Cyclic polyribonucleotide and pharmaceutical composition of the same

Abstract

To provide a pharmaceutical composition and preparation of cyclic polyribonucleotide, and use thereof.SOLUTION: In one embodiment, pharmaceutical preparation of a cyclic polyribonucleotide molecule includes a linear polyribonucleotide molecule of less than a predetermined threshold level when measured by a specified method. For example, the preparation includes a linear polyribonucleotide molecule of a level satisfying a drug release specification, and the preparation includes a linear polyribonucleotide molecule of a level satisfying a standard described below in this specification (e.g., w / v standard or w / w standard) for example. In some cases, the standard can be a level of less than a detection limit when measured by a specified method.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 813,666, filed Mar. 4, 2019 , U.S. Provisional Patent Application No. 62 / 825,683, filed Mar. 28, 2019, 201 U.S. Provisional Patent Application No. 62 / 840,174, filed Apr. 29, 2019, and 2020 U.S. Provisional Patent Application No. 62 / 967,545, filed Jan. 29, and all of their contents are incorporated herein by reference and are hereby incorporated by reference in their entirety.

Background Art

[0002] Certain cyclic polynucleotides are ubiquitous in human tissues and cells, including healthy human tissues and cells and are present throughout.

Summary of the Invention

Means for Solving the Problems

[0003] The present disclosure provides pharmaceutical compositions or formulations of cyclic polynucleotide molecules having a defined or reduced amount of linear polynucleotide molecules, and methods related thereto. The inventors have found that linear polynucleotide molecules in cyclic polynucleotide pharmaceutical compositions or formulations should be detected, monitored, and / or controlled, e.g., reduced or purified, from the cyclic polynucleotide pharmaceutical compositions or formulations. The inventors have found that linear polynucleotide molecules in cyclic polynucleotide pharmaceutical compositions or formulations should be detected, monitored, and / or controlled, e.g., reduced or purified, from the cyclic polynucleotide pharmaceutical compositions or formulations. from the cyclic polynucleotide pharmaceutical compositions or formulations. For example, it has been found that they should be reduced or purified.

[0004] Pharmaceutical Formulations In one aspect, a pharmaceutical formulation of a cyclic polynucleotide molecule comprises a level of linear polynucleotide molecules below a predetermined threshold when measured by a defined method, e.g., the formulation meets pharmaceutical release specifications ​ contains a linear polynucleotide molecule at a level that satisfies the specification (e.g., pharmaceutical formulation). The specification is satisfied at a level of linear polynucleotide molecule as described herein below (e.g., w / v specification or w / w specification). In some cases, the specification may be at a level below the detection limit when measured by a defined method.

[0005] In another aspect, a pharmaceutical formulation of a cyclic polynucleotide molecule contains 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 1 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1 mg / ml, 1.5 mg / ml, or 2 mg / ml or less of a linear polynucleotide molecule.

[0006] In another aspect, a pharmaceutical formulation of a cyclic polynucleotide molecule is at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w) with respect to all ribonucleotide molecules in the pharmaceutical formulation. )、99% (w / w), 99.1% (w / w), 99.2% (w / w), 99.3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w), 99. 7% (w / w), 99.8% (w / w), 99.9% (w / w), or 100% (w / w ). In certain embodiments, at least 30% (w / w), 40% (w / w), 50% (w / w) of the total ribonucleotide molecules in the pharmaceutical formulation, 60% (w / w), 70% (w / w), 80% (w / w) , 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w ), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w ), 99% (w / w), 99.1% (w / w), 99.2% (w / w), 99.3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w), 99.7% (w / w), 99.8% (w / w), 99.9% (w / w), or 100% (w / w) are cyclic ribonucleotide molecules. In certain embodiments, at least 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 pharmaceutical formulation are cyclic ribonucleotide molecules. In another aspect, the pharmaceutical formulation of cyclic ribonucleotide molecules has a level of linear ribonucleotide molecules in the formulation before the purification step compared to after the purification step (e.g., after one or multiple purification steps), of at least 30% (w / w), at least 40% (w / w), at least

[0007] 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 99.1% (w / w), 99.2% (w / w), 99.3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w), 99.7% (w / w), 99.8% (w / w), 99.9% (w / w), or 100% (w / w) of the total ribonucleotide molecules in the pharmaceutical formulation are cyclic ribonucleotide molecules. In certain embodiments, at least 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 pharmaceutical formulation are cyclic ribonucleotide molecules. In another aspect, the pharmaceutical formulation of cyclic ribonucleotide molecules has a level of linear ribonucleotide molecules in the formulation before the purification step compared to after the purification step (e.g., after one or multiple purification steps), of at least 30% (w / w), at least 40% (w / w), at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 99.1% (w / w), 99.2% (w / w), 99.3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w), 99.7% (w / w), 99.8% (w / w), 99.9% (w / w), or 100% (w / w) of the total ribonucleotide molecules in the pharmaceutical formulation are cyclic ribonucleotide molecules. In certain embodiments, at least 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 pharmaceutical formulation are cyclic ribonucleotide molecules. In another aspect, the pharmaceutical formulation of cyclic ribonucleotide molecules has a level of linear ribonucleotide molecules in the formulation before the purification step compared to after the purification step (e.g., after one or multiple purification steps), of at least 30% (w / w), at least 40% (w / w), at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 99.1% (w / w), 99.2% (w / w), 99.3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w), 99.7% (w / w), 99.8% (w / w), 99.9% (w / w), or 100% (w / w) of the total ribonucleotide molecules in the pharmaceutical formulation are cyclic ribonucleotide molecules. In certain embodiments, at least 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 pharmaceutical formulation are cyclic ribonucleotide molecules. In another aspect, the pharmaceutical formulation of cyclic ribonucleotide molecules has a level of linear ribonucleotide molecules in the formulation before the purification step compared to after the purification step (e.g., after one or multiple purification steps), of at least 30% (w / w), at least 40% (w / w), at least At least 50% (w / w), at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), at least 90% (w / w), or at least 95% (w / w) of the level of linear polynucleotide molecules that are reduced.

[0008] In another aspect, a pharmaceutical formulation of a cyclic polynucleotide molecule is 5% (w / w) or less of the nick poly ribonucleotide molecules of the cyclic polynucleotide molecule and all ribonucleotide molecules in the pharmaceutical formulation. In certain embodiments, the pharmaceutical composition is 9% (w / w), 8% (w / w), 7% (w / w), 6% (w / w ), 5% (w / w), 4% (w / w), 3% (w / w), 2% (w / w), 1% (w / w ), or 0.5% (w / w) or less of the nick polyribonucleotide molecules of all ribonucleotide molecules in the pharmaceutical formulation. In certain embodiments, the pharmaceutical composition is 2% (w / w) or less of the nick polyribonucleotide molecules of all ribonucleotide molecules in the pharmaceutical formulation.

[0009] In another aspect, a pharmaceutical formulation of a cyclic polynucleotide molecule is 0.5% (w / w), 1% (w / w) , 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w) , 7% (w / w), 8% (w / w), 9% (w / w), or 10% (w / w) or less of the linear polyribonucleotide molecules of the cyclic polynucleotide molecule and all ribonucleotide molecules in the formulation.

[0010] In certain embodiments of each aspect listed above, a pharmaceutical formulation of a cyclic polynucleotide molecule is 0.5% ​​(w / w), 1% (w / w), 2% (w / w), or 5% (w / w) or less of linear polyribo nucleotide molecules.

[0011] In certain embodiments of each of the aspects listed above, the cyclic polyribonucleotide molecule is a sequence encoding an expression product, or a plurality of sequences, e.g., encoding a therapeutic protein or nucleic acid including, for example, a therapeutic expression product. In certain embodiments of each of the aspects listed above, the cyclic polyribonucleotide molecule includes a sequence containing a scaffold, or a plurality of sequences (e.g., aptamer sequences).

[0012] In certain embodiments of each of the aspects listed above, the level of the linear polyribonucleotide molecule in the pharmaceutical formulation of the cyclic polyribonucleotide molecule is determined by microscopy, spectrophotometry, fluorescence analysis, denaturing urea polyacrylamide gel electrophoresis imaging, UV-Vis spectrophotometry, R NA electrophoresis, RNAse H analysis, UV spectroscopic or fluorescence detectors, light scattering techniques, H PLC using or not using surface plasmon resonance (SPR ), HPLC, use or not use of derivatization methods before or after separation, chip or gel-based electrophoresis, detection of linear polyribonucleotide molecules using detection methods using silver or dye staining or radioactive decay, or microscopy methods, visual methods or methods using a spectrophotometer, or any suitable combination thereof, by any preferred method.

[0013] In certain embodiments of each of the aspects listed above, the pharmaceutical formulation of the cyclic polyribonucleotide molecule also reduces the linear polyribonucleotide compared to before the purification step, for medical When the pharmaceutical preparation is subjected to an enrichment or purification step (or multiple purification steps), after administration to a subject, , it gives rise to one or more markers at a reduced level of immune or inflammatory response. In certain embodiments wherein, one or more markers of immune or inflammatory response are cytokine or immunogenic-related gene expression. In certain embodiments, one or more markers of immune or inflammatory response are RI G-I, MDA5, PKR, IFN-β, OAS, and OASL selected from the group consisting of gene expression.

[0014] In certain embodiments of each of the aspects listed above, the pharmaceutical preparation of the cyclic polynucleotide molecule further substantially comprises no impurities, such as process-related impurities or product-related substances. In certain embodiments, the process-related impurities include proteins (e.g., cellular proteins such as host cell proteins), deoxyribonucleic acids (e.g., cellular deoxyribonucleic acids such as host cell deoxyribonucleic acids), monodeoxyribonucleotide or dideoxyribonucleotide molecules, enzymes (e.g., nucleases or ligases), reagent components, gel components, or chromatography materials. In certain embodiments, the impurities are selected from buffer reagents, ligases, nucleases (e.g., exonucleases or endonucleases), RNas e inhibitors, RNase R, deoxyribonucleotide molecules, acrylamide gel fragments, and monodeoxyribonucleotide molecules. In certain embodiments, the pharmaceutical preparation contains, per milligram (mg) of the cyclic polynucleotide molecule, 0.1 ng, 1 ng , 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng of one or more impurities selected from the group consisting of buffer reagents, ligases, nucleases (e.g., exonucleases or endonucleases), RNase e inhibitors, RNase R, deoxyribonucleotide molecules, acrylamide gel fragments, and monodeoxyribonucleotide molecules. In certain embodiments, the pharmaceutical preparation contains, per milligram (mg) of the cyclic polynucleotide molecule, 0.1 ng, 1 ng , 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or 500 ng of protein contamination.

[0015] In certain embodiments of each of the aspects recited above, the pharmaceutical formulation may further comprise a pharmaceutical impurity or Substantially free of contaminants, e.g., the pharmaceutical preparation is When tested, it contains less than 10 EU / kg endotoxin or is In one embodiment, the pharmaceutical formulation has a bacterial count of less than 100 CFU / 100 ml prior to sterilization. or a bioburden of less than 10 CFU / 100 ml. The formulation is a sterile pharmaceutical formulation. In certain embodiments, the sterile pharmaceutical formulation is tested under sterility conditions. In one embodiment, the pharmaceutical formulation is a reference to the United States Pharmacopeia as published as of the filing date of this application. copeia) chapter 71 (USP <71> ) standard. In the United States Pharmacopoeia (USP), pharmaceutical preparations are defined as those prescribed by the United States Pharmacopoeia (USP) as published as of the filing date of this application. Harmacopeia Chapter 85 (USP <85> ) standards.

[0016] In certain embodiments of each of the aspects recited above, the linear polyribonucleotide molecule of the formulation is a linear polyribonucleotide molecule equivalent of a circular polyribonucleotide molecule or a circular polyribonucleotide molecule equivalent of a linear polyribonucleotide molecule It includes fragments of linear polyribonucleotide molecules equivalent to ribonucleotide molecules. In certain embodiments of each of the aspects recited in claim 1, the linear polyribonucleotide molecule of the formulation is A linear polyribonucleotide molecule equivalent of a circular polyribonucleotide molecule (e.g., a circularized In some embodiments, the circularized version is In this case, the linear polyribonucleotide molecule is a linear poly of a circular polyribonucleotide molecule ribonucleotide molecule equivalent or a fragment thereof, a linear polyribonucleotide molecule non-equivalent of a circular polyribonucleotide molecule or a fragment thereof, or a combination thereof. In certain embodiments, the linear polyribonucleotide molecule is a linear polyribonucleotide molecule equivalent (e.g., the pre-circularized form) of a circular polyribonucleotide molecule, a linear polyribonucleotide molecule non-equivalent of a circular polyribonucleotide molecule, or a combination thereof. In certain embodiments of each aspect listed above, the linear polyribonucleotide molecule fragment is a fragment having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 5 0, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2 000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1 0000, 11000, 12000, or more nucleotide lengths, or a fragment having any number of nucleotides therebetween.

