic polyribonucleotides to a mammal. providing (e.g., administering) the protein to a mammal, wherein the mammal expresses a second level of the protein. expressing a gene encoding the nucleotide sequence of the present invention, wherein the second level is changed by 20% or less of the first level; Thus, the expression of the protein in the mammal is maintained at least at a first level of the protein. In some embodiments, the method comprises the step of: The method of expression comprises administering a first composition comprising a cyclic polyribonucleotide encoding a protein. to a mammal, wherein the mammal is expressing a first level; and a second composition comprising a cyclic polyribonucleotide, providing (e.g., administering) to a mammal, wherein the mammal is and the second level is 1%, 5%, 15%, 20%, 25% or more of the first level. %, 30%, 35%, 40%, or 50% or less; thereby maintaining expression of the protein in the animal at least at a first level of the protein. Furthermore, the second composition may be administered after the first composition has been provided (e.g., administered). and the level of the protein produced by the first composition is substantially In some embodiments, the antibody may be provided (e.g., administered) before it is detectable. The second composition is administered after the first composition has been provided (e.g., administered) and after the first composition has been administered. before the first level of a protein expressed by In certain embodiments, the second composition is provided (e.g., administered) to the first After the compositions are provided (e.g., administered) and the proteins expressed by the first composition are The first level of protein provided before the body is reduced by 25% to 75% in mammals In certain embodiments, a second composition is provided (e.g., administered). The step of providing (e.g., administering) the first composition is performed after the first composition is provided (e.g., administered) and after the first composition is administered. The first level of the protein expressed by , before it drops by more than 20%, 25%, 30%, 35%, 40%, or 50%.

[0119] In some embodiments, the method for binding a target within a cell comprises using a cyclic polyribonucleotide. providing a first composition comprising the compound to the cells, wherein the cells are responsive to the compound provided with the first composition. and a second composition of cyclic polyribonucleotides, comprising a first level of conjugation after the first step. providing the cells with a second level of binding, the level is at least as high as the first level; thereby In one embodiment, the method comprises maintaining binding to the target at at least a first level. The method for binding to a target in a cell includes administering to the cell a first composition comprising a cyclic polyribonucleotide. wherein the cells exhibit a first level of binding after being provided with the first composition. and providing a second composition of cyclic polyribonucleotides to the cells. whereby the cells comprise a second level of binding, the second level of binding providing a second composition. and then changing the first level by no more than 20%; thereby In one embodiment, the method comprises maintaining binding to the target at at least a first level. The method for binding to a target in a cell includes administering to the cell a first composition comprising a cyclic polyribonucleotide. wherein the cells exhibit a first level of binding after being provided with the first composition. and providing a second composition of cyclic polyribonucleotides to the cells. whereby the cells comprise a second level of binding, the second level of binding providing a second composition. Then the first level is 1%, 5%, 15%, 20%, 25%, 30%, 35%, 40% or by 50% or less; thereby inhibiting binding to the target in the cell at a low level. maintaining the temperature at least at the first level.

[0120] Furthermore, the second composition may be administered after the first composition has been provided and after the first composition has been administered to the cells. The level of binding from the antibody can be provided before the antibody is substantially undetectable in the cell. In an embodiment, the step of providing the second composition is performed after the first composition is provided and before the first level of binding produced by the composition of claim 1 is reduced by more than 50% in the cells. In one embodiment, the step of providing the second composition is carried out after the first composition is provided. and a first level of binding produced by the first composition is detected in the cells. In some embodiments, the second composition is provided before the concentration is reduced by 5% to 75%. The step of performing the ... The levels of 1%, 5%, 15%, 20%, 25%, 30%, 35%, 40% %, or more than 50% reduction in the activity of the target within the cell. The method comprises providing a cell with a first composition comprising a cyclic polyribonucleotide. wherein the cells comprise a first level of binding after being provided with the first composition; and providing a second composition of polyribonucleotides to the cells, wherein the cells a second level of binding, the second level of binding being at least as good as the first level; thereby maintaining binding to the target in the cell at least at a first level. In one embodiment, the method of binding to an intracellular target comprises the step of: providing a first composition comprising a nucleotide to the cells, wherein the cells react with the first composition a step of providing a first level of conjugation after which the first level of conjugation of the cyclic polyribonucleotide is provided; and providing the composition of claim 2 to the cells, wherein the cells contain a second level of binding, the second level of changes by no more than 20% of the first level after providing the second composition. thereby maintaining binding to the target in the cell at least at a first level. In one embodiment, the method of binding to an intracellular target comprises the step of: providing a first composition comprising a nucleotide to the cells, wherein the cells react with the first composition a step of providing a first level of conjugation after which the first level of conjugation of the cyclic polyribonucleotide is provided; and providing the composition of claim 2 to the cells, wherein the cells contain a second level of binding, The second level of the first composition is then provided at 1%, 5%, 15%, 2% or more of the first level. a process that changes by no more than 0%, 25%, 30%, 35%, 40%, or 50%; Thus, the method includes maintaining target binding in the cell at at least a first level.

[0121] Furthermore, the second composition may be administered after the first composition has been provided and after the first composition has been administered to the cells. The level of binding from the antibody can be provided before the antibody is substantially undetectable in the cell. In an embodiment, the step of providing the second composition is performed after the first composition is provided and before the first level of binding produced by the composition of claim 1 is reduced by more than 50% in the cells. In one embodiment, the step of providing the second composition is carried out after the first composition is provided. and a first level of binding produced by the first composition is detected in the cells. In some embodiments, the second composition is provided before the concentration is reduced by 5% to 75%. The step of performing the ... The levels of 1%, 5%, 15%, 20%, 25%, 30%, 35%, 40% %, or before it drops by more than 50%.

[0122] In some embodiments, binding a target in a subject (e.g., a mammal, e.g., a human) The method includes administering a first composition comprising a cyclic polyribonucleotide to a subject (e.g., a mammal). ), wherein the subject (e.g., mammal) receives a first comprising a first level of binding after the composition is provided (e.g., administered); and A second composition of cyclic polyribonucleotides is provided (e.g., to a subject (e.g., a mammal) a step of administering to the subject (e.g., mammal) a second level of binding. wherein the second level of binding is at least as great as the first level; thereby maintaining target binding in a subject (e.g., a mammal) at at least a first level. In one embodiment, the method comprises administering to a subject (e.g., a mammal, e.g., a human) The method for binding to a target in the present invention comprises administering a first composition comprising a cyclic polyribonucleotide to a target ( providing (e.g., administering) a subject (e.g., a mammal) to animal) after being provided (e.g., administered) with the first composition, and and administering a second composition of cyclic polyribonucleotides to a subject (e.g., a mammal). ), wherein the subject (e.g., mammal) a second level of binding, wherein the second level of binding occurs when a second composition is provided (e.g., administered after the first level is changed by 20% or less; The method includes maintaining target binding in a mammal (e.g., a mammal) at at least a first level. In certain embodiments, a target in a subject (e.g., a mammal, e.g., a human) is bound to the target. The method comprises administering to a subject (e.g., a mammal) a first composition comprising a cyclic polyribonucleotide. providing (e.g., administering) a first set of a step of providing (e.g., administering) the composition, comprising a first level of binding; and A second composition of polyribonucleotides is provided to a subject (e.g., a mammal) (e.g., and administering to the subject (e.g., mammal) a second level of binding. The second level of binding is determined after the second composition is provided (e.g., administered) and after the first 1%, 5%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% or more of the level whereby binding to a target in a subject (e.g., a mammal) is altered. at least a first level.

[0123] Additionally, the second composition may be administered after the first composition has been provided (e.g., administered) and in combination with the first composition. The level of binding from the first composition in the subject (e.g., mammal) is can be provided (e.g., administered) before being substantially undetectable in mammals In some embodiments, providing (e.g., administering) the second composition is After the composition is provided (e.g., administered) and the bond produced by the first composition is performed before the first level of is reduced by more than 50% in a subject (e.g., a mammal). In certain embodiments, providing (e.g., administering) the second composition is After the composition is provided (e.g., administered) and the binding produced by the first composition is The first level occurs before the level is reduced by 25% to 75% in a subject (e.g., a mammal). In certain embodiments, the step of providing (e.g., administering) the second composition comprises: After the first composition is provided (e.g., administered) and The first level of binding is 1%, 5%, 15%, 20%, or 30% in a subject (e.g., a mammal). %, 25%, 30%, 35%, 40%, or 50% decrease. In one embodiment, a method of binding a target in a subject (e.g., a mammal, e.g., a human) comprises: Providing a first composition comprising a cyclic polyribonucleotide to a subject (e.g., a mammal) (e.g., administering) a first composition to a subject (e.g., a mammal) a step of forming a circular polyribonucleotide comprising a first level of binding after the polyribonucleotide has been bound (e.g., administered) to the target cell; A second composition of nucleotides is provided (e.g., administered) to a subject (e.g., a mammal). wherein the subject (e.g., mammal) comprises a second level of binding and a first level of binding. the second level is at least as high as the first level; thereby maintaining binding to the target in a mammal (e.g., a mammal) at at least a first level. In some embodiments, the target in a subject (e.g., a mammal, e.g., a human) The method of binding comprises administering a first composition comprising a cyclic polyribonucleotide to a subject (e.g., a mammal). providing (e.g., administering) a therapeutic agent to a subject (e.g., a mammal) comprising a first level of binding after one composition is provided (e.g., administered); and providing a second composition of cyclic polyribonucleotides to a subject (e.g., a mammal). (e.g., administering) a second level of binding, wherein the subject (e.g., mammal) and the second level of binding is determined after the second composition is provided (e.g., administered): the first level is changed by no more than 20%; thereby ) at least at a first level. In the method of binding to a target in a subject (e.g., a mammal), a circular polyribonucleic acid A first composition comprising a leutide is provided (e.g., administered) to a subject (e.g., a mammal). a subject (e.g., a mammal) to which a first composition has been provided (e.g., a step of administering a first level of binding to a cyclic polyribonucleotide after the first level of binding to a cyclic polyribonucleotide; providing (e.g., administering) the composition of claim 2 to a subject (e.g., a mammal); the subject (e.g., mammal) comprises a second level of binding, the second level of binding comprising: After the second composition is provided (e.g., administered), the first level of 1%, 5%, 15% , which varies by no more than 20%, 25%, 30%, 35%, 40%, or 50%; by detecting binding to a target in a subject (e.g., a mammal) at least at a first level. The method includes a step of maintaining the temperature.

[0124] Additionally, the second composition may be administered after the first composition has been provided (e.g., administered) and in combination with the first composition. The level of binding from the first composition in the subject (e.g., mammal) is can be provided (e.g., administered) before being substantially undetectable in mammals In some embodiments, providing (e.g., administering) the second composition is After the composition is provided (e.g., administered) and the bond produced by the first composition is performed before the first level of is reduced by more than 50% in a subject (e.g., a mammal). In certain embodiments, providing (e.g., administering) the second composition is After the composition is provided (e.g., administered) and the binding produced by the first composition is The first level occurs before the level is reduced by 25% to 75% in a subject (e.g., a mammal). In certain embodiments, the step of providing (e.g., administering) the second composition comprises: After the first composition is provided (e.g., administered) and The first level of binding is 1%, 5%, 15%, 20%, or 30% in a subject (e.g., a mammal). %, 25%, 30%, 35%, 40%, or 50% decrease.