[0017] In certain embodiments of each aspect listed above, the circular polyribonucleotide molecule comprises a pseudoknot structure. In certain embodiments, the circular polyribonucleotide molecule comprises a pseudodouble-stranded secondary structure. In certain embodiments of each aspect listed above, the circular polyribonucleotide molecule comprises one or more expression sequences and a stopper element at the 3' end of at least one expression sequence. In certain embodiments of each aspect listed above, the circular polyribonucleotide molecule comprises one or more aptamer sequences. In certain embodiments of each aspect listed above, In this case, the cyclic polynucleotide molecule has a sequence encoding an endogenous or natural cyclic polynucleotide. It has a sequence encoding the sequence.

[0018] In certain embodiments of each of the aspects listed above, the pharmaceutical formulation is an intermediate pharmaceutical formulation for the final cyclic polynucleotide drug product. In certain embodiments, the pharmaceutical formulation is a drug or active pharmaceutical ingredient (API). In certain embodiments, the pharmaceutical formulation is a drug product for administration to a subject.

[0019] In certain embodiments of each of the aspects listed above, the pharmaceutical formulation contains at least 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / m L, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL 60 μg / mL, 70 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, 1 mg / mL, 2 mg / mL, 3 mg / m L, 4 mg / mL, 5 mg / mL, 10 mg / mL, 100 mg / mL, 200 mg / m L, or a concentration of 500 mg / mL of the cyclic polynucleotide molecule.

[0020] In certain embodiments of each of the aspects listed above, the pharmaceutical formulation has zero DNA, substantially does not contain DNA, or contains 1 pg / ml, 10 pg / ml, 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 2 00 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, or 1 μg / mL or less of DNA. In certain embodiments, the DNA is a monodeoxyribonucle otide, dideoxyribonucleotide molecule, polydeoxyribonucleotide molecule, or a combination of any of these. In certain embodiments, the pharmaceutical formulation has an A260 / A280 absorbance ratio of about 1.6 - 2.3 when measured by a spectrophotometer. In certain embodiments, the DNA concentration of the pharmaceutical formulation is measured after total DNA digestion with an enzyme that digests nucleosides by quantitative liquid chromatography - mass spectrometry (LC - MS), where

[0021] the amount of DNA is back - calculated from the standard curve of each base (i.e., A, C, G, T) when measured by LC - MS. In certain embodiments of each aspect listed above, the amount of linear polyribonucleotide molecules as compared to circular polyribonucleotide molecules is determined using the method of Example

[0022] 2 or Example 3. In certain embodiments, the amount of linear polyribonucleotide molecules in the pharmaceutical formulation is determined using the method of Example 2. In certain embodiments, the amount of circular polyribonucleotide molecules in the pharmaceutical formulation is determined using the method of Example 3. comprising the step of generating a pharmaceutical composition for pharmaceutical use. In certain embodiments, the step d) further processing comprises i) treating the formulation to substantially remove DNA and / or protein (e.g., cellular proteins such as host cell proteins and / or endotoxin; ii) evaluating the amount of DNA and / or protein (e.g., cellular proteins such as host cell proteins in the formulation and / or endotoxin; iii) formulating the formulation with a pharmaceutical excipient; and iv) optionally concentrating the formulation including one or more of the steps.

[0023] In another aspect, a method of making a pharmaceutical drug substance comprises a) providing a formulation of a cyclic polynucleotide molecule; b) evaluating the amount of linear polynucleotide molecules in the formulation; and c) treating the formulation of the cyclic polynucleotide molecule as a pharmaceutical drug substance if the formulation meets a reference standard (e.g., a pharmaceutical release standard, such as a pharmaceutical release standard or reference standard described herein) for the amount of linear polynucleotide molecules present in the formulation.

[0024] In another aspect, a method of making a pharmaceutical drug substance comprises a) providing a plurality of linear polynucleotide molecules; b) cyclizing the plurality of linear polynucleotide molecules to provide a formulation of a cyclic polynucleotide molecule; c) evaluating the amount of linear polynucleotide molecules remaining in the formulation; and d) treating the formulation of the cyclic polynucleotide molecule as a pharmaceutical drug substance if the formulation meets a reference standard for the amount of linear polynucleotide molecules present in the formulation.

[0025] ​​In another aspect, a method of making a pharmaceutical comprises: a) providing a plurality of linear polynucleotide molecules; b) circularizing the plurality of linear polynucleotide molecules to provide a formulation of circular polynucleotide molecules; c) assessing the amount of linear and / or nicked polynucleotide molecules remaining in the formulation; and d) treating the formulation of circular polynucleotide molecules as a pharmaceutical if the formulation meets a reference standard for the amount of linear and / or nicked polynucleotide molecules present in the formulation.

[0026] In another aspect, a method of making a pharmaceutical drug prod uct comprises: a) providing a plurality of linear polynucleotide molecules and ; b) circularizing the plurality of linear polynucleotide molecules to provide a formulation of circular polynucleotide molecules ; c) measuring the amount of linear and / or nicked polynucleotide molecules in the formulation ; d) formulating the formulation of circular polynucleotide molecules as a pharmaceutical drug product if the formulation meets a reference standard for the amount of linear and / or nicked polynucleotide molecules present in the formulation ; and e) labeling and shipping the pharmaceutical drug product if it meets a reference standard for the amount of linear polynucleotide molecules present in the pharmaceutical drug product.

[0027] In another aspect, a method of making a pharmaceutical drug product comprises: a) providing a formulation of circular polynucleotide molecules, b) formulating the formulation of circular polynucleotide molecules as a pharmaceutical drug product if it meets a reference standard for the amount of linear polynucleotide molecules present in the formulation, c) measuring the amount of linear polynucleotide molecules in a sample of the pharmaceutical drug product, and d) if it meets a reference standard for the amount of linear polynucleotide molecules present in the pharmaceutical drug product molecules present in the pharmaceutical drug product For a pharmaceutical product, when the amount of the linear polynucleotide molecule meets the reference standard, formulating, labeling, and / or shipping the pharmaceutical product are included. And / or the step of shipping.

[0028] In another aspect, a method for preparing a pharmaceutical product includes: a) providing a plurality of linear polynucleotide molecules; b) cyclizing the plurality of linear polynucleotide molecules to provide a formulation of cyclic polynucleotide molecules; c) measuring the amount of linear polynucleotide molecules in the formulation; d) when the formulation meets the reference standard for the amount of linear polynucleotide molecules present in the formulation, formulating the formulation of cyclic polynucleotide molecules as a pharmaceutical product; e) when it meets the reference standard for the amount of linear polynucleotide molecules present in the pharmaceutical product, labeling and shipping the pharmaceutical product. And the step of shipping. In another aspect, a method for preparing a pharmaceutical composition includes: a) providing a plurality of linear polynucleotide molecules; And the step of shipping.

[0029] b) cyclizing the linear polynucleotide molecules to obtain a formulation of cyclic polynucleotide molecules; c) treating the formulation to substantially remove the linear polynucleotide molecules remaining in the formulation; d) optionally, evaluating the amount of linear polynucleotide molecules in the formulation remaining after the treatment step; e) further treating the formulation to generate a pharmaceutical composition for pharmaceutical use. In certain embodiments, the method includes: f) treating the formulation to substantially remove deoxyribonucleotide molecules; g) evaluating the amount of deoxyribonucleotide molecules in the formulation; h) formulating the formulation with a pharmaceutical excipient; i) concentrating the formulation; j) and recording, on a printed or digital medium, the amount of deoxyribonucleotide molecules in the formulation. In one embodiment, the method further comprises one or more of the steps of: f) extracting a protein; g) treating the formulation to substantially remove the contaminants; and h) formulating the formulation with pharmaceutical excipients; and i) formulating the formulation. In one embodiment, the further processing of step d) further comprises: f) concentrating the g) treating the formulation to substantially remove endotoxins; and assessing the amount of toxin; h) formulating the formulation with pharmaceutical excipients; i) and concentrating the formulation.

[0030] In certain embodiments of each of the above aspects, the circularization step comprises splint ligation. In certain embodiments of each of the above aspects, the cyclic polyribonucleic acid is The step of formulating the formulation of the nucleotide molecule comprises formulating the formulation of the circular polyribonucleotide molecule for use in a pharmaceutical Including combining with pharmaceutical excipients.

[0031] In certain embodiments of each of the above aspects, the method comprises: tide molecules (e.g., linear polyribonucleotide molecules and / or cyclic polyribonucleotide molecules) Record the amount of the compound (molecule) in a printed or digital medium, e.g., in a certificate of analysis for the formulation. The method further includes the step of:

[0032] In certain embodiments of each of the above aspects, the formulating step comprises the step of: The formulation includes combining the tide molecule with a pharmaceutical excipient.

[0033] In certain embodiments of each of the above aspects, the reference standard is a pharmaceutical release standard for a formulation of a cyclic polynucleotide molecule. For example, the reference standard can be one or more of the following: (a) the amount of linear polynucleotide molecules present in the pharmaceutical formulation is a specific amount, such as 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml, 600 mg / ml, 700 mg / ml or 750 mg / ml or less of linear polynucleotide molecules; (b) the pharmaceutical product or medicament is at least a specific amount, such as 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 0.1 μg / mL, 0.comprising a concentration of cyclic polynucleotide molecules of 5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL, 100 mg / mL, 200 mg / mL, 500 mg / mL, 600 mg / ml, 700 mg / ml, or 750 mg / ml; or (c) the pharmaceutical product or medicament comprises at least a specific amount, for example, at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 99.1% (w / w), 99.2% (w / w), 99.3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w), 99.7% (w / w), 99.8% (w / w), 99.9% (w / w), or 100% (w / w) of cyclic polynucleotide molecules, based on the total ribonucleotide molecules in the pharmaceutical formulation.

[0034] In certain embodiments of each of the above aspects, the reference for the amount of linear and / or nicked polyribonucleotide molecules present in the formulation is a) linear polyribonucleotide molecules that are 20%, 15%, 10%, 5%, 2%, 1%, or 0.5% (w / w) or less, based on the total ribonucleotide molecules in the formulation; b) nicked polyribonucleotide molecules that are 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% (w / w) or less, based on the total ribonucleotide molecules in the formulation; or c) a combination that is 20 %, 15%, 10%, 5%, 2%, 1%, or 0.5% (w / w) or less, based on the total ribonucleotide molecules in the formulation. thereof. Selected from the resulting linear and nicked polynucleotide molecules.

[0035] In certain embodiments of each of the above aspects, all ribonucleotide molecules in the pharmaceutical formulation at least 80% (w / w) are cyclic polynucleotide molecules. In certain embodiments of each of the above aspects the pharmaceutical composition comprises 2 0% (w / w) or less of linear polynucleotide molecules of all ribonucleotide molecules in the formulation. In certain embodiments of each of the above aspects the pharmaceutical composition comprises 10% (w / w ) or less of linear polynucleotide molecules of all ribonucleotide molecules in the formulation.

[0036] In certain embodiments of each of the above aspects, the cyclic polynucleotide molecules in the pharmaceutical formulation (e.g., relative to all ribonucleotide molecules) are determined using microscopy, spectrophotometry, fluorescence analysis, denaturing urea polyacrylamide gel electrophoresis imaging, UV-Vis spectrophotometry , RNA electrophoresis, RNAse H analysis, UV or fluorescence detectors, light scattering techniques , surface plasmon resonance (SPR) with or without the use of separation methods including HPLC, HPLC, derivatization methods before or after separation, chip - or gel-based electrophoresis, detection methods using silver or dye staining or radioactive decay for the detection of linear polynucleotide molecules, or by microscopy, visual methods or methods using a spectrophotometer. For example, the amount of cyclic polynucleotide relative to all ribonucleotide molecules can be determined using the method of Example 2 or Example 3. or by methods using a spectrophotometer. For example, the amount of cyclic polynucleotide relative to all ribonucleotide molecules can be determined using the method of Example 2 or Example 3.

[0037] ​​​In certain embodiments of each of the above-described aspects, the pharmaceutical product or drug further: (a) contains less than 10 EU / kg of endotoxin or is endotoxin-free as measured by the Limulus amoebocyte lysate test; (b) contains a bioburden of less than 100 CFU / 100 ml or less than 10 CFU / 100 ml prior to sterilization; (c) is a sterile pharmaceutical product or drug; (d) supports the growth of less than 100 viable microorganisms when tested in a sterile state; and / or (e) meets the standards of USP<71> or USP<85>.

[0038] In certain embodiments of each of the above-described aspects, the cyclic polynucleotide molecule contains one or more expression sequences and a stagger element at the 3' end of at least one expression sequence.

[0039] In certain embodiments of each of the above-described aspects, the formulation further meets a reference standard for the amount of DNA (e.g., cellular DNA such as host cell DNA) present in the formulation. In certain embodiments, the reference standard for the amount of DNA molecules present in the formulation is the presence below a specific amount, e.g., the DNA molecules are zero, substantially free of DNA molecules, or the presence of DNA molecules at 1 pg / ml, 10 pg / ml, 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml 400 ng / ml, or 500 ng / ml, 1 μg / mL, 5 μg / mL, 10 μ g / mL, or 100 μg / mL or less.

[0040] In certain embodiments of each of the above-described aspects, the formulation contains a protein present in the formulation Further meet the reference standards for the amount of contamination (e.g., cellular proteins such as host cell proteins or process-related proteins impurities, e.g., enzymes). In certain embodiments , the reference standard for the amount of protein contamination present in the formulation is less than a specific amount, e.g., per milligram (mg) of the cyclic polynucleotide molecule, 0.1 ng, 1 ng, 5 n g, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 n g, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or less than 500 ng of protein contamination. In certain embodiments of each of the above aspects, the protein contamination includes an enzyme.

[0041] In certain embodiments of each of the above aspects, the pharmaceutical product or medicament includes an A260 / A280 absorbance ratio of about 1.6 - 2.3 when measured by a spectrophotometer.

[0042] In certain embodiments of each of the above aspects, the linear polynucleotide molecule is a linear polynucleotide molecule equivalent of the cyclic polynucleotide molecule or a fragment of the linear polynucleotide molecule equivalent of the cyclic polynucleotide molecule. In certain embodiments of each of the above aspects, the linear polynucleotide molecule includes a linear polynucleotide molecule equivalent of the cyclic polynucleotide molecule (e.g., the pre-cyclization form). In certain embodiments of each of the above aspects, the linear polynucleotide molecule is a linear polynucleotide molecule equivalent of the cyclic polynucleotide molecule or a fragment thereof, a non-equivalent linear polynucleotide molecule of the cyclic polynucleotide molecule or a fragment thereof, a non-equivalent linear polynucleotide molecule of the cyclic polynucleotide molecule or a fragment thereof, a non-equivalent linear polynucleotide molecule of the cyclic polynucleotide molecule or a fragment thereof, a non-equivalent linear polynucleotide molecule of the cyclic polynucleotide molecule or a fragment thereof, a non-equivalent linear polynucleotide molecule of the cyclic polynucleotide molecule or a fragment thereof, a non-equivalent linear polynucleotide molecule of the cyclic polynucleotide molecule or a It includes fragments, or combinations thereof. In certain embodiments of each of the above aspects wherein, the linear polynucleotide molecule is a linear polynucleotide molecule equivalent (e.g., pre-cyclized form) of a circular polynucleotide molecule, a linear polynucleotide molecule non-equivalent of a circular polynucleotide molecule, or a combination thereof. It includes fragments, or combinations thereof. In certain embodiments of each of the above aspects wherein, the circular polynucleotide molecule includes a sequence encoding an expression product, or a plurality of sequences, e.g., a therapeutic protein or a nucleic acid encoding a therapeutic expression product. In certain embodiments of each of the above aspects

[0043] wherein, the circular polynucleotide molecule has a sequence including a scaffold (e.g., an aptamer sequence). In certain embodiments of each of the above aspects wherein, the circular polynucleotide molecule has a sequence encoding an endogenous or natural circular polynucleotide sequence. In such embodiments the pharmaceutical preparation may further meet the reference criteria for a circular polynucleotide molecule having a sequence with at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, 100%, or any percentage therebetween) sequence identity to a reference sequence, e.g., a reference sequence encoding an expression product. wherein, the circular polynucleotide molecule has a sequence including a scaffold (e.g., an aptamer sequence). In certain embodiments of each of the above aspects wherein, the circular polynucleotide molecule has a sequence encoding an endogenous or natural circular polynucleotide sequence. In such embodiments the pharmaceutical preparation may further meet the reference criteria for a circular polynucleotide molecule having a sequence with at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, 100%, or any percentage therebetween) sequence identity to a reference sequence, e.g., a reference sequence encoding an expression product. the pharmaceutical preparation may further meet the reference criteria for a circular polynucleotide molecule having a sequence with at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, 100%, or any percentage therebetween) sequence identity to a reference sequence, e.g., a reference sequence encoding an expression product. at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, 100%, or any percentage therebetween) sequence identity to a reference sequence, e.g., a reference sequence encoding an expression product. the pharmaceutical preparation may further meet the reference criteria for a circular polynucleotide molecule having a sequence with at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, 100%, or any percentage therebetween) sequence identity to a reference sequence, e.g., a reference sequence encoding an expression product. the pharmaceutical preparation may further meet the reference criteria for a circular polynucleotide molecule having a sequence with at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, 100%, or any percentage therebetween) sequence identity to a reference sequence, e.g., a reference sequence encoding an expression product.

[0044] Method of Use In another aspect, a method of delivering a cyclic polynucleotide molecule to a cell or tissue of a subject, or to a subject, comprises administering to the cell or tissue of the subject, or to the subject, a pharmaceutical formulation described herein, a pharmaceutical composition described herein, a medicament described herein, or a pharmaceutical product described herein, wherein the cyclic polynucleotide molecule is detected in the cell, tissue, or subject, for example, at least 3 days (e.g., at least 4, 5, 6, 7, 10, 12, 15, 20, 24 days or more, or after any day therebetween) after the administration step.

[0045] In another aspect, a method of delivering a cyclic polynucleotide molecule to a cell or tissue of a subject, or to a subject, comprises administering to the cell or tissue of the subject, or to the subject, a pharmaceutical formulation described herein, a pharmaceutical composition described herein, a medicament described herein, or a pharmaceutical product described herein, wherein the cyclic polynucleotide or a product translated from the cyclic polynucleotide is detected in the cell, tissue, or subject at least 3 days after the administration step.

[0046] In another aspect, a method of delivering a therapeutic product to a cell or tissue of a subject in need thereof, or to the subject, comprises administering to the cell or tissue of the subject, or to the subject, a pharmaceutical formulation described herein, a pharmaceutical composition described herein, a medicament described herein, or a pharmaceutical product described herein. In certain embodiments of each of the above aspects, the circular polynucleotide molecule of the composition or formulation comprises a circular polynucleotide molecule having a sequence that includes a therapeutic product, and the therapeutic product transcribed or translated from the circular polynucleotide molecule is detected in the cell, tissue, or subject, for example, at least 3 days (e.g., at least 4, 5, 6, 7, 10, 12, 15, 20, 24 days or more, or after any day in between) after the administration step. In certain embodiments of this aspect, the circular polynucleotide molecule of the composition or formulation comprises a circular polynucleotide molecule having a sequence that includes an aptamer, and the circular polynucleotide molecule is detected in the cell, tissue, or subject at least 3 days (e.g., at least 4, 5, 6, 7, 10, 12, 15, 20, 24 days or more, or after any day in between) after the administration step. In certain embodiments of this aspect, the circular polynucleotide of the composition or formulation comprises a circular polynucleotide molecule having an endogenous or native circular polynucleotide molecule sequence, and the endogenous or native circular polynucleotide molecule is detected in the cell, tissue, or subject at least 3 days (e.g., at least 4, 5, 6, 7, 10, 12, 15, 20, 24 days or more, or after any day in between) after the administration step.

[0047] In another aspect, a parenteral nucleic acid delivery system comprises (i) a pharmaceutical formulation described herein, a pharmaceutical composition described herein, a medicament described herein, or a pharmaceutical product described herein, and (ii) a parenterally acceptable diluent. In certain embodiments of this aspect, the pharmaceutical formulation, pharmaceutical composition, medicament, or pharmaceutical product does not include any carrier.

[0048] In another aspect, a method of delivering a cyclic polynucleotide comprises parenterally administering to a subject in need thereof a pharmaceutical formulation described herein, a pharmaceutical composition described herein, a medicament described herein, or a pharmaceutical product described herein. In certain embodiments of this aspect, the cyclic polynucleotide is in an amount effective to cause or induce a biological response in the subject. In certain embodiments of this aspect, the cyclic polynucleotide is in an amount effective to exert a biological effect on cells or tissues in the subject. In certain embodiments of this aspect, parenteral administration is effected intravenously, intramuscularly, intraocularly or locally.

[0049] In another aspect, a method of delivering a cyclic polynucleotide to a cell or tissue of a subject comprises parenterally administering to the cell or tissue a pharmaceutical formulation described herein, a pharmaceutical composition described herein, a medicament described herein, or a pharmaceutical product described herein. In certain embodiments of this aspect, parenteral administration is effected intravenously, intramuscularly, intraocularly or locally.

[0050] In certain embodiments of each of the above aspects, the method further comprises assessing the presence of a cyclic polynucleotide molecule or a product translated from a cyclic polynucleotide molecule in the cell, tissue or subject prior to the administration step. In certain embodiments of each of the above aspects, the method further comprises assessing the presence of a cyclic polynucleotide molecule or a product translated from a cyclic polynucleotide molecule in the cell, tissue or subject after the administration step (e.g., 24 hours, 48 hours, 72 hours, 4 days, 7 days, 14 days or more after the administration step, or after any day in between). In certain embodiments of each of the above aspects, the pharmaceutical formulation, pharmaceutical composition, medicament, or pharmaceutical product comprises a diluent (e.g., a parenterally acceptable diluent) and no optional carrier.

[0051] Definitions The present invention is described with reference to specific embodiments and specific figures, but the present invention It is not limited to these, but is only limited by the scope of the claims. The terms described hereinafter should generally be understood in their general meanings, unless otherwise specifically indicated. The terms such as "obtainable by", "generatable by" are used to indicate that the claims or embodiments refer to the compound, composition, product, etc. itself, that is, the compound, composition, product, etc. can be obtained or generated by the method described for the manufacture of the compound, composition, product, etc., but it is also possible that the compound, composition, product, etc. can be obtained or generated by methods other than the described method. The terms such as "obtained by", "generated by" indicate that the compound, composition, product is obtained or generated by the specific method described. The terms such as "obtainable by", "generatable by" should be understood to also disclose, as preferred embodiments, the terms such as "obtained by", "generated by".

[0052] The expression "compound, composition, product, etc. for treatment, regulation, etc." should be understood to refer to the compound, composition, product, etc. itself that is suitable for the indicated purpose of treatment, regulation, etc. The expression "compound, composition, product, etc. for treatment, regulation, etc." further discloses, as a preferred embodiment, that such compound, composition, product, etc. are for use in treatment, regulation, etc. The terms such as "compound, composition, product, etc. for use in", "agent, pharmaceutical composition, etc. for"

[0053]

[0054] ​​​​​​​​​​​​​​"Use of a compound, composition, product, etc. in the manufacture of a veterinary composition, diagnostic composition, etc." and such expressions indicate that such compounds, compositions, products, etc. are used in a method of treatment that can be practiced on the body of a human or animal. They are considered to be equivalent disclosures of embodiments and claims relating to methods such as treatment. When an embodiment or claim refers to a "compound for use in treating a human or animal suspected of suffering from a disease" as above, this is also considered to be a disclosure of the "use of a compound in the manufacture of a medicament for treating a human or animal suspected of suffering from a disease" or the "method of treatment by administering a compound to a human or animal suspected of suffering from a disease". The expression "compound, composition, product, etc. for treatment, regulation, etc." should be understood to refer to the compound, composition, product, etc. itself that is suitable for the indicated purposes of treatment, regulation, etc. The term "pharmaceutical composition" is also intended to disclose that the cyclic polynucleotide contained within the pharmaceutical composition can be used for the treatment of the body of a human or animal by therapy. Therefore, it is intended that it be equivalent to a "cyclic polynucleotide for use in treatment". As described herein, cyclic polynucleotide molecules, compositions containing such cyclic polynucleotide molecules, methods of making and using such cyclic polynucleotides, etc. are related to the effects of linear RNA molecules in cyclic RNA formulations (e.g., Examples 1-12), and (e.g., see Example 13) different elements such as replication elements, expression sequences, stager elements for treatment, regulation, etc." is understood to refer to the compound, composition, product, etc. itself that is suitable for the indicated purposes of treatment, regulation, etc. purposes of treatment, regulation, etc. It should be understood that the expression "compound, composition, product, etc. for treatment, regulation, etc." refers to the compound, composition, product, etc. itself that is suitable for the indicated purposes of treatment, regulation, etc.

[0055] The term "pharmaceutical composition" is also intended to disclose that the cyclic polynucleotide contained within the pharmaceutical composition can be used for the treatment of the body of a human or animal by therapy. Therefore, it is intended that it be equivalent to a "cyclic polynucleotide for use in treatment". As described herein, cyclic polynucleotide molecules, compositions containing such cyclic polynucleotide molecules, methods of making and using such cyclic polynucleotides, etc. are related to the effects of linear RNA molecules in cyclic RNA formulations (e.g., Examples 1-12), and (e.g., see Example 13) different elements such as replication elements, expression sequences, stager elements for treatment, regulation, etc." is understood to refer to the compound, composition, product, etc. itself that is suitable for the indicated purposes of treatment, regulation, etc.