[0125] In one embodiment, a first composition and a second composition are administered in a staggered administration regimen or method. The composition may be followed by one or more additional compositions of cyclic polyribonucleotides. In embodiments, the one or more additional compositions are a third, fourth, fifth, sixth, seventh, eighth, A ninth, tenth or more composition is included.

[0126] In some embodiments, the third composition of cyclic polyribonucleotides is and then provided (e.g., administered) to a cell or subject (e.g., a mammal, e.g., a human). , thereby providing a third composition, followed by the addition of a cyclic polyribonucleotide, a protein or maintain the level of binding at least at a first level. The third composition is administered after the second composition and in a cell or subject (e.g., a mammal) to the first and second compositions. and a second composition of a circular polyribonucleotide, protein, or bond produced by the second composition. levels are substantially undetectable in a cell or subject (e.g., a mammal). In some embodiments, the third composition is administered to a subject. After the substance is provided (e.g., administered) and the cyclic polynucleotide produced by the first composition is ribonucleotides, proteins expressed by the first composition, or proteins expressed by the first composition The first level of binding produced by the In one embodiment, the third set is provided (e.g., administered) before the blood glucose level drops by more than 100%. The composition is administered after the first composition is provided (e.g., administered) and the circular polyribonucleotide produced, the protein expressed by the first composition, or The first level of binding produced by the first composition is expressed in a cell or subject (e.g., a mammalian provided (e.g., administered) before the antibody titer is reduced by 25% to 75% in the animal. In some embodiments, the third composition is administered to the subject in the same manner as the first composition was provided (e.g., administered). ) and the cyclic polyribonucleotides produced by the first composition, a protein expressed by the first composition, or a first level of binding produced by the first composition, , 1%, 5%, 15%, 20%, 25%, 3% in cells or subjects (e.g., mammals) It is provided (eg, administered) before a decrease of more than 0%, 35%, 40%, or 50%. In staggered administration, one or more additional compositions are administered after a previous composition is provided and after a previous composition is administered. The level of cyclic polyribonucleotides, bonds, or proteins produced by the cells or may be provided beforehand so that it is substantially undetectable in a subject (e.g., a mammal). In some embodiments, one or more additional compositions may be added to the same composition as a previous composition provided (e.g., The circular polyribonucleotides, conjugates, or is a protein whose levels are reduced by more than 50% in a cell or subject (e.g., a mammal) In some embodiments, one or more additional combinations of The composition is produced after the previous composition has been provided (e.g., administered) and by the previous composition. The levels of cyclic polyribonucleotide, binding, or protein detected in a cell or subject (e.g., provided (e.g., administered) before being reduced by 25% to 75% in mammals In some embodiments, one or more additional compositions may be added to the same composition as the previous composition. Circular polyribonucleotides produced by the post- and pre-compositions (e.g., administered) , binding, or protein levels are less than 1% in a cell or subject (e.g., mammal), Offered before it declines by more than 5%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% provided (e.g., administered).

[0127] In certain embodiments, the second composition is a compound that is capable of inhibiting the growth of a cell or a cell surface in a subject (e.g., a mammal). The protein level is approximately the protein level before administering or providing the first composition. administered or provided to a cell or a subject (e.g., a mammal) before returning to the cell. In the method, the second composition is used to detect the level of a protein in a cell or a subject (e.g., a mammal). before the level of protein returns to approximately the level before administering or providing the first composition. administered or provided to a cell or a subject (e.g., a mammal). or (b) administering to a subject (e.g., a mammal) additional doses of the third composition to the subject. The protein level is approximately the protein level before administering or providing the first composition. The antibody is administered or provided to a cell or a subject (e.g., a mammal) before returning to the cell. and one or more further compositions fourth, fifth, sixth, seventh, eighth, ninth, tenth or thereabouts. The above compositions are used to detect the level of a protein in a cell or a subject (e.g., a mammal) The cells or subject may then return to approximately the protein level prior to administering or providing the composition of claim 1. (e.g., a mammal)

[0128] In some embodiments, the composition is administered to a cell or a subject (e.g., a mammal) by injecting the composition. and then, after a time interval, administering or providing the compound to a cell or a subject (e.g., a mammal). For example, the second composition may be used to administer the first composition to a cell or a subject (e.g., a mammal). After a first time interval, the cell or subject (e.g., mammal) the third composition may be administered or provided to a cell or subject (e.g., a mammal) and after a second time interval, the cell or subject (e.g., mammal) the fourth composition may be administered or provided to a cell or subject (e.g., a mammal) by administering the third composition to the cell or subject (e.g., a mammal) and after a third time interval, administering or providing the compound to the cell or subject (e.g., mammal) or a fifth, sixth, seventh, eighth, ninth, or more compositions may be administered or provided to: The fourth, fifth, sixth, seventh, eighth, ninth, or more compositions are administered to a cell or subject (e.g., and administering or providing the compound to a mammal (or mammals) on the fourth, fifth, sixth, seventh, eighth, ninth, or subsequent day. After the above time intervals, the first antibody may be administered or provided to a cell or a subject (e.g., a mammal). The time interval may be determined by measuring the time interval between administration or provision of the first composition to the cell or subject (e.g., mammal) before administration or provision of the first composition. The amount of time required for the protein level in It could be shorter.

[0129] In some embodiments, the second time interval is longer than the first time interval. In one embodiment, the third time interval is longer than the first time interval. In one embodiment, the fifth, sixth, seventh, eighth, and seventh time intervals are longer than the first time interval. The ninth or more time intervals are longer than the first time interval. The second time interval is the same as the first time interval. In one embodiment, the third time interval In one embodiment, the fourth time interval is the same as the first time interval. In some embodiments, the fifth, sixth, seventh, eighth, ninth, or any of the preceding intervals is the same as the interval between adjacent cells. The above time interval is the same as the first time interval. In some embodiments, the second time interval In one embodiment, the third time interval is shorter than the first time interval. In one embodiment, the fourth time interval is shorter than the first time interval. In some embodiments, the fifth, sixth, seventh, eighth, ninth, or more time intervals are In one embodiment, the second time interval is longer than the first time interval. In one embodiment, the third time interval is longer than the second time interval. In the fourth time interval, the fourth time interval is longer than the third time interval.

[0130] In one embodiment, the first level of cyclic polyribonucleotide comprises a first composition The highest level of cyclic polyribonucleotides was measured 1 to 2 days after administration of the first composition. The highest level of cyclic polyribonucleotide 1-2 days after providing, e.g., the first composition 24 to 48 hours (e.g., 1 to 2 days) after providing cyclic polyribonucleotides In one embodiment, the first level of cyclic polyribonucleotide is , 1-2 days after providing the first composition, the highest level of cyclic polyribonucleotides was 40 %, 50%, 60%, 70%, 80%, or 90%. The second level of polyribonucleotides is added at least 1, 2, or 3 times after providing the second composition. , 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40 The maximum amount of cyclic polyribonucleotides was measured 1-2 days after providing the first composition. of levels of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10 0%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 18 In one embodiment, the polyribonucleic acid is 0%, 190%, or 200%. The second level of the method may be performed after providing the second composition by at least 1, 2, 3, 4, 5, 6, For 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40 days, The maximum level of cyclic polyribonucleotides was less than 1-2 days after administration of the composition of claim 1. In one embodiment, the cyclic The third level of polyribonucleotides is provided after at least 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40 The maximum amount of cyclic polyribonucleotides was measured 1-2 days after providing the first composition. of levels of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10 0%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 18 In one embodiment, the polyribonucleic acid is 0%, 190%, or 200%. The third level of the method comprises providing the third composition and then administering at least 1, 2, 3, 4, 5, 6, For 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40 days, The maximum level of cyclic polyribonucleotides was less than 1-2 days after administration of the composition of claim 1. In one embodiment, the first For each subsequent composition provided after the composition, the ring expressed after each subsequent composition Subsequent levels of polyribonucleotides are added at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or cyclic polyribonucleotide 1-2 days after providing the first composition over a 40-day period At least 30%, 40%, 50%, 60%, 70%, 80%, 90% of the highest level of , 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170% , 180%, 190%, or 200%. For each subsequent composition provided in the Subsequent levels of nucleotides are administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 Within 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40 days The highest level of cyclic polyribonucleotides was observed 1-2 days after the first composition was administered. at least 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than the

[0131] In one embodiment, after providing the second composition, the average cyclic polyribonucleotide The level is at least 40%, 50%, 60%, 70%, 80%, or 9% of the first level. 0%, and the average level of cyclic polyribonucleotides is 1 day after providing the second composition. The measurement is made from the day when cyclic polyribonucleotides are substantially undetectable. In an embodiment, the circular polyribonucleic acid is added after providing each subsequent composition after the first composition. The average level of the octide is at least 40%, 50%, 60%, 70%, 80% or more of the first level. 0%, or 90%, and the average level of cyclic polyribonucleotide in each subsequent composition Measured from one day after donation until the day when cyclic polyribonucleotides are virtually undetectable will be done.

[0132] In one embodiment, the first level of cyclic polyribonucleotide is at least 6 hours. 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 35 days The second composition of cyclic polyribonucleotides is provided and maintained for a period of days. In one embodiment, the first level of cyclic polyribonucleotide is administered for at least 6 hours. , 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 35 days The second composition of cyclic polyribonucleotides is maintained for a period of time. In some embodiments, the first level of cyclic polyribonucleotide is administered for at least 6 hours. 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, or 35 days After providing the third composition of cyclic polyribonucleotides, the In an embodiment, after providing the second composition, The second level of cyclic polyribonucleotide in the cell or a first level of cyclic polyribonucleotide in a subject (e.g., a mammal) In some implementations, the In some embodiments, after providing the third composition, The third level of cyclic polyribonucleotide is provided by providing the first composition followed by a plurality of At least 5%, 10%, 20%, 30% above the first level of cyclic polyribonucleotide , 40%, 50%, or 60% higher.

[0133] In one embodiment, the second composition of cyclic polyribonucleotides is provided for 1 hour. 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days , 15 days, 20 days, 25 days, 30 days, 40 days, or 45 days after The second level of cyclic polyribonucleotides is obtained by the first level of cyclic polyribonucleotides after providing the first composition of cyclic polyribonucleotides. At least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the In one embodiment, the third composition of cyclic polyribonucleotides is provided within 1 hour. , 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 1, 15, 20, 25, 30, 40, or 45 days after The third level of the method comprises providing a first composition of cyclic polyribonucleotides after the first level. At least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% lower than the expensive.