[0056] The cyclic polynucleotide molecules, compositions containing such cyclic polynucleotide molecules, methods of making and using such cyclic polynucleotides, etc. described herein are related to the effects of linear RNA molecules in cyclic RNA formulations (e.g., Examples 1-12), and (e.g., see Example 13) different elements such as replication elements, expression sequences, stager elements in cyclic RNA formulations (e.g., Examples 1-12), and (e.g., see Example 13) different elements such as replication elements, expression sequences, stager elements and (e.g., see Example 13) different elements such as replication elements, expression sequences, stager elements Elements and encryptogens (see, e.g., Example 13) or, for example, expression arrays, stager elements Circular polynucleotides containing elements and regulatory elements (see, e.g., Examples 34 and 44) Production and use of effectors, and their technical effects (e.g., increased translation efficiency compared to linear equivalents in Examples 43 and 44 and increased half-life compared to linear equivalents in Examples 33 and 60) are based in part on the Examples shown. It is to be understood that the following description contemplates various modifications of the specific discoveries and combinations contemplated in the Examples, particularly based on these Examples. When used herein, the term "total ribonucleotide molecule" means the total amount of any ribonucleotide molecule, including linear polynucleotide molecules, circular polynucleotide molecules, monomeric ribonucleotides, other polynucleotide molecules, fragments thereof, and modified forms thereof, as measured based on the total mass of the ribonucleotide molecule. When used herein, the terms "circular RNA (circRNA)", "circular polynucleotide", "circular RNA (circular RNA)", or "circular polynucleotide molecule" are used synonymously and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), e.g., a polynucleotide molecule that forms a circular or endless structure via covalent or non-covalent bonds. When used herein, the term "fragment" refers to a nucleotide molecule

[0057] When used herein, the term "total ribonucleotide molecule" means the total amount of any ribonucleotide molecule, including linear polynucleotide molecules, circular polynucleotide molecules, monomeric ribonucleotides, other polynucleotide molecules, fragments thereof, and modified forms thereof, as measured based on the total mass of the ribonucleotide molecule. When used herein, the terms "circular RNA (circRNA)", "circular polynucleotide", "circular RNA (circular RNA)", or "circular polynucleotide molecule" are used synonymously and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), e.g., a polynucleotide molecule that forms a circular or endless structure via covalent or non-covalent bonds. When used herein, the term "fragment" refers to a nucleotide molecule When used herein, the term "total ribonucleotide molecule" means the total amount of any ribonucleotide molecule, including linear polynucleotide molecules, circular polynucleotide molecules, monomeric ribonucleotides, other polynucleotide molecules, fragments thereof, and modified forms thereof, as measured based on the total mass of the ribonucleotide molecule.

[0058] When used herein, the terms "circular RNA (circRNA)", "circular polynucleotide", "circular RNA (circular RNA)", or "circular polynucleotide molecule" are used synonymously and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), e.g., a polynucleotide molecule that forms a circular or endless structure via covalent or non-covalent bonds. When used herein, the terms "circular RNA (circRNA)", "circular polynucleotide", "circular RNA (circular RNA)", or "circular polynucleotide molecule" are used synonymously and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), e.g., a polynucleotide molecule that forms a circular or endless structure via covalent or non-covalent bonds. When used herein, the terms "circular RNA (circRNA)", "circular polynucleotide", "circular RNA (circular RNA)", or "circular polynucleotide molecule" are used synonymously and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), e.g., a polynucleotide molecule that forms a circular or endless structure via covalent or non-covalent bonds. When used herein, the terms "circular RNA (circRNA)", "circular polynucleotide", "circular RNA (circular RNA)", or "circular polynucleotide molecule" are used synonymously and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), e.g., a polynucleotide molecule that forms a circular or endless structure via covalent or non-covalent bonds. When used herein, the terms "circular RNA (circRNA)", "circular polynucleotide", "circular RNA (circular RNA)", or "circular polynucleotide molecule" are used synonymously and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), e.g., a polynucleotide molecule that forms a circular or endless structure via covalent or non-covalent bonds. When used herein, the terms "circular RNA (circRNA)", "circular polynucleotide", "circular RNA (circular RNA)", or "circular polynucleotide molecule" are used synonymously and refer to a polynucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), e.g., a polynucleotide molecule that forms a circular or endless structure via covalent or non-covalent bonds.

[0059] When used herein, the term "fragment" refers to a nucleotide molecule ​Refers to any portion of a nucleotide molecule that is at least one nucleotide shorter . For example, the nucleotide molecule can be a linear polyribonucleotide molecule, and its fragment can be a monoribonucleotide or any number of consecutive polyribonucleotides that are part of the linear polyribonucleotide molecule. As another example, the nucleotide molecule can be a cyclic poly ribonucleotide molecule, and its fragment can be a polyribonucleotide or any number of consecutive polyribonucleotides that are part of the cyclic polyribonucleotide molecule . .

[0060] As used herein, the term "cryptogen" refers to a nucleic acid sequence or structure of a cyclic polyribonucleotide that serves to reduce, avoid, and / or evade detection by immune cells and / or reduce the induction of an immune response against the cyclic polyribonucleotide . .

[0061] As used herein, the term "expression sequence" refers to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide or a regulatory nucleic acid. Exemplary expression sequences that encode a peptide or polypeptide can include a plurality of nucleotide triplets, each of which can encode an amino acid and is called a "codon" . .

[0062] As used herein, the term "immune protein binding site" refers to a nucleotide sequence that binds to an immune protein. In certain embodiments, the immune protein binding site serves to mask the cyclic polyribonucleotide as being exogenous. For example, the immune protein binding site can prevent the cyclic polyribonucleotide from being bound by an immune protein . .​​​​ It can be bound by a protein (e.g., a competitive inhibitor) that recognizes and prevents binding, thereby reducing or avoiding an immune response to the cyclic polynucleotide. As used in this specification, the term "immune protein" refers to, for example, any protein or peptide related to an immune response to an immunogen, such as a cyclic polynucleotide. Non-limiting examples of immune proteins include T cell receptors (TCRs), antibodies (immunoglobulins), major histocompatibility complex (MHC) proteins, complement proteins, and RNA-binding proteins.

[0063] As used in this specification, the terms "linear RNA" or "linear polynucleotide" or "linear polynucleotide molecule" are used synonymously and mean a polynucleotide molecule having 5' and 3' ends. One or both of the 5' and 3' ends may be free ends or may be bound to another moiety. As used in this specification, linear RNA has not undergone cyclization (e.g., is pre-cyclization) and can be used as a starting material for cyclization, for example, by splint ligation, or by chemical, enzymatic, ribozyme, or splicing-catalyzed cyclization methods.

[0064] As used in this specification, the terms "nick RNA" or "nick linear polynucleotide" or "nick linear polynucleotide molecule" are used synonymously and mean a polynucleotide molecule having 5' and 3' ends resulting from the cleavage or degradation of circular RNA.

[0065] As used herein, the term "non-circular RNA" refers to all nicked RNA and linear RNA.

[0066] As used herein, the term "modified ribonucleotide" is a nucleotide having at least one modification to a sugar, nucleobase or internucleoside linkage.

[0067] As used herein, the phrase "pseudocircular structure" is a higher-order structure of a circular polynucleotide, where at least a portion of the circular polynucleotide is folded into a pseudostructure.

[0068] As used herein, the phrase "pseudodouble-stranded secondary structure" is a higher-order structure of a circular polynucleotide, where at least a portion of the circular polynucleotide forms an internal double strand.

[0069] As used herein, the term "regulatory element" is a moiety such as a nucleic acid sequence that regulates the expression of an expression sequence within a circular polynucleotide.

[0070] As used herein, the term "repeated nucleotide sequence" is a repeated nucleic acid sequence within a stretch of DNA or RNA or throughout the genome. In certain embodiments, the repeated nucleotide sequence includes a polyCA or polyTG(UG) sequence. In certain embodiments, the repeated nucleotide sequence includes a repeated sequence in the Alu family of introns.

[0071] As used herein, the term "replication element" is useful for replication or is a sequence and / or motif that initiates transcription of circular polynucleotides.

[0072] As used herein, the term "stagger element" refers to a portion such as a nucleotide sequence that induces pausing of the ribosome during translation . In certain embodiments, the stagger element is a non-conserved sequence of amino acids having a strong alpha helix tendency, followed by a consensus sequence -D (V / I)ExNPG P (where x is any amino acid). In certain embodiments , the stagger element may include chemical moieties such as glycerol, non-nucleic acid linking moieties, chemical modifications, modified nucleic acids, or any combination thereof.

[0073] As used herein, the term "substantially resistant" means at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8 5%, 90%, 95%, 96%, 97%, 98% or 99% resistance to the vector compared to the control .

[0074] As used herein, the term "stoichiometric translation" refers to substantially equivalent production of the expression product translated from circular polynucleotides. For example, in a circular polynucleotide having two expression sequences , stoichiometric translation of the circular polynucleotide means that the expression products of the two expression sequences have substantially equal amounts, e.g., the difference in amount (e.g., molar difference) between the two expression sequences is about 0 or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or less than 20%, or any percentage therebetween .

[0075] As used herein, the term "translation initiation sequence" is a nucleic acid sequence that initiates translation of an expression sequence in a circular polynucleotide.

[0076] As used herein, the term "termination element" is a moiety such as a nucleic acid sequence that stops translation of an expression sequence in a circular polynucleotide.

[0077] As used herein, the term "translation efficiency" is the rate or amount of protein or peptide production from a ribonucleotide transcript. In certain embodiments, translation efficiency can be represented, for example, as the amount of protein or peptide produced per given amount of a single transcript encoding the protein or peptide, in a given period of time, in a given translation system, such as an in vitro translation system like rabbit reticulocyte lysate or an in vivo translation system such as a eukaryotic or prokaryotic cell.

[0078] As used herein, the term "circularization efficiency" is a measure of the resulting circular polynucleotide compared to its non-circular starting material.

[0079] As used herein, the term "immunogenicity" is the potential to induce an immune response against a substance. In certain embodiments, an immune response can be induced when an immune system of an organism or a certain type of immune cell is exposed to an immunogenic substance. The term "non-immunogenic" is the absence or non-existence of an immune response that exceeds a detectable threshold against a substance. In certain embodiments, no immune response is detected when an immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic substance. In certain embodiments, the non-immunogenic circular polynucleotides provided herein ​​​​​​​​​​​​​Ribonucleotides are not considered to be toxic to humans, as they are not known to cause immune deficiencies above a certain threshold as measured by an immunogenicity assay. For example, using an immunogenicity assay, When measuring antibodies elicited against inflammatory markers, the non-immune The proteolytic polyribonucleotides do not induce the production of antibodies or markers at levels below a predetermined threshold. The predetermined threshold may be, for example, a threshold value for the level of antibody or marker induced by a control. In another example, the immunogenicity of the antibody may be 1.5, 2, 3, 4, or 5 times lower than the antibody level. When measuring the innate immune response to cyclic polyribonucleotides using the inflammatory matrix The non-immunogenic polyribonucleotides provided herein are A level below a predetermined threshold may result in the occurrence of an innate immune response. The predetermined threshold may be, for example, , 1.5-fold, 2-fold, 3-fold the level of the marker produced by the natural response to the control reference. The amount of oxidized cellulose may be less than 1, 2, 3, or 4 times.

[0080] As used herein, the term "impurity" refers to any impurity in a composition, e.g., In one embodiment, the impurities are unwanted substances present in the pharmaceutical composition described in are process-related impurities. In certain embodiments, impurities are undesirable in the final composition. Other than the products of, for example, the active pharmaceutical ingredients described herein, e.g., cyclic polyribonucleic acid As used herein, "process-related" refers to a product-related substance other than leutidine. The term "impurities" refers to any impurities other than the linear polyribonucleotides described herein that are present in the final composition. Undesirable in the composition, formulation, or product, In one embodiment, the process The related impurities are enzymes used in the synthesis or cyclization of polynucleotides. As used herein, the term "product-related substance" refers to a substance or by-product generated during the synthesis of a composition, formulation, or product, or any intermediate thereof. In certain embodiments, the product-related substance is a deoxyribonucleotide fragment. In certain embodiments, the product-related substance is a deoxyribonucleotide monomer. In certain embodiments, the product-related substance is a derivative or fragment of the polynucleotide described herein, e.g., a fragment of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acids, monoribonucleic acid, diribonucleic acid, or triribonucleic acid, among others. One or more.

[0081] As used herein, the term "substantially free of" refers to a level of a component in a composition, formulation, or product, or any intermediate thereof, that is lower than the level necessary to induce a biological, chemical, physical, and / or pharmacological effect. In certain embodiments, the level of the component is only detectable in trace amounts, or the level is determined using detection techniques based on mass spectrometry, UV-visible light, fluorescence, light scattering, refractive index, or those using silver or dye staining or radioactive decay for detection, with or without derivatization methods before or after separation, using related detection techniques (e.g., chromatography (using columns, paper, gels, HPLC, UHPLC, etc., or by IC, SEC, reverse phase, anion exchange, mixed mode, etc.) or electrophoresis (urea PAGE, chip-based, polyacrylamide gel, RNA, capillary, c-I ​ If it is below the detectable level by, for example, EF, the composition, formulation, or product substantially does not contain the component Instead, whether the composition, formulation, or product substantially does not contain the component can be determined without using separation techniques by mass spectrometry, microscopy, circular dichroism (CD) spectrometry, UV or UV- vis spectrophotometry, fluorescence analysis (e.g., Qubit), RNase H analysis, surface plas mon resonance (SPR), or methods using silver or dye staining or radioactive decay for detection .