[0134] In one embodiment, the first level of binding is At least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and 14 days after providing the product In some embodiments, the administration of the active ingredient is maintained for 21 days, 28 days, or 35 days. The first level of binding was maintained to provide a third composition of cyclic polyribonucleotide. After at least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, In some embodiments, the second composition is provided. a second level of binding in the cell or subject (e.g., mammal) after the first composition; less than the first level of binding in a cell or subject (e.g., mammal) after providing the substance. At least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher. In an embodiment, in a cell or a subject (e.g., a mammal) after providing the third composition A third level of administration in the first composition provides the first level of binding in the plurality. at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the

[0135] In one embodiment, the first level of protein is obtained from 1 to 2 The highest protein level was 1-2 days after administration of the first composition. The highest level of quality, e.g., peak amount of protein, was achieved in the first 24 hours. After about 48 hours (e.g., 1-2 days), the gene was expressed from the circular polyribonucleotide. In the embodiment, the first level of protein is measured 1 to 2 days after providing the first composition. The protein content can be 40%, 50%, 60%, 70%, 80%, or 90% of the highest protein level. In one embodiment, the second level of protein is at least 100 mg / mL after providing the second composition. At most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 3 The highest protein levels were observed 1-2 days after providing the first composition over a 5 or 40 day period. Level at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 %, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180 %, 190%, or 200%. In some embodiments, the second level of the protein After providing the second composition, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 12, 15, 20, 25, 30, 35, or 40 days. The highest protein levels after 1-2 days of treatment were at least 1-, 5-, 10-, or 100-fold higher than the highest levels after treatment. fold, or 1000 times higher. In one embodiment, the third level of protein is a third After providing the composition, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 1 1 to 2, in which the first composition was provided for 5, 20, 25, 30, 35, or 40 days. At least 30%, 40%, 50%, 60%, 70% of the highest protein levels after 2 days ,80%,90%,100%,110%,120%,130%,140%,150%,1 60%, 170%, 180%, 190%, or 200%. a third level of protein is provided after providing the third composition, said third level of protein being provided after providing the third composition, and Over 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40 days Thus, the protein level is at least 1-2 days higher than the highest level of protein 1-2 days after providing the first composition. fold, 5 times, 10 times, 100 times, or 1000 times higher. For each subsequent composition provided after the composition, the protein expressed after each subsequent composition Subsequent levels of quality may be achieved after providing each subsequent composition for at least 1, 2, 3, 4, 5, Over 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40 days , at least 30% of the highest protein level 1-2 days after providing the first composition; 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 1 30%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% In some embodiments, for each subsequent composition provided after the first composition, The subsequent levels of protein expressed after each subsequent composition provide After serving, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, Tanning was performed 1-2 days after application of the first composition for 25, 30, 35, or 40 days. At least 1, 5, 10, 100, or 1000 times higher than the highest protein level stomach.

[0136] In one embodiment, the average level of the protein after providing the second composition is at least 40%, 50%, 60%, 70%, 80%, or 90% of the level of The average level of protein was measured from day 1 after providing the second composition, and the protein was substantially undetectable. In some embodiments, the first composition is followed by each subsequent composition until the day when the first composition is inoperable. the average protein level after providing the composition is at least 40% of the first level, 0%, 60%, 70%, 80%, or 90%, and the average level of protein is The composition is measured from one day after application until the day when the protein is substantially undetectable. do.

[0137] In one embodiment, the first level of protein is determined after the first composition is provided: At least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 The first composition and the second composition of cyclic polyribonucleotides are administered over a period of 35 days or 35 days. In one embodiment, the first level of the protein is maintained after providing the composition. At least 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days after the first composition is applied for 14 days, 21 days, 28 days, or 35 days. The first composition, the second composition, and the third composition are provided and then maintained.

[0138] In some embodiments, the cell or subject (e.g., mammal) after providing the second composition. ) is measured after the first composition has been provided to the cell or subject ( at least 1%, 5%, 10%, or more than the first level of the protein in a mammal (e.g., a mammal). %, 20%, 30%, 40%, 50%, or 60% higher. A third level of the protein in the cell or subject (e.g., mammal) after providing the composition. the level is less than the first level of protein in the plurality after the first composition is provided. at least 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher.

[0139] In one embodiment, the second composition of cyclic polyribonucleotides is provided for 1 hour. 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days , 15 days, 20 days, 25 days, 30 days, 40 days, or 45 days later, the second level of protein is at least 1%, 5%, or more than the first level after the protein first composition is provided. 10%, 20%, 30%, 40%, 50%, or 60% higher. A third composition of polyribonucleotides was provided at 1 hour, 12 hours, 18 hours, 1 day, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 15th, 20th, 25th, A third level of protein after 30, 40, or 45 days is determined based on the first composition provided. at least 1%, 5%, 10%, 20%, 30% higher than the initial level of protein after 40%, 50%, or 60% higher.

[0140] In one embodiment, the level of protein in the first composition is maintained at 100% or higher for at least 1 hour. 2 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, or 45 days The second composition of cyclic polyribonucleotides is maintained for a period of time. In an embodiment, the level of the protein in the first composition is maintained for at least 1 hour, 12 hours, or , 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days , 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, or 45 days Thus, the third composition of cyclic polyribonucleotides is provided and then maintained. In some embodiments, the level of the protein in the first composition is maintained for at least 1 hour, 12 hours, 18 hours, or more. Hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 for 15, 20, 25, 30, 40, or 45 days, Fourth, fifth, sixth and seventh compositions of cyclic polyribonucleotides , an eighth composition, a ninth composition, a tenth composition or more are provided, and then maintained. The maintained protein level is measured by measuring the level of the cells or tissues one day after the first composition is provided. is the level of the protein in a subject (e.g., a mammal). , the first composition, followed by the circular polyribonucleoside in a cell or a subject (e.g., a mammal). Tide levels are at least 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, and 4 days 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days the second cyclic polyribonucleotide for 30 days, 40 days, or 45 days. In one embodiment, the cells or The level of cyclic polyribonucleotides in a subject (e.g., a mammal) is at least 1 Hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, or After providing the third composition of cyclic polyribonucleotides for 45 days, the In some embodiments, the first composition is administered to a cell or a subject (e.g., a mammal) The level of cyclic polyribonucleotides in the culture medium was maintained for at least 1 hour, 12 hours, 18 hours, and 1 hour. Days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 Cyclic polypropylene for 5, 20, 25, 30, 40, or 45 days The fourth composition, the fifth composition, the sixth composition, the seventh composition, the eighth composition of ribonucleotides a ninth composition, a tenth composition or more are provided and then maintained.

[0141] In one embodiment, the cells after providing the second composition of cyclic polyribonucleotides. or the level of the protein in a subject (e.g., a mammal) provided with the first composition. 1% to 5%, 5% or more of the protein level in the cell or subject (e.g., mammal) after %~10%, 10%~15%, 15%~20%, 20%~25%, 25%~30%, 30 %~35%, 35%~40%, 40%~45%, 45%~50%, 50%~55%, 55 %~60%, 60%~65%, 65%~70%, 70%~75%, 75%~80%, 80 %~85%, 85%~90%, 90%~92%, 92%~94%, 94%~95%, 95 %~96%, 96%~97%, 97%~98%, 98%~99%, 10%~30%, 10 %~40%, 10%~50%, 10%~60%, 10%~70%, 10%~80%, 10 %~90%, 10%~95%, 40%~50%, 40%~60%, 40%~70%, 40 %~80%, 40%~90%, 40%~95%, 60%~80%, 60%~90%, 60 In some embodiments, the circular polyribonucleic acid is 60% to 95%, or 60% to 98% higher. and the like. The level of the polynucleotide is measured in the cell or subject (e.g., mammal) after providing the first composition. 1%-5%, 5%-10%, 10% higher than the level of cyclic polyribonucleotides in ~15%, 15%~20%, 20%~25%, 25%~30%, 30%~35%, 35% ~40%, 40%~45%, 45%~50%, 50%~55%, 55%~60%, 60% ~65%, 65%~70%, 70%~75%, 75%~80%, 80%~85%, 85% ~90%, 90%~92%, 92%~94%, 94%~95%, 95%~96%, 96% ~97%, 97%~98%, 98%~99%, 10%~30%, 10%~40%, 10% ~50%, 10%~60%, 10%~70%, 10%~80%, 10%~90%, 10% ~95%, 40%~50%, 40%~60%, 40%~70%, 40%~80%, 40% ~90%, 40%~95%, 60%~80%, 60%~90%, 60%~95%, or 6 0% to 98% higher.

[0142] Readministration After providing the cells with at least two compositions of cyclic polyribonucleotides, to generate a cyclic polyribonucleotide level, a binding level, or to express a protein. Disclosed herein are methods for re-administering cyclic polyribonucleotides to cells for the purpose of inhibiting the growth of cells. and administering to a subject (e.g., a mammal, e.g., a human) at least two compositions of to generate a cyclic polyribonucleotide level, a binding level, or to express a protein. Disclosed herein are methods of re-administration for the treatment of rheumatoid arthritis.

[0143] In one embodiment, at least two compositions of cyclic polyribonucleotides are the same. In one embodiment, the composition comprises at least two pairs of cyclic polyribonucleotides. In some embodiments, the same composition may contain the same protein. In some embodiments, the cyclic polyribonucleotide encoding the same or containing the same binding site. In this case, the different compositions encode different proteins or contain different binding sites. In one embodiment, the first composition comprises a first protein. The second composition comprises a first cyclic polyribonucleotide encoding a second protein. a second circular polyribonucleotide encoding a protein, wherein the first protein In one embodiment, the first composition and the second protein are the same protein. comprises a first circular polyribonucleotide encoding a first protein and a second composition The article includes a second circular polyribonucleotide encoding a second protein, wherein The first protein and the second protein are different proteins. wherein the first composition comprises a first cyclic polyribonucleotide comprising a first binding site; The second composition comprises a second cyclic polyribonucleotide comprising a second binding site, wherein In one embodiment, the first binding site and the second binding site are the same binding site. The first composition comprises a first cyclic polyribonucleotide comprising a first binding site and a second The composition includes a second cyclic polyribonucleotide comprising a second binding site, wherein the first The first binding site and the second binding site are different binding sites. The composition comprises a first circular polyribonucleotide encoding a protein, and a second The composition includes a second circular polyribonucleotide that includes a binding site.

[0144] In one embodiment, the expression of a protein in a mammal (e.g., a human) is maintained. The method includes (a) providing a first composition comprising a cyclic polyribonucleotide encoding a protein; to a mammal; and (b) 6 hours to 9 hours after step (a). After 0 days, a second composition containing a cyclic polyribonucleotide encoding a protein is administered to the mammal. and providing (e.g., administering) the protein to the mammal, thereby increasing the expression of the protein in the mammal. In certain embodiments, a second composition is provided (e.g., administered) The step of providing (e.g., administering) the first composition is performed after the first composition is provided (e.g., administered) and after the first composition is administered. The first level of protein expressed by the In one embodiment, the method is performed after the third step of the cyclic polyribonucleotide. is provided (e.g., administered) to the mammal after the second composition, thereby The method further comprises recovering the protein in the mammal.

[0145] In one embodiment, a method for maintaining expression of an antigen in a mammal (e.g., a human) (a) administering a first composition comprising a cyclic polyribonucleotide encoding an antigen to a mammal and (b) 6 hours to 90 days after step (a), providing (e.g., administering) the antigen to the subject. providing to the mammal a second composition comprising a cyclic polyribonucleotide encoding the (e.g., administering) thereby maintaining expression of the protein in the mammal. .

[0146] In some embodiments, the step of providing (e.g., administering) the second composition comprises the steps of: After a), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 7 In some embodiments, the time period is 0, 75, 80, 85, or 90 days, or any time period therebetween. In the method, the step of providing (e.g., administering) the second composition is performed 1, 2 or 3 times after step (a). , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, In one embodiment, the period is 85, 90, or any time therebetween. The step of providing (e.g., administering) the second composition may be performed 1, 2, 3, 4, or 5 days after step (a). , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , or 20 years, or any time therebetween. The step of providing (e.g., administering) the composition may be performed within 6 hours to 45 days after step (a). In some embodiments, the step of providing (e.g., administering) the second composition comprises the steps of: After step (a), the period is 6 hours to 30 days. The step of administering (e.g., administering) is 6 hours to 65 days after step (a). In some embodiments, the step of providing (e.g., administering) the second composition is performed 3 days after step (a). In some embodiments, a second composition is provided (e.g., administration). The step of administering the therapeutic agent is performed for 14 to 30 days after step (a). The step of providing (e.g., administering) the second composition may be performed 14 days to 45 days after step (a). In certain embodiments, providing (e.g., administering) the second composition is 30 to 65 days after step (a). The step of providing (eg, administering) is 30 to 90 days after step (a).

[0147] In some embodiments, the first level of protein is determined by the amount of protein present in the first composition provided (e.g., For example, the first composition and the second composition are administered for 6 hours to 90 days after the administration of the first composition and the second composition. In one embodiment, the first portion of the protein is provided (e.g., administered) and then maintained. The level of 1 is between 6 hours and 270 days after the first composition is provided (e.g., administered). Over a period of time, a first composition, a second composition, and a third composition of cyclic polyribonucleotides are In one embodiment, the composition is provided (e.g., administered) and then maintained. The first level of quality is measured within 6 hours to 3 hours after the first composition is provided (e.g., administered). After providing (e.g., administering) the first composition and the second composition over a period of 5 days, Virtually undetectable.

[0148] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and The level of the circular polyribonucleic acid in the cells after administration of the composition of claim 2 is compared to the level of the protein in the cells after administration of the composition of claim 2. After providing the cells with the first composition and the second composition of nucleotides, the cells are subjected to the synthesis of proteins. The method for expressing a level of quality is to first synthesize a protein-encoding cyclic polyribonucleotide. providing a composition of the formula (I) to a cell, wherein the cell receives a first sequence of the cyclic polyribonucleotide After the composition is provided, it contains a certain level of protein; and providing a second composition of the compound to the cells after the first composition, wherein the cells At least, the level of protein after the second composition of cyclic polyribonucleotides is provided. thereby forming a first composition of linear equivalents of cyclic polyribonucleotides. and the level of the protein in the cells after the second composition has been provided. The expression of the level of the protein in the first composition of cyclic polyribonucleotide and the second composition of cyclic polyribonucleotide are After the composition is provided, the step of maintaining the composition is also included. in cells after providing a first composition and a second composition of linear equivalents of nucleotides. The first composition and the second composition of cyclic polyribonucleotides are compared to the level of the protein. The method for expressing the level of a protein in a cell after the composition is provided to the cell is providing to the cell a first composition of cyclic polyribonucleotides encoding a protein; and wherein the cells express a certain level of cyclic polyribonucleotides after being provided with the first composition of cyclic polyribonucleotides. and mixing a second composition of cyclic polyribonucleotides with the first composition of cyclic polyribonucleotides. providing the composition to cells after which the cells react with a second set of cyclic polyribonucleotides. the level of the protein changes by no more than 20% of the level after the composition is provided. thereby forming a first composition and a second composition of linear equivalents of cyclic polyribonucleotides; The level of the circular polyribonucleoside is compared to the level of the protein in the cells after delivery. The level of the protein in the cells after the first and second compositions of the peptides are provided The step of maintaining expression of

[0149] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and and comparing the protein levels in a subject (e.g., a mammal) after providing the two compositions. and administering the first and second compositions of cyclic polyribonucleotides to a subject (e.g., a mammal). and expressing a certain level of the protein in the subject (e.g., mammal) after providing the protein to the subject (e.g., mammal). The method comprises: providing a first composition of cyclic polyribonucleotides encoding a protein to a subject; providing (e.g., administering) a subject (e.g., a mammal) with a mammal) to which a first composition of cyclic polyribonucleotides has been provided (e.g., administered) and a second step of cyclic polyribonucleotides, This composition is provided (e.g., administered) to the subject (e.g., mammal) after the first composition. a process for translating a circular polyribonucleic acid sequence into a target protein (e.g., a mammal) The second composition of the method comprises measuring the level of the protein after the second composition is provided (e.g., administered). , whereby a first composition of linear equivalents of a cyclic polyribonucleotide and a second composition of linear equivalents of a cyclic polyribonucleotide are obtained. The composition is provided (e.g., administered) to a subject (e.g., a mammal) and then the subject is treated with the composition. The first and second compositions of cyclic polyribonucleotides are compared to the level of the protein. After the substance is provided (e.g., administered), the protein in the subject (e.g., mammal) In one embodiment, the method further comprises maintaining the level of expression of the circular polyribonucleic acid protein. a subject (e.g., a mammal) after providing the subject with a first composition and a second composition of linear equivalents of a botulinum toxin; The first composition of cyclic polyribonucleotides compared to the level of proteins in the animal After providing the product and the second composition to the subject (e.g., mammal), A method for expressing a level of protein in a circulating polynucleotide encoding the protein is A first composition of ribonucleotides is provided (e.g., administered) to a subject (e.g., a mammal). a step of administering to a subject (e.g., a mammal) a first portion of a cyclic polyribonucleotide; The composition contains a certain level of protein after being provided (e.g., administered). and administering a second composition of cyclic polyribonucleotide to the subject (e.g., providing (e.g., administering) a subject (e.g., mammal) to However, after the second composition of cyclic polyribonucleotide is provided (e.g., administered) the level of the protein changes by no more than 20% of the level of the normal protein; A first composition and a second composition of linear equivalents of ribonucleotides were provided (e.g., compared to the level of the protein in a subject (e.g., a mammal) after administration Thus, a first composition and a second composition of cyclic polyribonucleotides are provided (e.g., maintains the level of expression of the protein in a subject (e.g., a mammal) after administration The method includes the step of:

[0150] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and The level of the circular polyribonucleic acid in the cells after administration of the composition of claim 2 is compared to the level of the protein in the cells after administration of the composition of claim 2. After providing the cells with the first composition and the second composition of nucleotides, The method for expressing a level of quality is to first synthesize a protein-encoding cyclic polyribonucleotide. providing a composition of the formula (I) to a cell, wherein the cell receives a first sequence of the cyclic polyribonucleotide and providing the first composition to the cells, comprising the level of the protein after the composition is provided; providing a second composition of cyclic polyribonucleotides after the cells have been subjected to 1%, 5%, 15% of the level after the second composition of cyclic polyribonucleotides is provided , of proteins that vary by less than 20%, 25%, 30%, 35%, 40%, or 50% a step of generating a first set of linear equivalents of a circular polyribonucleotide, and comparing the levels of the protein in the cells after providing the composition and the second composition. After providing the first and second compositions of polyribonucleotides, the transcription factor in the cells is The method includes maintaining the level of expression of the protein.

[0151] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and and comparing the protein levels in a subject (e.g., a mammal) after providing the two compositions. and administering the first and second compositions of cyclic polyribonucleotides to a subject (e.g., a mammal). and expressing a certain level of the protein in the subject (e.g., mammal) after providing the protein to the subject (e.g., mammal). The method comprises: providing a first composition of cyclic polyribonucleotides encoding a protein to a subject; providing (e.g., administering) a subject (e.g., a mammal) with a mammal) to which a first composition of cyclic polyribonucleotides has been provided (e.g., administered) and a second step of cyclic polyribonucleotides, This composition is provided (e.g., administered) to the subject (e.g., mammal) after the first composition. a step of chromatinizing a second set of cyclic polyribonucleotides in a subject (e.g., a mammal) The composition may be at 1%, 5%, 15%, 20%, or 30% of the level after delivery (e.g., administration). including protein levels that change by less than 25%, 30%, 35%, 40%, or 50%. thereby producing a first composition of linear equivalents of cyclic polyribonucleotides and in a subject (e.g., a mammal) after the composition of claim 2 has been provided (e.g., administered) The first composition and the second composition of cyclic polyribonucleotides are compared to the level of the protein. After the composition is provided (e.g., administered), the tumor in the subject (e.g., mammal) The method includes maintaining the level of expression of the protein.

[0152] Furthermore, the second composition may be provided after the first composition is provided and after the first composition is produced. The level of the protein to be detected is substantially undetectable in a cell or a subject (e.g., a mammal). In some embodiments, the second composition may be provided after the first composition has been administered. The level of a protein in a cell or subject (e.g., a mammal) produced by At least 1 minute, 1 hour, 1 day, 2 days, 3 days, or 4 days after becoming qualitatively undetectable 12 months, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months month, 5 months, 6 months, 8 months, 10 months, or 1 year, the cell or subject (e.g., a mammal) In one embodiment, the second composition is a compound produced by the first composition. The level of the protein in the cell or subject (e.g., mammal) is substantially undetectable. After becoming functional, cells or subjects (e.g., In one embodiment, the second composition is provided to a mammal after the first composition. , at least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks , 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months , 10 months, or 1 year, and after the first composition, 20 years, 15 years, or 10 years In one embodiment, the second set is provided to a cell or a subject (e.g., a mammal). The composition may be administered to a cell or subject (e.g., a mammal) for a period of 1 minute to 20 years, or any time in between. provided to animals.