[0082] As used herein, the term "linear equivalent" refers to a polynucleotide molecule (and fragments thereof) having the same or a similar nucleotide sequence (e.g., 100%, 95%, 90%, 85% , 80%, 75%, or any percentage sequence similarity therebetween) as the circular polynucleotide and having two free ends (i.e., the non-circularized form of the circularized polynucleotide (and fragments thereof)). In certain embodiments, the linear equivalent (e.g., the pre-circularized form) has the same or a similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80 %, 75%, or any percentage sequence similarity therebetween) and the same or a similar nucleic acid modification as the circular polynucleotide and has two free ends (i.e., the non-circularized form of the circularized polynucleotide (and fragments thereof)). In certain embodiments, the linear equivalent has the same or a similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80% as the circular polynucleotide and has two free ends (i.e., the non-circularized form of the circularized polynucleotide (and fragments thereof)). In certain embodiments, the linear equivalent is a polynucleotide molecule (and fragments thereof) having the same or a similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80% , 75%, or any percentage sequence similarity therebetween) as the circular polynucleotide and having two free ends (i.e., the non-circularized form of the circularized polynucleotide (and fragments thereof)). In certain embodiments, the linear equivalent has the same or a similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80% as the circular polynucleotide and has two free ends (i.e., the non-circularized form of the circularized polynucleotide (and fragments thereof)). In certain embodiments, the linear equivalent is a polynucleotide molecule (and fragments thereof) having the same or a similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80% as the circular polynucleotide and having two free ends (i.e., the non-circularized form of the circularized polynucleotide (and fragments thereof)). In certain embodiments, the linear equivalent has the same or a similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80% , 75%, or any percentage sequence similarity therebetween) and have different nucleic acid modifications either have or have no nucleic acid modifications, and have two free ends, a polyribonucleotide molecule ( and fragments thereof) (i.e., the non-circularized form of a circularized polyribonucleotide (and its fragments)). In certain embodiments, a fragment of a polyribonucleotide molecule that is a linear equivalent is a linear equivalent that is shorter than the linear equivalent polyribonucleotide molecule and is any portion of the linear equivalent polyribonucleotide molecule. In certain embodiments, the linear equivalent further comprises a 5' cap. In certain embodiments, the linear equivalent further comprises a polyadenosine tail . In certain embodiments, the linear equivalent further comprises a 3' UTR. In certain embodiments, the linear equivalent further comprises a 5' UTR.

[0083] As used herein, the term "aptamer sequence" refers to a non-natural or synthetic oligonucleotide that specifically binds to a target molecule. Typically, an aptamer is 20 to 500 nucleotides. Typically, an aptamer binds to its target by secondary structure rather than sequence homology. In certain embodiments, a synthetic oligonucleotide can have the same sequence as a natural oligonucleotide that specifically binds to a target molecule.

[0084] As used herein, the term "carrier" refers to a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., a circular polyribonucleotide) into a cell by covalent modification of the circular polyribonucleotide, partially or completely by an encapsulating agent, or a combination thereof. Non-limiting examples of carriers include carbohydrate carriers ​​​A body (e.g., an anhydride-modified phyto-glycogen or glycogen-like material), a nanoparticle ( e.g., a nanoparticle encapsulating or covalently bound to a cyclic polynucleotide ), a liposome, fusosome, ex vivo differentiated reticulocyte, exosome, tamp protein carrier (e.g., a protein covalently bound to a cyclic polynucleotide), or a cat ionic carrier (e.g., a cationic lipopolymer or transfection reagent) is exem plified.

[0085] As used herein, the term "naked delivery" refers to a formulation for delivery to cells that does not use a carrier and has no covalent modification to a moiety that aids in delivery to the cells. A naked delivery formulation does not contain any transfection reagent, cationic carrier, carbohydrate carrier, nanoparticle carrier, or protein carrier. For example, a naked delivery formulation of a cyclic polynucleotide is a formulation that contains a cyclic polynucleotide having no covalent modification and does not contain a carrier.

[0086] The term "diluent" refers to a vehicle containing an inert solvent in which the compositions described herein (e.g., a composition containing a cyclic polynucleotide) can be diluted or dissolved. A diluent can be an RNA solubilizing agent, buffer, isotonic agent, or a mixture thereof. A diluent can be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, cane oil, etc.) Sorghum oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran furfuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, 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 term "parenterally acceptable diluent" refers to a composition Diluents used for parenteral administration of (e.g., compositions containing cyclic polyribonucleotides) be.

[0088] Incorporation by Reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. Each patent or patent application is specifically and individually indicated to be incorporated by reference. and is incorporated by reference herein 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 purposes of illustrating the invention, the embodiments illustrated herein will be described in detail with reference to the accompanying 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 description of the drawings]

[0090]

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Mode for Carrying Out the Invention

[0091] The present invention generally relates to circular polynucleotide pharmaceutical compositions and preparations and their use. To do.

[0092] In one aspect, the invention described herein relates to circular RNA compositions, formulations, and methods of using and making circular RNA compositions and formulations, particularly pharmaceutical circular RNA compositions and formulations, having reduced, controlled, or specified levels of linear RNA. As described in Examples 1 to 12 herein, for example, the presence of linear RNA in circular RNA formulations affects, for example, the expression level, persistence, half-life, and / or stability of circular RNA; and / or having a level of linear RNA; including methods of using and making circular RNA compositions and formulations, particularly pharmaceutical circular RNA compositions and formulations. As described herein, for example, as described in Examples 1 to 12, the presence of linear RNA in circular RNA formulations affects, for example, the expression level, persistence, half-life, and / or stability of circular RNA; and / or For example, as described in Examples 1 to 12, the presence of linear RNA in circular RNA formulations affects, for example, the expression level, persistence, half-life, and / or stability of circular RNA; and / or For example, the expression level, persistence, half-life, and / or stability of circular RNA; and / or It can affect the immune response to the formulation.

[0093] Table 1 is intended to provide a brief overview of the content of the detailed description, which is in no way exclusive or limiting. Certain aspects of the detailed description may not be reflected in the overview of the detailed description in Table 1.

[0094] **Table 1**

[0095] **Table 2**

[0096] Circular polynucleotide In certain embodiments, the circular RNA has a sequence, or sequences, that encode an expression product, such as a therapeutic expression product. For example, the circular RNA encodes a therapeutic protein or nucleic acid. In certain embodiments, the circular RNA has a sequence, or sequences, that include an aptamer. In certain embodiments, the circular RNA has a sequence that encodes a sequence having at least 80% (e.g., 85%, 90%, 95%, 97% %, 99%, 100% or any percentage therebetween) sequence identity to an endogenous or native circular polynucleotide sequence. In certain embodiments, the circular RNA and the formulation do not cause an undesirable immune response in a mammal, such as a human.

[0097] In certain embodiments, the circular polynucleotide has a half-life that is at least that of its linear equivalent, such as a linear expression sequence, or a linear polynucleotide. In certain embodiments ​​​​​​​​In certain embodiments, the cyclic polynucleotide has a longer half-life than its linear equivalent. In certain embodiments, the half-life is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more, or any percentage therebetween longer. In certain embodiments, the cyclic polynucleotide has a half-life or persistence in cells of at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 2 4 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days , 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days , 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days , 27 days, 28 days, 29 days, 30 days, 60 days, or more, or any time therebetween. In certain embodiments, the cyclic polynucleotide has a half-life or persistence in cells of about 10 minutes or less to about 7 days, or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours , 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours , 21 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4 days , 5 days, 6 days, 7 days or less, or any time therebetween. In certain embodiments, the cyclic polynucleotide has a half-life or persistence in cells while the cells are dividing. In certain embodiments, the cyclic polyribo nucleotide has a half-life or persistence in the cells after division. In certain embodiments, the cyclic polynucleotide is greater than about 10 minutes to about 30 days, or at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 o'clock minutes, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 o'clock minutes, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 day, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 1 9 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 2 7 days, 28 days, 29 days, 30 days, 60 days, or more or any time in between has a half-life or persistence in the divided cells. Over any time, it has a half-life or persistence in the divided cells.

[0098] In certain embodiments, the cyclic polynucleotide regulates cell function, e.g., temporarily or for a long period. In certain embodiments, for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or more or any time in between, the cell function such as regulation is stably changed. In certain embodiments, e.g., about 3 0 minutes or less to about 7 days, or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 o'clock minutes, 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, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days or less, or Cell functions such as regulation that persist over any time between them are temporarily altered .

[0099] In certain embodiments, the cyclic polynucleotide is at least about 20 nucleotides , at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nu cleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nu cleotides, 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 8,0 00 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nu cleotides, at least about 12,000 nucleotides, at least about 14,000 nucleo tides, at least about 15,000 nucleotides, at least about 16,000 nucleotide s, 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 polynucleotide can be of a size sufficient to correspond to the binding site for ribosomes. One of ordinary skill in the art will understand that the maximum size of the cyclic polynucleotide can be of a size within the technical constraints of generating and / or using the cyclic polynucleotide. It is possible. Although not restricted by theory, multiple segments of RNA are annealed to their 5' and 3' free ends to generate the "sequences" of DNA and RNA such that they can be generated from those 5' and 3' free ends which, ultimately, can be circularized when only one 5' free end and one 3' free end remain. In certain embodiments, the maximum size of the circular polyribonucleotide can be limited by the ability to package and deliver the RNA to a target. In certain embodiments, the size of the circular polyribonucleotide is of sufficient length to encode a useful polypeptide and thus is at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides in length may be useful. In certain embodiments, the circular polyribonucleotide comprises one or more elements described elsewhere herein. In certain embodiments, the elements can be separated from each other by spacer sequences or linkers. In certain embodiments, the elements are 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides In certain embodiments, the circular polyribonucleotide comprises one or more elements described elsewhere herein. In certain embodiments, the elements can be separated from each other by spacer sequences or linkers. In certain embodiments, the elements are 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides In certain embodiments, the circular polyribonucleotide comprises one or more elements described elsewhere herein. In certain embodiments, the elements can be separated from each other by spacer sequences or linkers. In certain embodiments, the elements are 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides In certain embodiments, the circular polyribonucleotide comprises one or more elements described elsewhere herein. In certain embodiments, the elements can be separated from each other by spacer sequences or linkers. In certain embodiments, the elements are 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides In certain embodiments, the circular polyribonucleotide comprises one or more elements described elsewhere herein. In certain embodiments, the elements can be separated from each other by spacer sequences or linkers. In certain embodiments, the elements are 1 ribonucleotide, 2

[0100] In certain embodiments, the circular polyribonucleotide comprises one or more elements described elsewhere herein. In certain embodiments, the elements can be separated from each other by spacer sequences or linkers. In certain embodiments, the elements are 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides In certain embodiments, the circular polyribonucleotide comprises one or more elements described elsewhere herein. In certain embodiments, the elements can be separated from each other by spacer sequences or linkers. In certain embodiments, the elements are 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides Nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 2 00 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides , about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000 nucleotides, separated from each other by any amount of nucleotides therebetween obtainable. In certain embodiments, one or more elements are contiguous with each other, for example, lacking spacer elements. In certain embodiments, one or more elements in the cyclic polynucleotide have conformational flexibility. In certain embodiments, the conformational flexibility is due to the substantially lack of secondary structure of the sequence. In certain embodiments, the cyclic polynucleotide has a secondary or tertiary structure corresponding to one or more desired functions or properties described herein, for example, a binding site for ribosomes , for example, translation, for example, rolling circle type translation.

[0101] In certain embodiments, the cyclic polynucleotide comprises specific sequence characteristics. For example , the cyclic polynucleotide can comprise a specific nucleotide composition. In certain such embodiments, the cyclic polynucleotide can comprise one or more purine-rich regions (adenine or guanosine). In certain such embodiments, the cyclic polynucleotide can comprise one or more purine-rich regions (adenine or guanosine). In certain embodiments, the cyclic polynucleotide can comprise one or more AU-rich regions or elements (ARE). In certain embodiments, the cyclic polynucleotide can comprise one or more a ​​​It may contain a dinucleotide-rich region.

[0102] In certain embodiments, the circular polynucleotide may contain one or more repetitive elements described elsewhere herein.

[0103] In certain embodiments, the circular polynucleotide contains one or more modifications described elsewhere herein.

[0104] In certain embodiments, the circular polynucleotide contains one or more expression sequences and is configured for sustained expression in the cells of a subject in vivo. In certain embodiments, the circular polynucleotide is configured such that the expression of one or more expression sequences in the cell at a later time point is equal to or greater than that at an earlier time point. In such embodiments, the expression of one or more expression sequences can be maintained at a relatively stable level or can increase over time. The expression of the expression sequence can be relatively stable over a long period. For example, in certain cases, the expression of one or more expression sequences in the cell over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 days or more does not decrease by 50 %, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In certain cases, in certain cases, the expression of one or more expression sequences in the cell is maintained at a level that does not change by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15 %, 10%, or 5% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or more.