[0153] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and Levels of linear equivalents of cyclic polyribonucleotides in cells after providing the composition of claim 2 providing a first composition and a second composition of cyclic polyribonucleotides to the cells, compared to The method for producing a level of cyclic polyribonucleotides in cells after the providing a first composition of nucleotides to the cells, wherein the cells ingest the first composition. providing a step of providing a level of a circular polyribonucleotide; and providing a second composition of nucleotides to the cells, wherein the cells are capable of absorbing at least the second composition of nucleotides. a step of increasing the level of cyclic polyribonucleotides after providing the composition; After providing the first and second compositions of linear equivalents of ribonucleotides, The first composition and the second composition of cyclic polyribonucleotides are compared to the level of their linear counterparts in the cells. and maintaining the level of cyclic polyribonucleotides in the cells after providing the second composition. In one embodiment, the first linear equivalent of the cyclic polyribonucleotide is Linear synthesis of circular polyribonucleotides in cells after providing the composition and the second composition. The first and second compositions of cyclic polyribonucleotides are compared to equivalent levels. A method for producing a level of cyclic polyribonucleotides in a cell after providing the cell with the cyclic polyribonucleotides comprises: providing a first composition of cyclic polyribonucleotides to the cells, after the composition of claim 1 is provided, comprising a level of cyclic polyribonucleotide; and providing a second composition of cyclic polyribonucleotides to the cells, wherein the cells and providing a second composition, wherein the level of the polyribonucleotides is changed by 20% or less. a step of synthesizing the linear structure of the circular polyribonucleotide, thereby Level of linear equivalent in cells after providing first and second compositions of equivalents after the first and second compositions of cyclic polyribonucleotides are provided, , including maintaining the level of cyclic polyribonucleotides in the cell.

[0154] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and cyclic polyribonucleotides in a subject (e.g., a mammal) after providing the composition of claim 2 the first composition of cyclic polyribonucleotides and the second composition of cyclic polyribonucleotides compared to the level of their linear equivalents. After providing the composition to a subject (e.g., a mammal), A method for producing a level of cyclic polyribonucleotides includes: providing (e.g., administering) the first composition to a subject (e.g., a mammal); Thus, a subject (e.g., a mammal) is provided (e.g., administered) with a first composition. after the step of containing a level of circular polyribonucleotide; and providing (e.g., administering) a second composition of the invention to a subject (e.g., a mammal); and wherein the subject (e.g., a mammal) is provided with at least a second composition (e.g., , administered) to the patient; thereby A first composition and a second composition of linear equivalents of ribonucleotides were provided (e.g., compared to the linear equivalent level in a subject (e.g., a mammal) after Thus, a first composition and a second composition of cyclic polyribonucleotides are provided (e.g., (administered) in a subject (e.g., a mammal) In one embodiment, the linear equivalent of the cyclic polyribonucleotide is maintained. and administering to a subject (e.g., a mammal) a first composition of matter and a second composition of matter. of cyclic polyribonucleotides compared to the levels of their linear counterparts After providing the first composition and the second composition to a subject (e.g., a mammal), the subject (e.g., The method for producing levels of cyclic polyribonucleotides in a mammal (e.g., a mammal) includes the steps of: A first composition of polyribonucleotides is provided to a subject (e.g., a mammal, e.g., a human). providing (e.g., administering) a first composition to a subject (e.g., a mammal) is provided (e.g., administered) after which a certain level of cyclic polyribonucleotides is and administering a second composition of cyclic polyribonucleotides to a subject (e.g., a mammal). providing (e.g., administering) a cyclic polylysine monophosphate (PPMP) to a subject (e.g., a mammal) A second composition was provided (e.g., a 20% or less variation in the level of polynucleotides). , administered) to the circular polyribonucleic acid; A first composition and a second composition of linear equivalents of methicillin-3-one are provided (e.g., administered the level of the cyclic polylysine as compared to the level of its linear equivalent in a subject (e.g., a mammal) after A first composition and a second composition of polynucleotides are provided (e.g., administered) and then maintaining the level of cyclic polyribonucleotides in the subject (e.g., mammal). Includes the process.

[0155] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and Levels of linear equivalents of cyclic polyribonucleotides in cells after providing the composition of claim 2 providing a first composition and a second composition of cyclic polyribonucleotides to the cells, compared to The method for producing a level of cyclic polyribonucleotides in cells after the providing a first composition of nucleotides to a cell, wherein the cell detects that the first composition and providing a step of providing a cyclic polyribonucleotide containing a level of cyclic polyribonucleotide; providing a second composition of nucleotides to the cells, wherein the cells 1%, 5%, 15%, 20%, 25%, 30%, 35%, 40% of nucleotide levels or the level of the protein after the second composition is provided changes by 50% or less. , whereby a first composition of linear equivalents of a cyclic polyribonucleotide and a second composition of linear equivalents of a cyclic polyribonucleotide are obtained. the level of circular polyribonucleic acid compared to the linear equivalent in cells after providing the composition Circular polyribonucleic acid in cells after providing the first and second compositions of ribonucleotides. The method includes maintaining the level of leutinone in the patient.

[0156] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and cyclic polyribonucleotides in a subject (e.g., a mammal) after providing the composition of claim 2 the first composition of cyclic polyribonucleotides and the second composition of cyclic polyribonucleotides compared to the level of their linear equivalents. After providing the composition to a subject (e.g., a mammal), the subject (e.g., a mammal, e.g., A method for producing levels of cyclic polyribonucleotides in humans is A first composition of nucleotides is provided (e.g., administered) to a subject (e.g., a mammal). a subject (e.g., a mammal) to which a first composition has been provided (e.g., and administering to the subject a level of cyclic polyribonucleotides containing the cyclic polyribonucleotides; A second composition of polynucleotides is provided (e.g., administered) to a subject (e.g., a mammal). wherein the subject (e.g., mammal) is 1%, 5%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% or less of The level of the protein after the second composition is provided (e.g., administered) changes. thereby producing a first composition of linear equivalents of cyclic polyribonucleotides; and and the level of linear equivalents in the subject (e.g., mammal) after providing the second composition. In comparison, after providing the first and second compositions of cyclic polyribonucleotides, the subject maintaining levels of cyclic polyribonucleotides in a mammal (e.g., a mammal). .

[0157] In certain embodiments, the cyclic polyribonucleotide is an exogenous synthetic cyclic polyribonucleotide. In one embodiment, the cyclic polyribonucleotide is a polyribonucleotide having a polyA sequence, a polynucleotide having ... lacking the manufacturing element, or both.

[0158] In one embodiment, the first composition comprises a first cyclic polyribonucleotide, The second composition comprises a second circular polyribonucleotide, wherein the first circular polyribonucleotide The nucleotide and the second cyclic polyribonucleotide are the same. The first composition comprises a first cyclic polyribonucleotide and the second composition comprises a second cyclic polyribonucleotide. a first circular polyribonucleotide and a second circular polyribonucleotide, Polyribonucleotides are different.

[0159] Additionally, the second composition may be administered after providing the first composition and after removing the cells or The level of cyclic polyribonucleotides in a subject (e.g., a mammal, e.g., a human) , substantially undetectable in a cell or subject (e.g., a mammal, e.g., a human) In some embodiments, the second composition may be provided by the first composition. The level of cyclic polyribonucleotides in a cell or subject (e.g., a mammal) is , after which it becomes virtually undetectable for at least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year, cells or subjects (e.g., mammals) In one embodiment, the antibody is provided to (e.g., administered to) an animal, e.g., a human. The second composition may be used in a cell or subject (e.g., a mammal) produced by the first composition. After the level of cyclic polyribonucleotides in the 0 years, or any time therebetween, cells or subjects (e.g., mammals, e.g., humans) In some embodiments, the second composition is provided (e.g., administered) to the first After applying the composition, apply it for at least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year, and after the first composition, 20 years, 15 years, or The gene is provided to a cell or a subject (e.g., a mammal, e.g., a human) for less than 10 years. In embodiments, the second composition may be used for 1 minute to 20 years, or any time therebetween. provided (e.g., administered) to a cell or a subject (e.g., a mammal, e.g., a human) .

[0160] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and and the level of binding in the cells after providing the circular polyribonucleic acid composition of claim 2. A method of providing a first composition and a second composition of peptide to a cell and then binding a target within the cell. The method comprises providing to a cell a first composition of cyclic polyribonucleotides encoding a protein. the cells are provided with a first composition of cyclic polyribonucleotides, and providing the first composition to the cells, followed by administering the circular polyribonucleic acid to the cells. providing a second composition of peptides, wherein the cells are capable of expressing at least the circular polyribonucleic acid a step of providing a second composition of oxidase and then attaching the oxidase to the second composition; after providing the cells with a first composition and a second composition of linear equivalents of ribonucleotides. the level of binding in the first and second compositions of cyclic polyribonucleotides compared to the level of binding in the first and second compositions of cyclic polyribonucleotides. After providing the composition, the step of maintaining expression of the level of binding in the cells is included. In this state, a first composition and a second composition of linear equivalents of a cyclic polyribonucleotide are The level of cyclic polyribonucleotides in the cells after delivery was compared to the level of proteins in the cells after delivery. The method of providing a first composition and a second composition to a cell and then binding to a target within the cell includes the steps of: providing a cell with a first composition of cyclic polyribonucleotides encoding a protein; The method further comprises providing a cell with a first composition of cyclic polyribonucleotides followed by a step of: and providing the first composition to the cells, followed by the step of: providing a second composition of cyclic polyribonucleotides, wherein the cells the level of binding changes by no more than 20% of the level after providing the composition; This allows for the formation of a first composition and a second composition of linear equivalents of cyclic polyribonucleotides. The level of cyclic polyribonucleotides in the cells after delivery was compared to the level of proteins in the cells after delivery. maintaining the level of binding in the cells after providing the first composition and the second composition. Includes:

[0161] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and and comparing the level of binding in the subject (e.g., mammal) after providing the two compositions. A first composition and a second composition of cyclic polyribonucleotides are administered to a subject (e.g., a mammal). and subsequently binding a target in a subject (e.g., a mammal, e.g., a human). a first composition of cyclic polyribonucleotides encoding a protein, providing (e.g., administering) a subject (e.g., a mammal, e.g., a human) with a mammal) to which a first composition of cyclic polyribonucleotides has been provided (e.g., administered and a second step of attaching the circular polyribonucleotide to the cyclic polyribonucleotide, which includes a level of attachment after the cyclic polyribonucleotide is attached to the cyclic polyribonucleotide. The composition is provided (e.g., administered) to the subject (e.g., mammal) after the first composition. ) a step of administering to a subject (e.g., a mammal) at least a cyclic polyribonucleotide a step of forming a circular polyribonucleotide, the step comprising: A first composition and a second composition of linear equivalents of nucleotides are provided (e.g., administration the level of binding in the subject (e.g., mammal) after After the first and second compositions of nucleotides are provided (e.g., administered), In some embodiments, the method includes maintaining expression of the level of binding in the subject (e.g., mammal). In embodiments, a first composition and a second composition of linear equivalents of cyclic polyribonucleotides are the level of the protein in the subject (e.g., mammal) after providing the a first composition of polyribonucleotides and a second composition of polyribonucleotides in a subject (e.g., a mammal); After providing, the method of binding to a target in a subject (e.g., a mammal) comprises: A first composition of cyclic polyribonucleotides to be loaded is administered to a subject (e.g., a mammal, e.g., providing (e.g., administering) a therapeutic agent to a subject (e.g., a mammal) comprising: After the first composition of cyclic polyribonucleotides is provided (e.g., administered), and a second composition of cyclic polyribonucleotides, After the first composition is provided (e.g., administered) to a subject (e.g., a mammal). Thus, the subject (e.g., mammal) receives a second composition of cyclic polyribonucleotides, The level of binding varies by no more than 20% of the level after administration (e.g., administration) , whereby a first composition of linear equivalents of a cyclic polyribonucleotide and a second composition of linear equivalents of a cyclic polyribonucleotide are obtained. and determining whether the protein is present in a subject (e.g., a mammal) after providing (e.g., administering) the composition. The first and second compositions of cyclic polyribonucleotides are compared to the level of the protein. After providing (e.g., administering), the level of binding in the subject (e.g., mammal) is maintained. The method includes a step of holding the

[0162] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and and the level of binding in the cells after providing the circular polyribonucleic acid composition of claim 2. After providing the cells with the first and second compositions of peptides, a certain level of The method for generating the linkage comprises providing a first composition of cyclic polyribonucleotides comprising a linkage site. to a cell, wherein the cell detects a first composition of cyclic polyribonucleotides after providing, including a level of binding; and after providing the first composition to the cell, Providing a second composition of cyclic polyribonucleotides, wherein the cells 1%, 5%, 15%, 20% of the level after the second composition of polynucleotides is provided; including levels of binding that vary by no more than 25%, 30%, 35%, 40%, or 50%. whereby a first composition and a second composition of linear equivalents of cyclic polyribonucleotides are obtained. The level of binding in cells after administration of the cyclic polyribonucleotide was compared to that after administration of the composition. After providing the first composition and the second composition, maintaining the expression of the level of binding in the cells. This includes the step of:

[0163] In one embodiment, a first composition of linear equivalents of a cyclic polyribonucleotide and and comparing the level of binding in the subject (e.g., mammal) after providing the two compositions. A first composition and a second composition of cyclic polyribonucleotides are administered to a subject (e.g., a mammal). and subsequently binding a target in a subject (e.g., a mammal, e.g., a human). a first composition of cyclic polyribonucleotides encoding a protein, providing (e.g., administering) a subject (e.g., a mammal, e.g., a human) with a mammal) to which a first composition of cyclic polyribonucleotides has been provided (e.g., administered and a second step of attaching the circular polyribonucleotide to the cyclic polyribonucleotide, which includes a level of attachment after the cyclic polyribonucleotide is attached to the cyclic polyribonucleotide. The composition is provided (e.g., administered) to the subject (e.g., mammal) after the first composition. ) a step of administering to a subject (e.g., a mammal) at least a cyclic polyribonucleotide comprising the level of binding after the second composition has been provided (e.g., administered); Thereby, a first composition and a second composition of linear equivalents of cyclic polyribonucleotides the level of binding in a subject (e.g., a mammal) after providing the cyclic polylysine After providing the first and second compositions of polynucleotides, a subject (e.g., a mammal) In one embodiment, the cyclic polynucleotide comprises a step of maintaining the expression of the level of binding in the cyclic polynucleotide. After providing the subject (e.g., The level of cyclic polyribonucleotides is higher than that of proteins in mammals (e.g., mammals). After providing the first composition and the second composition to a subject (e.g., a mammal), the subject (e.g., The method for binding to a target in a mammal (e.g., a mammal) comprises the steps of: A first composition of leutide is provided (e.g., administered) to a subject (e.g., a mammal). a subject (e.g., a mammal) administering a first composition of cyclic polyribonucleotides to a subject (e.g., a mammal) contains a certain level of protein after being provided (e.g., administered); and A second composition of cyclic polyribonucleotides is administered to the subject (e.g., mammal) after the first composition. providing (e.g., administering) a cyclic Levels after the second composition of polyribonucleotides is provided (e.g., administered) the level of binding varies by no more than 20% of the total binding of the circular polyribonucleic acid;

[0023] The subject (e.g., a mammal) is administered a first composition of a linear equivalent of methicillin-3 (methicillin-3) and a second composition of methicillin-3 (methicillin-3) after administration of the first composition and the second composition of methicillin-3 (methicillin-3). The first set of cyclic polyribonucleotides is compared to the level of proteins in mammals (mammals). and a second composition, and then determining the level of binding in the subject (e.g., mammal). The method includes a step of maintaining the

[0164] Furthermore, the second composition may be provided after the first composition is provided and after the first composition is produced. The level of the protein to be detected is substantially undetectable in a cell or a subject (e.g., a mammal). In some embodiments, the second composition may be provided after the first composition has been administered. the level of binding in a cell or subject (e.g., a mammal) produced by After becoming undetectable, the device will not be detected for at least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year, the cells or the subject (e.g., a mammal, e.g., In one embodiment, the second composition is provided to (e.g., administered to) a subject (e.g., a human). is a measure of binding in a cell or subject (e.g., a mammal) produced by the first composition. After levels become virtually undetectable, for 1 minute to 20 years, or any time in between provided (e.g., administered) to a cell or a subject (e.g., a mammal, e.g., a human) In some embodiments, the second composition is applied for at least 1 minute after the first composition. Hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks 12 months, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year and for less than 20 years, 15 years, or 10 years after the first composition, the cell or subject (e.g., For example, the antibody may be provided (e.g., administered) to a mammal, e.g., a human. The second composition may be administered to cells or subjects for 1 minute to 20 years, or any time therebetween. provided to (e.g., administered to) (e.g., a mammal, e.g., a human).

[0165] In one embodiment, the re-administration regimen or method comprises a first composition and a second composition. The composition may be followed by one or more additional compositions of cyclic polyribonucleotides. In some embodiments, the one or more additional compositions may be a third, fourth, fifth, sixth, seventh, eighth, ninth, or In the re-administration, one or more additional compositions may be administered in the same manner as in the previous administration. Circular polyribonucleotides produced by the composition after providing the composition and by the composition before providing the composition, binding or the level of the protein is substantially undetectable in multiple cells (e.g., a subject). For example, one or more additional compositions may be provided after a previous composition has been produced. the cyclic polyribonucleotide formed, the bond formed by the previous composition, or the previous composition at least after the level of the protein expressed by the Also 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months , or multiple times over a period of one year. In re-administration, one or more additional compositions may be administered in addition to the previous composition. For example, one or more additional compositions may be provided after providing a previous composition. At least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, or 7 days after administration , 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months The cells may be provided to a subject (e.g., a mammal) for 1, 8 months, 10 months, or 1 year. In some embodiments, one or more additional compositions may be administered within 1 minute to 30 minutes after providing the previous composition. In one embodiment, the antibody is provided to a cell or a subject (e.g., a mammal) for 20 years. One or more additional compositions are administered at least one minute, one hour, one day after administering the previous composition. , 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks , 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year, provided to a cell or subject (e.g., a mammal) for 20 years, 15 years, or 10 years or less do.

[0166] In certain embodiments, the second composition is a compound that is capable of inhibiting the growth of a cell or a cell surface in a subject (e.g., a mammal). The protein level is approximately the protein level before administering or providing the first composition. administered or provided to a cell or subject (e.g., a mammal, e.g., a human) after returning to the cell. In certain embodiments, the second composition is a cytosolic agent in a cell or a subject (e.g., a mammal). The level of the protein is approximately the level of the protein before administering or providing the first composition. After returning to the room, days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 provided to a cell or subject (e.g., a mammal) for 1 month, 8 months, 10 months, or 1 year In one embodiment, the third composition of the one or more additional compositions is a cell or subject (e.g., For example, the level of a protein in a mammal (e.g., a mammal) is determined before administering or providing the first composition. After the level of the protein has returned to approximately In one embodiment, a third composition of the one or more additional compositions is provided. The level of the protein in the cell or subject (e.g., mammal) is determined after administering the first composition or After returning to approximately pre-serving protein levels, continue for at least 1 minute, 1 hour, and 1 Days, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year, cells or is administered or provided to a subject (e.g., a mammal). In some embodiments, one or more a fourth, fifth, sixth, seventh, eighth, ninth, tenth or more of the additional compositions The level of the protein in the cell or subject (e.g., mammal) is determined by administering the first composition. After the protein level returns to approximately the level before administration or provision, the cells or subject (e.g., mammal) In some embodiments, the first of the one or more additional compositions is administered or provided to a mammal. The fourth, fifth, sixth, seventh, eighth, ninth, tenth or more compositions are administered to a cell or subject (e.g., The level of the protein in a mammal (e.g., a mammal) is determined based on the level of the protein in the mammal (e.g., a mammal) prior to administering or providing the first composition. After returning to approximately normal protein levels, continue for at least 1 minute, 1 hour, 1 day, 2 days, 3 days. days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year, cells or subjects (e.g., In one embodiment, the composition described above is administered or provided to a cell. or the level of the protein in the subject (e.g., mammal) is determined after administering the first composition or After returning to approximately the pre-donation protein level, cells or subjects (e.g., a mammal)

[0167] In certain embodiments, the second composition is administered or administered to the cells after providing the first composition. In one embodiment, the second composition is provided at least a short time after the first composition is provided. At least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 In some embodiments, the one or more additional compositions are provided to the cells for a period of time, such as one month, or one year. The third composition of matter is administered or provided to the cells after providing the first composition. In some embodiments, the third composition of one or more additional compositions is provided after the first composition. , at least 1 minute, 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks , 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months In some embodiments, one or more a fourth, fifth, sixth, seventh, eighth, ninth, tenth or more compositions of the additional composition is administered or provided to the cells after providing the first composition. a fourth, fifth, sixth, seventh, eighth, ninth, tenth or more of one or more additional compositions The composition is applied for at least 1 minute, 1 hour, 1 day, 2 days, 3 days after applying the first composition. 12 months, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months administered or provided to cells for one month, four months, five months, six months, eight months, ten months, or one year In one embodiment, the composition described above is applied for 1 minute to 2 minutes after providing the first composition. Administered or provided to a cell or subject (e.g., a mammal) for 20 years.

[0168] In some embodiments, the composition is administered to a cell or a subject (e.g., a mammal) by injecting the composition. and then, after a time interval, administering or providing the compound to a cell or a subject (e.g., a mammal). For example, the second composition may be used to administer the first composition to a cell or a subject (e.g., a mammal). After a first time interval, the cell or subject (e.g., mammal) the third composition may be administered or provided to a cell or subject (e.g., a mammal) and after a second time interval, the cell or subject (e.g., mammal) the fourth composition may be administered or provided to a cell or subject (e.g., a mammal) by administering the third composition to the cell or subject (e.g., a mammal) and after a third time interval, administering or providing the compound to the cell or subject (e.g., mammal) or a fifth, sixth, seventh, eighth, ninth, or more compositions administering a fourth, fifth, sixth, seventh, eighth, ninth, or more compositions to a cell or subject (e.g., mammal) after administration or provision thereto, and After a time interval of 10 or more, the compound is administered or provided to a cell or a subject (e.g., a mammal). The first time interval is a time interval during which the level of a protein in a cell or a subject (e.g., a mammal) is measured. The amount of protein required for the blood to return to approximately the level prior to administering or providing the first composition. The amount of time may be longer than the recommended amount.