[0105] ​​​​​In certain embodiments, the cyclic polynucleotide is non-immunogenic in mammals, such as humans. In certain embodiments, the cyclic polynucleotide replicates in cells derived from aquaculture animals (such as fish, crabs, shrimp, oysters, etc.), mammalian cells, such as cells derived from pets or zoo animals (such as cats, dogs, lizards, birds, lions, tigers, and bears), cells derived from livestock or draft animals (such as 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, blastocysts, adult cells, mitotic cells, non-mitotic cells, or any combination thereof, or replicates. In certain embodiments, the invention includes cells comprising the cyclic polynucleotides described herein, wherein the cells are cells derived from aquaculture animals (such as fish, crabs, shrimp, oysters, etc.), mammalian cells, such as cells derived from pets or zoo animals (such as cats, dogs, lizards, birds, lions, tigers, and bears), cells derived from livestock or draft animals (such as 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, blastocysts, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In certain embodiments, the cells are modified to contain the cyclic polynucleotide.

[0106] Methods for producing circular RNAs In certain embodiments, the production of the cyclic polynucleotide is non-natural and recombinant. Techniques (the methods described below; e.g., obtained in vitro using a DNA plasmid ) or the preparation of deoxyribonucleic acid sequences that can be generated using chemical synthesis. In certain embodiments , the circularization of linear polynucleotides is performed by splint ligation .

[0107] It is within the scope of the present invention that the DNA molecules used to generate the RNA ring can include the DNA sequence of the natural original nucleic acid sequence, its modified forms, or DNA sequences encoding synthetic polypeptides not normally found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using various techniques including, but not limited to, site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and / or mutagenesis of selected regions of nucleic acid sequences, synthesis of oligonucleotide mixtures and ligation of mixtures of nucleic acid molecules to "construct" mixtures thereof, and combinations thereof, such as classical mutagenesis techniques and recombinant techniques. The circular polynucleotides can be prepared according to any available technique including, but not limited to, chemical synthesis and enzymatic synthesis. In certain embodiments, linear primary constructs or linear

[0108] mRNA can be circularized or concatenated to generate the circular polynucleotides described herein. The mechanism of cyclization or concatenation can be performed by methods including, but not limited to, chemical, enzymatic, splint ligation, or ribozyme-catalyzed methods. The newly formed 5'- / 3'-bonds can be intramolecular or intermolecular bonds . 。

[0109] The methods for preparing the cyclic polynucleotides described in this specification are, for example, described in Khud yakov & Fields, Artificial DNA: Methods an d Applications, CRC Press (2002); Zhao, Synt hetic Biology: Tools and Applications, (Fi rst Edition), Academic Press (2013); and Egli & Herdewijn, Chemistry and Biology of Ar tificial Nucleic Acids, (First Edition), W iley-VCH (2012).

[0110] Various methods for synthesizing cyclic polynucleotides are also described in the art (e.g., U.S. Patent No. 6,210,9 31, U.S. Patent No. 5,773,244, U.S. Patent No. 5,766,903 , U.S. Patent No. 5,712,128, U.S. Patent No. 5,426,180, U.S. Patent App lication Publication No. 2010 / 0137407, International Publication No. 1992 / 001813 Pamphlet; International Publication No. 2016 / 197121 Pamphlet; International Publication No. 2010 / 08437 1 Pamphlet, the entire contents of each of which are hereby incorporated by reference).

[0111] For example, Example 13 described herein (see below) describes a method for preparing and characterizing a pharmaceutical cyclic RNA formulation .

[0112] Detection of linear and cyclic RNAs The inventors have found that the presence of linear RNA in pharmaceutical circular RNA formulations can have unexpected and sometimes undesirable effects. Accordingly, the present invention particularly relates to pharmaceutical compositions and formulations in which circular RNA is enriched, isolated, and / or purified compared to linear RNA; methods by which linear RNA can be monitored, evaluated, and / or controlled (e.g., methods of manufacturing circular RNA formulations); and methods of delivering effectors (e.g., aptamer sequences) such as therapeutic effectors or scaffolds to cells, tissues, or subjects using such pharmaceutical compositions and formulations. In certain embodiments, the circular RNA formulation has linear RNA below a threshold level, e.g., the circular RNA formulation is enriched relative to linear RNA and purified to reduce linear RNA.

[0113] Generally, the detection and quantification of elements in a pharmaceutical formulation involves the use of a reference standard that is either the component of interest (e.g., circular RNA, linear RNA, fragments, impurities, etc.) or a similar material (e.g., a linear RNA structure having the same sequence as the circular RNA structure for use as a standard for circular RNA), or involves the use of signals from internal standards or test samples. In certain embodiments, the standard is used to establish the response from the detector for the amount of material in a process or relative amount of material (response coefficient). In certain embodiments, the response coefficient is determined from the standard at one or multiple concentrations (e.g., using linear regression analysis). In certain embodiments, the response coefficient is then used to determine the amount of the material of interest from the signal by that component. In certain embodiments, the response coefficient has a value of 1 or is assumed to have a value of 1.

[0114] ​​​​​​​​​​​​​​​​​Detection and quantification of linear RNA relative to circular RNA in a pharmaceutical composition is determined using a comparison with the linear form of the circular polynucleotide. In certain embodiments the mass of total ribonucleotides in a pharmaceutical composition is determined using a standard curve generated using the linear form of the circular polynucleotide and assuming a response coefficient of 1. In certain embodiments, the w / w percentage of circular polynucleotide in a pharmaceutical formulation is determined by comparison to a standard curve generated by the band intensity of the circular polynucleotide in the pharmaceutical formulation and the band intensities of multiple known amounts of the linear form of the circular poly ribonucleotide. In certain embodiments, the bands are generated during gel-based electrophoresis and the band intensities are measured by a gel imaging device (e.g., E-gel Imager). For example the amount of linear polynucleotide compared to circular polynucleotide can be determined using the methods of Example 2 and / or Example 3. In certain embodiments, the circular polynucleotide formulation contains linear polynucleotide molecules below a threshold amount (e.g., here the threshold amount is a reference standard for the circular polynucleotide formulation such as a pharmaceutical release specification) when evaluated as described herein.

[0115] In certain embodiments, the detection and quantification of nicked RNA relative to total RNA in a pharmaceutical composition is determined by sequencing after gel extraction of the formulation containing circular RNA. In certain embodiments the detection and quantification of nicked RNA relative to linear RNA in a pharmaceutical composition is determined by sequencing after gel extraction of the formulation containing circular RNA. For example, total R ​The amount of nicked polynucleotides compared to NA can be determined using the method of Example 5. For example, the amount of nicked polynucleotides compared to linear RNA can be determined using the method of Example 5. In certain embodiments, the cyclic polynucleotide formulation contains nicked RNA, linear RNA, or a combination of linear and nicked RNA that is below a threshold amount (e.g., here, the threshold amount is a reference standard for the cyclic polynucleotide formulation, such as a pharmaceutical release specification) when evaluated as described in the present specification. For example, the reference standard for the amount of linear polynucleotide molecules present in the formulation is 30%, 20%, 15%, 10%, 1%, 0.5%, or 0.1% or less of linear polynucleotide molecules, or any percentage therebetween, relative to the total nucleotide molecules in the formulation. In certain embodiments, the reference standard for the amount of nicked polynucleotide molecules present in the formulation is 30%, 20%, 1 5%, 10%, 1%, 0.5%, or 0.1% or less, or any percentage therebetween, of nicked polynucleotide molecules relative to the total nucleotide molecules in the formulation. In certain embodiments, the reference standard for the amount of linear and nicked polynucleotide molecules present in the formulation is 40%, 30%, 20%, 15%, 10%, 1%, 0 .5%, or 0.1% or less, or any percentage therebetween, of combined linear polynucleotide and nicked polynucleotide molecules relative to the total nucleotide molecules in the formulation. In certain embodiments, the standard is experimented under the same conditions as the sample. For example, the standard is

[0116] , gels of the same type as the sample, the same buffer, and exposure are used for the experiment. Further embodiments In, the standard is experimented in parallel with the sample. In certain embodiments, the quantification of the element is repeated in multiple samples from the formulation of the present invention to obtain average results (e.g for example, twice or in triplicate). In certain embodiments, the quantification of linear RNA is para llel capillary electrophoresis (e.g., using a fragment ment analyzer or analytical HPLC with UV detection) is used for measurement.

[0117] Purification of circular RNA Circular polynucleotides can be separated, enriched, or purified from unwanted substances (unwanted (e.g., linear) RNA, enzymes, DNA, etc.). In certain embodiments, the unwanted substances are present during or result from the process of making and / or manufacturing the circular polynucleotide. The circular polynucleotides described herein can be enriched and / or purified before formulation in a pharmaceutical formulation, pharmaceutical composition, pharmaceutical drug, or pharmaceutical product. The circular polynucleotides described herein can be enriched and / or purified after formulation in a pharmaceutical formulation, pharmaceutical composition, pharmaceutical drug, or pharmaceutical product.

[0118] In certain embodiments, circular RNA is purified during or after production to remove unwanted elements, such as linear RNA, nick ed RNA, as well as recognized impurities, such as free ribonucleic acids (e.g., mono-ribo nucleic acids, di-ribonucleic acids, or tri-ribonucleic acids), DNA (e.g., cellular DNA such as host cell DNA), cell or process-related protein impurities (e.g., cell or process-related impurities), etc. In certain embodiments the impurities are process-related impurities. In certain embodiments, the process-related impurities are. In certain embodiments, the process-related impurities The substance is a protein (e.g., a cellular protein), a nucleic acid (e.g., a cellular nucleic acid), a buffer, or a buffer reagent, an enzyme, a medium / reagent component (e.g., a medium, a medium additive, a transition metal, or a vitamin), a preparatory or analytical gel component (e.g., an acrylamide fragment), DNA, or a chromatography material. The buffer reagent can be MgCl 2, DTT, ATP, SDS, Na, glycogen, Tris-HCl, or EtOH. Examples of buffer reagents include, but are not limited to, acetate, Tris, bicarbonate, phosphate, citrate, lactate, or TEA. The enzyme can be a ligase. The ligase can be T4 RNA ligase 2. In certain embodiments, the impurities are buffer reagents, medium / reagent components, salts, ligases, nucleases, RNase inhibitors, RNase R, linear polynucleotide molecules, deoxyribonucleotide molecules, acrylamide fragments, or mononucleotide molecules.

[0119] In certain embodiments, the circular polynucleotide can be enriched or purified by any known method commonly used in the art. Examples of non-limiting purification methods include column chromatography, gel exclusion, size exclusion, and the like.

[0120] In certain embodiments, the circular polynucleotide is purified, for example, by gel purification, such as UREA gel separation, as described in Example 3. For example, circular RNA can be degraded in denaturing PAGE, the band corresponding to the circular RNA can be excised, and the circular RNA can be eluted from the band using known methods. The eluted circular RNA can then be analyzed.

[0121] In certain embodiments, the cyclic polynucleotide is purified by chromatography, such as, hydrophobic interaction chromatography (HIC), mixed mode chromatography, liquid chromatography, such as, reversed phase ion pair chromatography (IP-RP), ion exchange chromatography (IE), affinity chromatography (AC), and size exclusion chromatography (SEC), and any combination thereof.

[0122] In certain embodiments, the cyclic polynucleotide is purified by taking advantage of the structural features of the cyclic polynucleotide to separate it from linear RNA or impurities. In certain embodiments, the cyclic polynucleotide is purified by taking advantage of structural features such as those described in Example 9 (e.g., lack of free ends). For example, circular RNA is enriched from a formulation containing a mixed pool of circular RNA and linear RNA equivalents having the same nucleotide sequence by using polyadenylation of the linear RNA equivalents or fragments thereof. The 3' end of the linear RNA equivalents or fragments thereof can be polyadenylated using poly(A) polymerase to effect addition of a 3' polyadenine tail. In certain embodiments, the 3' polyadenine tail enables pull-down of linear RNA and fragments thereof using a column, such as an affinity column, to enrich for This addition enables the pull-down of linear RNAs and their fragments in systems such as the biotin-streptavidin binding system. In contrast, circular RNAs, having no 3' ends, are not polyadenylated by poly(A) polymerase, do not have a polyadenylated tail for conjugation, and are not captured during pull-down . Thus, circular RNAs are enriched in the formulation after pull-down.

[0123] In certain embodiments, circular polynucleotides are purified by taking advantage of structural features of linear RNAs (e.g., the presence of free ends). For example, circular RNAs are enriched from a formulation containing a mixed pool of circular RNAs and linear RNA equivalents having the same nucleotide sequence using polyadenylation of the linear RNA equivalents. Exonucleases can be added to the mixed pool to hydrolyze the linear RNAs. In certain embodiments, the exonucleases can be 3' exonucleases or 5' exonucleases. In certain embodiments, both 3' exonucleases and 5' exonucleases can be used.