[0169] In some embodiments, the second time interval is longer than the first time interval. In one embodiment, the third time interval is longer than the first time interval. In one embodiment, the fifth, sixth, seventh, eighth, and seventh time intervals are longer than the first time interval. The ninth or more time intervals are longer than the first time interval. The second time interval is the same as the first time interval. In one embodiment, the third time interval In one embodiment, the fourth time interval is the same as the first time interval. In some embodiments, the fifth, sixth, seventh, eighth, ninth, or any of the preceding intervals is the same as the interval between adjacent cells. The above time interval is the same as the first time interval. In some embodiments, the second time interval In one embodiment, the third time interval is shorter than the first time interval. In one embodiment, the fourth time interval is shorter than the first time interval. In some embodiments, the fifth, sixth, seventh, eighth, ninth, or more time intervals are In one embodiment, the second time interval is longer than the first time interval. In one embodiment, the third time interval is longer than the second time interval. In the fourth time interval, the fourth time interval is longer than the third time interval.

[0170] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of the protein in the cell or subject (e.g., mammal) after provision is at least 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, or 30 days This is maintained.

[0171] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of the protein in the cell or subject (e.g., mammal) after provision is determined to be and a second composition of linear equivalents of ribonucleotides. is at least 5%, 10%, or 20%, 30%, 40%, 50%, or 60% higher.

[0172] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of the protein in the cell or subject (e.g., mammal) after provision is determined to be After providing the second composition of ribonucleotides, the patient is then given a 2-day, 3-day, 4-day, or days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 30 days, 40 days, or 45 days, the cell or subject (e.g., mammal) after providing the first and second compositions of the same ) at least 5%, 10%, 20%, 30%, 40% lower than the protein level in 50% or 60% higher.

[0173] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The release of cyclic polyribonucleotides in cells or subjects (e.g., mammals) after provision Bell is at least 1 hour, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days 1, 6, 7, 8, 9, 10, 15, 20, 25 days, or It will be maintained for 30 days.

[0174] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The release of cyclic polyribonucleotides in cells or subjects (e.g., mammals) after provision Bell provides a first composition and a second composition of linear equivalents of cyclic polyribonucleotides. Linear synthesis of circular polyribonucleotides in a cell or subject (e.g., a mammal) after At least 5%, 10%, 20%, 30%, 40%, 50%, or 60% lower than the equivalent level %expensive.

[0175] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of circular polyribonucleic acid in the plurality of cells after providing the for at least 1 day, 2 days, 3 days, 4 days, 5 days after administering the second composition of 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days , 40 days, or 45 days, The linear alignment of the circular polyribonucleotides in the plurality after providing the composition and the second composition. At least 5%, 10%, 20%, 30%, 40%, 50%, or 60% lower than the equivalent level %expensive.

[0176] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of binding in the cell or subject (e.g., mammal) after provision is at least 1 Hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, Maintained for 8, 9, 10, 15, 20, 25, or 30 days It will be held.

[0177] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of binding in the cells or subject (e.g., mammal) after provision is determined based on the circular polyribonucleotide. a cell or subject after providing a first composition and a second composition of linear equivalents of nucleotides; (e.g., mammals) %, 40%, 50%, or 60% higher.

[0178] In one embodiment, a first composition and a second composition of cyclic polyribonucleotides are The level of circular polyribonucleic acid in the plurality of cells after providing the for at least 1 day, 2 days, 3 days, 4 days, 5 days after administering the second composition of 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days , 40 days, or 45 days, at least 5% higher than the level of binding in the plurality after providing the composition and the second composition; 10%, 20%, 30%, 40%, 50%, or 60% higher.

[0179] cyclic polyribonucleotide The cyclic polyribonucleotides and compositions or pharmaceutical compositions thereof described herein may be used in combination with other cyclic polyribonucleotides. In multiple cells after providing at least two doses of cyclic polyribonucleotide to a number of at the level of cyclic polyribonucleotide, at the level of binding to the target, or at the level of the protein. The invention can be used in therapeutic and veterinary methods of administration to generate In some embodiments, the cyclic polyribonucleotide is non-immunogenic in mammals, e.g., humans. In one embodiment, the cyclic polyribonucleotide is derived from aquaculture animals (fish, crabs, cells derived from fish (e.g., shrimp, oysters, etc.), mammalian cells, e.g., pet or zoo animals Cells derived from animals (cats, dogs, lizards, birds, lions, tigers, and bears, etc.), livestock or or cells derived from working animals (horses, cows, pigs, chickens, etc.), human cells, cultured cells , primary cells or cell lines, stem cells, progenitor cells, differentiated cells, embryonic cells, cancer cells (e.g. , tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells capable of replicating in mitotic cells, non-mitotic cells, or any combination thereof. In one embodiment, the present invention provides a method for producing a cyclic nucleotide sequence as described herein. The present invention also includes cells containing polyribonucleotides, wherein the cells are derived from aquaculture animals (fish, crabs, larvae, etc.). cells derived from animals such as sea cucumbers, oysters, etc.; mammalian cells, e.g., from pet or zoo animals ( cells derived from animals such as cats, dogs, lizards, birds, lions, tigers, and bears, livestock, or cells derived from working animals (horses, cows, pigs, chickens, etc.), human cells, cultured cells, Primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g. , tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells In some embodiments, the cells are mitotic, non-mitotic, or any combination thereof. In this case, the cells are modified to contain the circular polyribonucleotide.

[0180] In certain embodiments, the cyclic polyribonucleotide comprises a sequence for an expression product. In some embodiments, the cyclic polyribonucleotide comprises a binding site for binding to a target. In certain embodiments, the cyclic polyribonucleotide is administered according to the methods described herein. In some embodiments, the antibody may be administered to multiple cells either by staggered administration or re-administration methods. In some embodiments, the cyclic polyribonucleotides described herein can be used to induce a response or In one embodiment, the cyclic polyribonucleotide The expression product encoded by the sequence is expressed in one or more of the cells in the plurality of cells. will be done.

[0181] In certain embodiments, the cyclic polyribonucleotide has at least one linear equivalent, e.g., For example, the half-life of a linear expression sequence or a linear circular polyribonucleotide. In embodiments, cyclic polyribonucleotides have increased half-lives over their linear counterparts. In certain embodiments, the half-life is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more longer. The cyclic polyribonucleotide is then allowed to stand for at least about 1 hour to about 30 days, or for at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, A half-life within cells of 60 days or more, or any time in between. In certain embodiments, the cyclic polyribonucleotide has a long-lasting action. Less than 1 hour to about 7 days, or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours Hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days or less, or It has a half-life or persistence within the cell over any time period therebetween. In this state, cyclic polyribonucleotides have a long half-life or In some embodiments, the cyclic polyribonucleotide is persistent after cleavage. In certain embodiments, the circular polyribonucleic acid has an intracellular half-life or persistence. The dose may be from more than about 10 minutes to about 30 days, or at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th for 30 days, 60 days, or more, or any time in between. It has a half-life or persistence within the cells in which it is administered.

[0182] In certain embodiments, cyclic polyribonucleotides can inhibit cellular function, e.g., temporarily or In certain embodiments, the therapeutic effect is controlled for a long period of time, from at least about 1 hour to about 30 days or less. At least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 1 4 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 2 2 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 3 Sustained for 0 days, 60 days or more, or any time in between In certain embodiments, a cellular function, such as regulating or inhibiting a cellular signal, is stably altered. 30 minutes or less to approximately 7 days or approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours Between, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days or less Cellular functions, such as regulation, that persist for any period of time between these events are temporarily altered. .v

[0183] In one embodiment, the cyclic polyribonucleotide is at least about 20 nucleotides. , at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides 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 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 00 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides 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 certain embodiments, the cyclic polyribonucleotide has a binding site for a ribosome. Those skilled in the art will appreciate that the size of the circular polyribonucleotide may be sufficient to accommodate The maximum size is the size that produces a circular polyribonucleotide and / or It is understood that the size can be somewhat large within the technical constraints of the code. Without being bound by theory, it is believed that multiple segments of RNA can be synthesized in a manner similar to DNA. and their 5' and 3' free ends annealed to produce a "string" of RNA (and These are finally circularized when only one 5' free end and one 3' free end remain. In some embodiments, the cyclic polyribonucleic acid may be produced from a cyclic polyribonucleic acid. The maximum size of a nucleotide is limited by its ability to package and deliver RNA to its target. In certain embodiments, the size of a circular polyribonucleotide may be limited to a useful polyribonucleotide. It is long enough to encode a peptide and therefore is at least 20,000 nucleotides long. nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides; at least 7,500 nucleotides or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides Nucleotide, at least 1,000 nucleotides, at least 500 nucleotides, At least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides A length of 100 nucleotides or at least 100 nucleotides may be useful.

[0184] In one embodiment, the cyclic polyribonucleotide comprises one or more expression sequences, In one embodiment, the vector is configured for sustained expression in the cells of a subject in vivo. The circular polyribonucleotides are then used to direct the expression of one or more expression sequences in the cell at a later time point. In such an embodiment, the time is configured to be equal to or greater than the previous time point. Expression of one or more expression sequences may be maintained at a relatively stable level or may decrease over time. The expression of the expression sequence may be relatively stable over an extended period of time. For example, In some cases, at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 Expression of one or more expression sequences in cells for 23 days or longer is 0%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% reduction In some cases, the expression of one or more expression sequences in a cell Expression continues for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, and 23 days. or more, 50%, 45%, 40%, 35%, 30%, 25%, 20%, It is maintained at a level that does not vary by more than 15%, 10%, or 5%.

[0185] Expression sequence After providing the cells with at least two compositions of cyclic polyribonucleotides, Methods of administration that produce levels of protein from expression sequences in the disclosed , where the circular polyribonucleotide encodes a protein.

[0186] In some embodiments, the cyclic polyribonucleotide encodes a peptide or polypeptide. Such peptides include, but are not limited to, However, small peptides, peptidomimetics (e.g., peptoids), amino acids, and amino acids Peptides may be linear or branched. The molecular weight of the polymer is less than about 5,000 grams per mole, and less than about 2,000 grams per mole. Molecular weight less than about 1,000 grams per mole, Molecular weight less than about 500 grams per mole and salts, esters and other pharmaceutically acceptable forms of such compounds. Such peptides include, but are not limited to, neurotransmitters, hormones, and the like. drugs, toxins, viral or microbial particles, synthetic molecules and their agonists or antagonists antagonists are included.

[0187] The polypeptide may be linear or branched. 00 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15, 000 amino acids, approximately 200 to approximately 10,000 amino acids, approximately 500 to approximately 5,000 amino acids The length may be about 1,000 to about 2,500 amino acids, or any range therebetween. In certain embodiments, the polypeptide is less than about 40,000 amino acids, about 35,000 less than 0 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, Less than 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids 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 2,0 less than about 1,000 amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 9 less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids Acid or shorter lengths may be useful.

[0188] Some examples of peptides or polypeptides include, but are not limited to, those with fluorescent tags or Markers, antigens, peptide therapeutics, synthetic or analogue peptides derived from natural bioactive peptides peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides peptides, bicyclic peptides, targeting or cytotoxic peptides, degradative or self-immolative peptides and and multiple degrading or self-immolating peptides. Peptides useful in this regard include antigen-binding peptides, e.g., antigen-binding antibodies or antibody-like fragments. Also included are antibodies, such as single chain antibodies, nanobodies (see, e.g., Steeland et al. al.2016.Nanobodies as therapeutics:big o Portunities for small antibodies.Drug D (See Iscov Today:21(7):1076-113.) The antigen-binding peptide may bind to a cytoplasmic antigen, a nuclear antigen, or an intraorganellar antigen.

[0189] In one embodiment, the cyclic polyribonucleotide comprises one or more RNA expression sequences. Each of these can encode a polypeptide. Thus, a polypeptide can be any protein molecule that can be produced. The polypeptide may be secreted from the cell or may be confined to the cytoplasm, nucleus, or membrane compartment of the cell. Some polypeptides may be polypeptides that can be localized. However, at least a portion of the viral envelope protein, metabolic regulatory enzymes (e.g., lipid or regulates steroid production), antigens, tolerogens, cytokines, toxins, and their absence can lead to disease enzymes associated with the polypeptides, and polypeptides that are not active in animals (e.g., in the animal intestine) until cleaved. Examples include peptides, and hormones.

[0190] In certain embodiments, the cyclic polyribonucleotide is a protein, e.g., a therapeutic protein. In one embodiment, the expression product of the expression sequence is a target protein. In some embodiments, the polypeptides disclosed herein are proteins, such as therapeutic proteins. Therapeutic proteins that can be expressed from cyclic polyribonucleotides containing ribonucleotides have antioxidant activity, binding , cargo receptor activity, catalytic activity, molecular carrier activity, molecular function regulator, molecular transducer activity, nutrient storage activity, protein tag, structural molecule activity, toxic activity, transcriptional regulation activity, translational regulation Some examples of therapeutic proteins include, but are not limited to, However, enzyme replacement proteins, replacement proteins, protein vaccinations, antigens (e.g. tumor antigens, viruses, bacteria), hormones, cytokines, antibodies, immunotherapy (e.g., cancer ), cell reprogramming / transdifferentiation factors, transcription factors, chimeric antigen receptors, transposases enzymes or nucleases, immune effectors (e.g., susceptibility to immune responses / signals) regulated death effector proteins (e.g., apoptosis or catabolism) death inducers), non-lytic inhibitors of tumors (e.g., inhibitors of oncoproteins), epigenetics Transcription factors, epigenetic enzymes, DNA or protein modifying enzymes enzymes, DNA intercalators, efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors enzyme inhibitors, competitive inhibitors for enzymes, protein synthesis effectors or inhibitors, nuclease inhibitors ases, protein fragments or domains, ligands or receptors, and CRs In some embodiments, the therapeutic target is an ISPR system or a component thereof. In certain embodiments, the antigen is a tumor antigen, a bacterial antigen, or a viral antigen. It is a virus antigen.

[0191] In some embodiments, the cyclic polyribonucleotides disclosed herein are expressed as Exemplary proteins that can be obtained include human proteins, e.g., receptor-binding proteins, proteins, growth factors, growth factor receptor modulators, and regenerative proteins (e.g., proliferation and proteins involved in cell differentiation, e.g., therapeutic proteins for wound healing). In certain embodiments, the cyclic polyribonucleotides disclosed herein are expressed An exemplary protein that can be used is EGF (epidermal growth factor). Exemplary polyribonucleotides that can be expressed from the cyclic polyribonucleotides disclosed herein are: Examples of proteins include enzymes, e.g., oxidoreductase enzymes, metabolic enzymes, and mitochondrial enzymes. Dehydrogenases, oxygenases, dehydrogenases, ATP-independent enzymes, and desaturases In certain embodiments, the circular polyribonucleases disclosed herein are Exemplary proteins that can be expressed from the tides include intracellular or cytoplasmic proteins. In certain embodiments, the cyclic polyribonucleotide is a phenylalanine or phenylalanine polyribonucleotide. In one embodiment, the cyclic polyribonucleotide expresses a cysteine ​​hydroxylase. , expressing NanoLuc® luciferase (nLuc). Exemplary proteins that can be expressed from the cyclic polyribonucleotides disclosed herein are: Proteins include secreted proteins, such as secreted enzymes. In some cases, the cyclic polyribonucleotide expresses erythropoietin. Polyribonucleotides are secreted proteins that may have a therapeutically short half-life in the blood. or expressing a protein having a subcellular localization signal or a secretion signal peptide. In one embodiment, the cyclic polyribonucleotide may be a protein having a cyclic nucleotide. In one embodiment, the vectors described herein express Gaussia luciferase (gLuc). Exemplary proteins that can be expressed from the cyclic polyribonucleotides disclosed in In some embodiments, the polyribonucleotides may be circular or transmembrane proteins. Nucleotides are expressed by transmembrane receptors, e.g., G protein-coupled receptors (GPCRs), receptors Expresses tyrosine kinase (RTK), antigen receptor, or chimeric antigen receptor. In the present invention, the cyclic polyribonucleotide is linked to a non-human protein, such as a fluorescent protein, Energy transfer receptors or protein tags such as Flag, Myc, or His In certain embodiments, exemplary sequences that can be expressed from a cyclic polyribonucleotide are: Suitable proteins include GFP. In one embodiment, the circular polyribonucleic acid The protease may be a tagged protein, e.g., a fusion protein containing a protein tag or Modified proteins, e.g., chitin-binding protein (CBP), maltose-binding protein (MBP), Fc tag, glutathione-S-transferase (GST), AviTa g(GLNDIFEAQKIEWHE), calmodulin-tag (KRRWKKNFIA VSAANRFKKISSSGAL); polyglutamate tag (EEEEEE); E-tag FLAG-tag (GAPVPYPDPLEPR); FLAG-tag (DYKDDDDK), HA-tag (YPYDVPDYA); His-tag (HHHHHH); Myc-tag (EQKLIS EEDL); NE-tag (TKENPRSNQEESYDDNES); S-tag (KET AAAKFERQHMDS);SBP-TAG(MDEKTTGWRGGHVVEGLAG ELEQLRARLEHHPQGQREP);Softag 1(SLAELLNAGL GGS);Softag 3(TQDPSRVG);Spot-tag(PDRVRAVS HWSS); Strep-tag (Strep-tag II: WSHPQFEK); TC-tag (CCPGCC);Ty tag (EVHTNQDPLD);V5 tag (GKPIPNPLL GLDST); VSV-tag (YTDIEMNRLGK); or Xpress-tag (DL YDDDDK).

[0192] In certain embodiments, the cyclic polyribonucleotide is an antibody, e.g., an antibody fragment. In one embodiment, the cyclic polyribonucleotide expresses the polypeptide, or a portion thereof. The antibodies expressed may be of any isotype, including IgA, IgD, IgE, IgG, and IgM. In some embodiments, the cyclic polyribonucleotide may be a portion of an antibody. , e.g., light chain, heavy chain, Fc fragment, CDR (complementarity determining region), Fv fragment In one embodiment, the cyclic The polyribonucleotide expresses one or more portions of an antibody. For example, a circular polyribonucleotide A polypeptide may contain two or more expression sequences, each of which expresses a portion of an antibody. In some cases, cyclic polyribonucleotides may be present, and the assembly of these may constitute an antibody. one expressed sequence encoding the heavy chain of the antibody and another expressed sequence encoding the light chain of the antibody In some cases, the circular polyribonucleotide is expressed in a cell or in a cell-free environment. When combined, the light and heavy chains undergo the proper modification, folding, or other post-translational processes to form a functional antibody. It can be subjected to modification.

[0193] After providing the cells with at least two compositions of cyclic polyribonucleotides, Disclosed herein are administration methods for producing certain levels of protein in cyclic Polyribonucleotides encode proteins.

[0194] The protein may be an intracellular protein, a membrane protein, or a secreted protein. Proteins can be secreted from the cell or localized to the cytoplasm, nucleus, or membrane compartment of the cell. The protein may be a polypeptide that is a viral envelope protein. at least a portion of a peptide protein, a metabolic regulatory enzyme (e.g., a protein that regulates lipid or steroid production), (see section 1), antigens, tolerogens, cytokines, toxins, enzymes whose absence is associated with disease, and Polypeptides and hormones that are not active in an animal (e.g., in the animal's intestine) until cleaved Examples include:

[0195] In certain embodiments, the protein is a therapeutic prote...

Claims

1. A kit for expressing chimeric antigen receptors, (i) a first composition comprising a first cyclic polyribonucleotide comprising an internal ribosome entry site (IRES) operably linked to a sequence encoding a chimeric antigen receptor (CAR); and (ii) A second composition comprising a second cyclic polyribonucleotide containing a sequence encoding IRES and CAR. Includes, The kit is configured such that the second composition is administered 48 to 96 hours after the administration of the first composition.

2. The kit according to claim 1, wherein the kit is configured such that the second composition is administered about 48 hours after the administration of the first composition.

3. The kit according to claim 1, wherein the kit is configured such that the second composition is administered about 72 hours after the administration of the first composition.

4. The kit according to claim 1, wherein the kit is configured such that the second composition is administered about 96 hours after the administration of the first composition.

5. The kit according to claim 1, further comprising a third composition comprising a cyclic polyribonucleotide containing an IRES operably linked to a sequence encoding a CAR.

6. The kit according to claim 5, wherein the kit is configured such that the third composition is administered 48 to 96 hours after the administration of the second composition.

7. The kit according to claim 6, wherein the kit is configured such that the third composition is administered about 48 hours after the administration of the second composition.

8. The kit according to claim 6, wherein the kit is configured such that the third composition is administered about 72 hours after the administration of the second composition.

9. The kit according to claim 1, wherein the cyclic polyribonucleotide comprises miRNA.

10. The kit according to claim 1, wherein the cyclic polyribonucleotide includes a spacer sequence.

11. The cyclic polyribonucleotide comprises first and second spacer elements, and the cyclic polyribonucleotide is 5' to 3': (i) First spacer element; (ii) The aforementioned IRES; (iii) Expression sequence encoding the CAR; and (iv) Second spacer element The kit according to claim 10, comprising:

12. The kit according to claim 1, wherein the IRES is EMCV IRES or CVB3 IRES.

13. The kit according to claim 1, wherein the cyclic polyribonucleotide of the first composition and the cyclic polyribonucleotide of the second composition contain the same nucleic acid sequence.

14. The kit according to claim 1, wherein the cyclic polyribonucleotide of the first composition and the cyclic polyribonucleotide of the second composition contain different nucleic acid sequences.

15. The kit according to claim 1, wherein the first composition and the second composition each further comprise a pharmaceutically acceptable carrier or excipient.

16. The kit according to claim 10, wherein the pharmaceutically acceptable carrier is lipid nanoparticles.

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