[0124] In one embodiment, a circular polynucleotide formulation (e.g., an intermediate in the production of a circular polynucleotide pharmaceutical formulation or composition or a circular polynucleotide formulation) is at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w ), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95 % (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w) on a mass basis. ​ w), or a purity of 100% (w / w). The purity can be determined by mass spectrometry, UV-visible light, fluorescence , light scattering, refractive index-based detection techniques, or those using silver or dye staining or radioactive decay for detection, using a pre-separation or post-separation derivatization method or without using it, but not limited to, chromatography (using columns, paper, gels, HPLC, UHPLC, etc., or IC, SEC, reverse phase, anion exchange, mixed mode, etc.) or electrophoresis (urea PAGE, chip-based, polyacrylamide gel, RNA, capillary, c-IEF, etc.) and other separation techniques, measured by any one of several analytical techniques known to those skilled in the art. Alternatively, the purity can be determined without using separation techniques such as mass spectrometry, microscopy, circular dichroism (CD) spectroscopy, UV or UV-vis spectroscopy colorimetry, fluorescence analysis (e.g., Qubit), RNAse H analysis, surface plasmon resonance (SPR), or methods using silver or dye staining or radioactive decay for detection. In certain embodiments, the purity can be measured by biological test methods (e.g., cell-based or receptor-based tests). In certain embodiments, at least 30% (w / w), 40% (w / w), 50 % (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 9

[0125] 4% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w of the total mass of ribonucleotides in the formulations described herein. ​​​​, 99% (w / w), or 100% (w / w) is present in the cyclic polynucleotide molecule The percentages are determined by mass spectrometry, UV-visible, fluorescence, light scattering, refractive index -based detection techniques, or by using silver or dye staining or radioactive decay for detection, with or without using derivatization methods before or after separation, and are not limited to chromatography (using columns, paper, gels, HPLC, UHPLC, etc., or by IC, SEC, reverse phase, anion exchange, mixed mode, etc.) or electrophoresis (urea PAGE, chip-based, polyacrylamide gel, RNA A, capillary, c-IEF, etc.), and can be measured by any one of several analytical techniques known to those skilled in the art. Alternatively, purity can be determined by mass spectrometry, microscopy, circular dichroism (CD) spectroscopy, UV or UV-vis spectrophotometry, fluorescence analysis (e.g., Qubit), RNAse H analysis, surface plasmon resonance (SPR), or by using separation techniques such as silver or dye staining or radioactive decay for detection (e.g., Qubit), RNAse H analysis, surface plasmon resonance (SPR), or by using separation techniques such as silver or dye staining or radioactive decay for detection and can be determined by using separation techniques such as silver or dye staining or radioactive decay for detection .

[0126] In one embodiment, the cyclic polynucleotide formulation (e.g., a cyclic polynucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polynucleotide formulation) is at least 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 n g / mL, 50 ng / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μ g / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 100 μ / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 100 μ g / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, 1000 μg / m L, 5000 μg / mL, 10,000 μg / mL, 100,000 μg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / m L, 650 mg / mL, 700 mg / mL, or 750 mg / mL of cyclic polyribonucle otide concentration. In one embodiment, the cyclic polyribonucleotide formulation (e.g., a cyclic polyribonucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polyribonucleotide formulation) is substantially free of mononucleotides or has a mononucleotide content of 1 pg / ml, 10 p g / ml, 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL , or 100,000 μg / mL or less. In one embodiment the cyclic polyribonucleotide formulation (e.g., a cyclic polyribonucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polyribonucleotide formulation) is from the limit of detection to 1 pg / ml, 10 pg / ml, 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml , 400 ng / ml, 500 ng / ml, 1000 μg / mL, 5000 μg / mL, 1 0,000 μg / mL, or up to 100,000 μg / mL of mononucleotide content to have.

[0127] In one embodiment, the cyclic polyribonucleotide formulation (e.g., an intermediate in the production of a cyclic polyribonucleotide pharmaceutical formulation or composition or cyclic polyribonucleotide formulation) is 0.1% (w / w), 0.2% (w / w), 0.3% (w / w), 0.4% (w / w), 0.5% (w / w), 0.6% (w / w), 0.7% (w / w), 0.8% (w / w), 0.9% (w / w), 1% (w / w), 2% (w / w), 3 % (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8 % (w / w), 9% (w / w), 10% (w / w), 15% (w / w), 20% (w / w ), 25% (w / w), 30% (w / w) or less, or any percentage in between of mononucleotide content, where the total nucleotide content is the total mass of deoxyribonucleotide molecules and ribonucleotide molecules.

[0128] In one embodiment, the cyclic polyribonucleotide formulation (e.g., an intermediate in the production of a cyclic polyribonucleotide pharmaceutical formulation or composition or cyclic polyribonucleotide formulation) 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, 2 ng / ml, ​00 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 1 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 g / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 5 mg / mL, 10 mg / mL, 50 mg / mL, 100 mg / m L, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 60 0 mg / mL, 650 mg / mL, 700 mg / mL, or 750 mg / mL or less of linear RNA content, e.g., having linear RNA equivalents or RNA fragments. In one embodiment the cyclic polynucleotide formulation (e.g., a cyclic polynucleotide pharmaceutical formulation or a composition or an intermediate in the production of a cyclic polynucleotide formulation) is from the limit of detection to 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 n g / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 n g / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 20 0 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 1 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 g / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 7 00 μg / ml, 800 μg / ml, 900 μg / ml, 1 mg / ml, 1.5 mg / m l, 2 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml , 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml, 600 linear R up to mg / ml, 650 mg / ml, 700 mg / ml, or 750 mg / ml NA content, e.g., having a linear RNA equivalent or RNA fragment.

[0129] In one embodiment, a cyclic polynucleotide formulation (e.g., an intermediate in the production of a cyclic polynucleotide pharmaceutical formulation or composition or a cyclic polynucleotide formulation) is from 10% (w / w), 9.9% (w / w), 9.8% (w / w), 9.7% (w / w), 9 .6% (w / w), 9.5% (w / w), 9.4% (w / w), 9.3% (w / w), 9 .2% (w / w), 9.1% (w / w), 9% (w / w), 8% (w / w), 7% (w / .w), 6% (w / w), 5% (w / w), 4% (w / w), 3% (w / w), 2% (w / .w), 1% (w / w), 0.5% (w / w), or 0.1% (w / w) or less, or a percentage of nick RNA content therebetween. In one embodiment, a cyclic poly ribonucleotide formulation (e.g., an intermediate in the production of a cyclic polynucleotide pharmaceutical formulation or composition or a cyclic polynucleotide formulation) has a nick RNA content of about zero or substantially free of nick RNA.

[0130] In one embodiment, a cyclic polynucleotide formulation (e.g., an intermediate in the production of a cyclic polynucleotide pharmaceutical formulation or composition or a cyclic polynucleotide formulation) is from 30% (w / w), 25% (w / w), 20% (w / w), 15% (w / w), 10%( w / w), 9% (w / w), 8% (w / w), 7% (w / w), 6% (w / w), 5%( w / w), 4% (w / w), 3% (w / w), 2% (w / w), 1% (w / w), 0.5 w / w), 4% (w / w), 3% (w / w), 2% (w / w), 1% (w / w), 0.5 (w / w), or 0.1% (w / w) or less, or a percentage combination therebetween has a combined linear RNA and nicked RNA content. In one embodiment, the cyclic poly ribonucleotide formulation (e.g., a cyclic polyribonucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polyribonucleotide formulation) has a combined nicked RNA and linear RNA content of about zero or is substantially free of nicked and linear RNA.

[0131] In certain embodiments, the cyclic polyribonucleotide formulation (e.g., a cyclic polyribonucle otide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polyribonucleotide formulation) is below the detection limit of an analytical method using a separation method including mass spectrometry, UV spectroscopy or fluorescence detector, light scattering techniques, HPLC, surface plasmon resonance (SPR) with or without the use of derivatization methods before or after separation, chip or gel based electrophoresis with or without the use of derivatization methods, detection methods using silver or dye staining or radioactive decay, or methods using microscopy, visual methods or spectrophotometers, etc., and has a linear RNA content, e.g., a linear RNA equivalent or RNA fragment.

[0132]

[0132] In one embodiment, the cyclic polyribonucleotide formulation (e.g., a cyclic polyribonucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polyribonucleotide formulation) is, e.g., 0.1% (w / w), 1% (w / w) , 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w) , 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 4 0% (w / w), 45% (w / w), 50% (w / w) or less of linear RNA.

[0133] In certain embodiments, the linear polynucleotide moiety of the cyclic polynucleotide formulation comprises a linear equivalent of the cyclic polynucleotide molecule or a fragment thereof. In certain embodiments, the linear polynucleotide molecule of the cyclic polynucleotide formulation comprises a linear equivalent (e.g., the pre-cyclized form). In certain embodiments, the linear polynucleotide molecule of the cyclic polynucleotide formulation comprises a non-equivalent to the cyclic polynucleotide or a fragment thereof. In certain embodiments, the linear polynucleotide molecule of the cyclic polynucleotide formulation comprises a non-equivalent to the cyclic polynucleotide. In certain embodiments, the linear polynucleotide molecule comprises a combination of an equivalent of the cyclic polynucleotide and a non-equivalent or a fragment thereof of the cyclic polynucleotide. In certain embodiments, the linear polynucleotide molecule comprises a combination of an equivalent of the cyclic polynucleotide and a non-equivalent of the cyclic polynucleotide. In certain embodiments the linear polynucleotide molecule fragment is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 200, 300, 400, 500, 1000, 2000, 3000, 4000, 500 0, 6000, 7000, 8000, 9000, 10000, 11000, 12000, or more nucleotide lengths, or a fragment of any number of nucleotides therebetween.

[0134] In certain embodiments, a cyclic polyribonucleotide formulation (e.g., (intermediates in the production of pharmaceutical preparations or compositions or cyclic polyribonucleotide preparations) For example, when measured by a spectrophotometer, the A260 / A280 ratio is about 1.6 to about 2.3. In one embodiment, the A260 / A280 absorbance ratio is about 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any number therebetween. oxidase (e.g., a pharmaceutical formulation or composition of a circular polyribonucleotide or The yield of 1.8 mg of octyldodecyl phosphate (an intermediate in the production of octyldodecyl phosphate) was approximately 1.8 mg as measured by spectrophotometer. In one embodiment, the A260 / A280 absorbance ratio is greater than or equal to 80Absorbance ratio is approximately 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3 , 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or higher.

[0135] In certain embodiments, a cyclic polyribonucleotide formulation (e.g., (intermediates in the production of pharmaceutical preparations or compositions or cyclic polyribonucleotide preparations) In various embodiments, the nucleic acid is substantially free of impurities. The level of at least one impurity in a composition containing the compound may be determined by purification or processing to remove the impurity. At least 30% (w / w), at least 40% (w / w) ), at least 50% (w / w), at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), at least 90% (w / w), or at least 9 It is reduced by 5% (w / w). In certain embodiments, the level of at least one process-related impurity is at least 30% (w / w), at least 40% (w / w), at least 50% (w / w), at least 60% (w / w), at least 70% (w / w), at least 80% (w / w ), at least 90% (w / w), or at least 95% (w / w) reduced compared to that of the composition before purification or treatment for removing impurities. In certain embodiments, the level of at least one product-related substance is at least 30% (w / w), at least 4 0% (w / w), at least 50% (w / w), at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), at least 90% (w / w), or at least 95% (w / w) reduced compared to that of the composition before purification or treatment for removing impurities. In certain embodiments, the cyclic polynucleotide preparation (e.g., an intermediate in the production of a cyclic polynucleotide pharmaceutical preparation or composition or a cyclic polynucleotide preparation) is further substantially free of process-related impurities . In certain embodiments, the process-related impurities include proteins (e.g., cellular proteins such as host cell proteins), deoxyribonucleic acids (e.g., cellular deoxyribonucleic acids such as host cell deoxyribonucleic acids), monodeoxyribonucleotide or dideoxyribonucleotide molecules, enzymes (e.g., nucleases such as endonucleases or exonucleases, or ligases), reagent components, gel components, or chromatography materials. In certain embodiments, the impurities include buffer reagents, ligases, nucleases, RNase inhibitors, RNase R, deoxyribonucleotide molecules, acrylamide gel fragments, and . In certain embodiments, the process-related impurities include proteins (e.g., cellular proteins such as host cell proteins), deoxyribonucleic acids (e.g., cellular deoxyribonucleic acids such as host cell deoxyribonucleic acids), monodeoxyribonucleotide or dideoxyribonucleotide molecules, enzymes (e.g., nucleases such as endonucleases or exonucleases, or ligases), reagent components, gel components, or chromatography materials. In certain embodiments, the impurities include buffer reagents, ligases, nucleases, RNase inhibitors, RNase R, deoxyribonucleotide molecules, acrylamide gel fragments, and . In certain embodiments, the impurities include buffer reagents, ligases, nucleases, RNase inhibitors, RNase R, deoxyribonucleotide molecules, acrylamide gel fragments, and chromatography materials. Selected from monodeoxyribonucleotide molecules. In certain embodiments, the pharmaceutical formulation is , per milligram (mg) of the cyclic polynucleotide molecule, 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng , less than 400 ng, or less than 500 ng of protein contamination (e.g., cellular proteins such as host cell proteins).

[0136] In one embodiment, a cyclic polynucleotide formulation (e.g., a cyclic polynucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polynucleotide formulation) is substantially free of DNA content, e.g., template DNA or cellular DNA (e.g., host cell DNA), or has a DNA content of about zero, or has a DNA content of 1 pg / ml, 10 pg / ml, 0 .1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 10 0 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL, or 100 ,000 μg / mL or less.

[0137] In one embodiment, a cyclic polynucleotide formulation (e.g., a cyclic polynucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polynucleotide formulation) is Substantially free of DNA, having a DNA content of about zero, or, on a mass basis, total nucleotides of 0.001% (w / w), 0.01% (w / w), 0.1% (w / w), 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w) , 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w) or less DNA content wherein the total nucleotide molecules are the total mass of deoxyribonucleotide content and ribonucleotide molecules. In one embodiment, a cyclic polyribonucleotide formulation (e.g., a cyclic polyribonucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polyribonucleotide formulation) is substantially free of DNA, has a DNA content of about zero, or, when measured after complete DNA digestion with an enzyme that digests nucleotides by quantitative liquid chromatography - mass spectrometry (LC - MS), has a DNA content of 0 .001% (w / w), 0.01% (w / w), 0.1% (w / w), 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 15% (w / w ), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40 % (w / w), 45% (w / w), 50% (w / w) or less on a mass basis, where the DNA content is calculated from the standard curve of each base (i.e., A, C, G, T ) when measured by LC - MS. ) is calculated inversely from the standard curve.

[0138] In one embodiment, the cyclic polynucleotide formulation (e.g., a cyclic polynucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polynucleotide formulation) has a protein (e.g., cellular protein (CP), e.g., an enzyme, a production-related protein, e.g., a carrier protein) contamination of 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 2 0 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 5 0 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 1 00 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, or 500 ng / ml or less. In one embodiment, the cyclic poly ribonucleotide (e.g., a cyclic polynucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic poly ribonucleotide) has a protein (e.g., cellular protein (CP), e.g., a production-related protein such as an enzyme) contamination of from the limit of detection to 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / m l, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / m l, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, or 500 ng / ml. In one embodiment, the cyclic polynucleotide formulation (e.g., a cyclic polynucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polynucleotide formulation) has, per milligram (mg) of cyclic polynucleotide, 0.1 ng, 1 ng, 5 ng, a protein (e.g., cellular protein (CP), e.g., a production-related protein such as an enzyme) contamination of from the limit of detection to

[0139] In one embodiment, the cyclic polynucleotide formulation (e.g., a cyclic polynucleotide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polynucleotide formulation) has a protein (e.g., cellular protein (CP), e.g., an enzyme, a production-related protein, e.g., a carrier protein) contamination of 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, or 500 ng / ml or less. 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 40 0 ng, or less than 500 ng of protein (e.g., cellular protein (CP), e.g., a protein related to production such as an enzyme) contamination. In one embodiment, the cyclic polyribo nucleotide (e.g., a cyclic polyribonucleotide pharmaceutical formulation or composition or an intermediate in the production of cyclic polyribo nucleotide) has, per milligram (mg) of cyclic polyribonucleotide, from 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, to 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or up to 500 ng of ta n protein (e.g., cellular protein (CP), e.g., a protein related to production such as an enzyme) contamination.

[0140] In one embodiment, a cyclic polyribonucleotide formulation (e.g., a cyclic polyribonucleo tide pharmaceutical formulation or composition or an intermediate in the production of a cyclic polyribonucleotide formulation) has a low level of endotoxin or is substantially endotoxin-free when measured, for example, by the Limulus amebocyte lysate (LAL) test. In certain embodiments, a pharmaceutical formulation or composition or an intermediate in the production of a cyclic polyribonucleotide contains less than 20 EU / kg (by weight), 10 EU / kg 5 EU / kg, 1 EU / kg of endotoxin or is endotoxin-free when measured by the Limulus amebocyte lysate test. In one embodiment, a cyclic polyribonucleotide composition has a low level of nucleo tide It has no protease or ligase, or does not contain nuclease or ligase.

[0141] In certain embodiments, the cyclic polynucleotide formulation (e.g., an intermediate in the production of a cyclic polynucleotide pharmaceutical formulation or composition or cyclic polynucleotide formulation) comprises at least one enzyme at about 50% (w / w), 45% (w / w), 40% (w / w), 35% (w / w), 30 % (w / w), 25% (w / w), 20% (w / w), 19% (w / w), 18% (w / w), 17% (w / w), 16% (w / w), 15% (w / w), 14% (w / w), 1 3% (w / w), 12% (w / w), 11% (w / w), 10% (w / w), 9% (w / w), 8% (w / w), 7% (w / w), 6% (w / w), 5% (w / w), 4% (w / w), 3% (w / w), 2% (w / w), 1% (w / w) or less, e.g., polymerase, e.g., RNA polymerase. For example, it includes polymerase, for example, RNA polymerase.

[0142] In one embodiment, the cyclic polynucleotide formulation (e.g., an intermediate in the production of a cyclic polynucleotide pharmaceutical formulation or composition or cyclic polynucleotide formulation) is sterilized or substantially free of microorganisms, e.g., the composition or formulation supports the growth of less than 100 viable microorganisms when tested under aseptic conditions, and the composition or formulation meets the standards of USP <71>, and / or the composition or formulation meets the standards of USP <85> . In certain embodiments, the pharmaceutical formulation has a bioburden of 100 CFU / 100 ml, 50 CF U / 100 ml, 40 CFU / 100 ml, 30 CFU / 100 ml, 200 CFU / 1 00 ml, 10 CFU / 100 ml, or less than 10 CFU / 100 ml prior to sterilization. It includes.

[0143] In certain embodiments, the cyclic polynucleotide formulation further comprises column chromatography or other techniques known in the art for removing impurities, such as pH / vial inactivation. It can be purified using techniques known in the art.

[0144] In certain embodiments, the cyclic polynucleotide formulation generates one or more markers at a reduced level of immune or inflammatory response after administration to a subject when the cyclic polynucleotide formulation is subjected to a purification step (or multiple purification steps) compared to before the purification step. Purification can be performed as described herein, for example, as in Examples 1-8. In certain embodiments, one or more markers of the immune or inflammatory response are cytokines or immune response-related genes. In certain embodiments, one or more markers of the immune or inflammatory response are the expression of genes such as RIG-I, MDA5, PKR, IFN-β, OAS, and OASL.

[0145] In one embodiment, a cyclic polynucleotide formulation (e.g., an intermediate in the production of a cyclic polynucleotide pharmaceutical formulation or composition or a cyclic polynucleotide formulation) expresses an expression product, e.g., a protein, e.g., in vitro translation activity, when measured by an assay described in Example 3, for example.

[0146] Pharmaceutical composition The present invention includes compositions combined with one or more pharmaceutically acceptable excipients. The pharmaceutical composition optionally may contain one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. The pharmaceutical composition may be a composition described herein (e.g., a composition approved by the US Food and Drug Administration ). approved by the United States Food and Drug Administration (FDA) and may optionally contain an inert substance that acts as a vehicle or medium for a composition containing a cyclic polynucleotide, such as any one of the inert ingredients listed in the Inactive Ingredient Database The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any approved by the United States Food and Drug Administration (FDA) and may optionally contain an inert substance that acts as a vehicle or medium for a composition containing a cyclic polynucleotide, such as any one of the inert ingredients listed in the Inactive Ingredient Database The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any approved by the United States Food and Drug Administration (FDA) and may optionally contain an inert substance that acts as a vehicle or medium for a composition containing a cyclic polynucleotide, such as any one of the inert ingredients listed in the Inactive Ingredient Database The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any approved by the United States Food and Drug Administration (FDA) and may optionally contain an inert substance that acts as a vehicle or medium for a composition containing a cyclic polynucleotide, such as any one of the inert ingredients listed in the Inactive Ingredient Database The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The pharmaceutical compositions of the present invention are sterilized and / or may be pyrogen-free. An overview of pharmaceutical formulation and / or manufacture can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, tannins, proteins, cells, hyaluronidase, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any

[0147] The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, but such compositions are generally understood by those skilled in the art to be suitable for administration to any other animal, e.g., a non-human animal, e.g., a non-human mammal. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals are well understood and would be routine for a veterinary pharmacologist having ordinary skill, if any Such modifications can be designed and / or made based solely on the experiments of The intended subjects for administration of the pharmaceutical composition include, but are not limited to, humans and / or other primates; commercially relevant mammals such as cows, pigs, sheep, goats, cats, dogs, mice, and / or rats; and / or commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0148] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed in the future. Generally, such preparation methods involve combining the active ingredient with excipients and / or one or more other auxiliary components, and then, if necessary and / or desirable, dividing, shaping, and / or packaging the product.

[0149] Method for manufacturing a pharmaceutical circular RNA formulation Methods for manufacturing the pharmaceutical compositions, medicaments, or pharmaceutical products disclosed herein can include treating a formulation of circular polynucleotides to reduce linear RNA and / or nicked RNA, evaluating the amount of remaining linear RNA and / or nicked RNA, and further treating the formulation to produce a pharmaceutical composition, medicament, or pharmaceutical product for pharmaceutical use.

[0150] Methods for manufacturing the pharmaceutical compositions, medicaments, or pharmaceutical products disclosed herein can include providing a formulation of circular polynucleotides, evaluating the formulation for the amount of linear RNA and / or nicked RNA, and, if the evaluation meets a predetermined reference standard, such as a pharmaceutical release specification, for linear RNA and / or nicked RNA, treating the formulation to produce a pharmaceutical composition, medicament, or pharmaceutical product for pharmaceutical use.

[0151] ​​​A method for testing a pharmaceutical composition, pharmaceutical agent, or pharmaceutical product disclosed herein may include providing a formulation of a cyclic polynucleotide, evaluating the formulation for the amount of linear RNA, and determining whether the evaluation meets a predetermined reference standard for the linear RNA, such as a pharmaceutical release specification.

[0152] A method for testing a pharmaceutical composition, pharmaceutical agent, or pharmaceutical product disclosed herein may include providing a formulation of a cyclic polynucleotide, evaluating the formulation for the amount of nicked RNA, and determining whether the evaluation meets a predetermined reference standard for the nicked RNA, such as a pharmaceutical release specification.

[0153] For example, the reference standard for the amount of linear polynucleotide molecules present in the formulation 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, 2 00 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 1 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 g / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1 mg / ml, 1.5 mg / ml, or the presence of linear polynucleotide molecules at 2 mg / ml or less.

[0154] For example, the reference standard for the amount of cyclic polynucleotide molecules present in the formulation is at least 30% (w / w), 40% (w / w) , 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w ), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98 % (w / w), 99% (w / w), 99.1% (w / w), 99.2% (w / w), 99 .3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w ), 99.7% (w / w), 99.8% (w / w), 99.9% (w / w), or 100 % (w / w) as the numerator.

[0155] For example, the reference standard for the amount of linear polynucleotide molecules present in the formulation is 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 2 5% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) or less of linear poly ribonucleotide molecules.

[0156] For example, the reference standard for the amount of nicked polynucleotide molecules present in the formulation is , 0.5% (w / w), 1% (w / w), 2% ( w / w), 5% (w / w), 10% (w / w), or 15% (w / w) or less of nicked poly ribonucleotide molecules.

[0157] For example, reference standards for the amount of combined nick and linear polynucleotide molecules present in a formulation are 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) or less of combined nick and linear polynucleotide molecules per total ribonucleotide molecules in a pharmaceutical formulation. In certain embodiments, the pharmaceutical formulation is an intermediate pharmaceutical formulation for a final cyclic polynucleotide drug product. In certain embodiments, the pharmaceutical formulation is a drug or pharmaceutical active ingredient (API). In certain embodiments, the pharmaceutical formulation is a drug product for administration to a subject.

[0158] In certain embodiments, the formulation of the cyclic polynucleotide is further processed to substantially remove DNA, protein contamination (e.g., cellular proteins such as host cell proteins or protein process impurities), endotoxin, mononucleotide molecules, and / or process-related impurities (before, during or after the reduction of linear RNA).

[0159] In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient, for example if it meets the specifications for the level of linear RNA. In certain embodiments, the pharmaceutical excipient includes an inorganic or organic buffer for controlling pH, a sugar, an amino acid or any other material for cyclic polynucleotide stability, sodium chloride or any other material for adjusting tonicity, or a surfactant such as a non-ionic surfactant. In certain embodiments, the pharmaceutical excipient includes a monosaccharide, a disaccharide (e.g., sucrose, lactose, or trehalose), a trisaccharide, a polysaccharide, an amino sugar (e.g., meglumine), a polyhydric alcohol, a salt (e.g., sodium bicarbonate, sodium phosphate, or sodium chloride), ​ thorium), magnesium stearate, amino acids (e.g., histidine or arginine ), surfactants (e.g., glycerol or polysorbate 80), chelating agents (e.g , EDTA), camphorsulfonic acid, or lyoprotectant (lyoprotect ant) (e.g., cyclodextrin). In certain embodiments, the pharmaceutical excipient comprises a citrate buffer. In certain embodiments, the pharmaceutical excipient comprises donor methyl group S -adenosylmethionine (SAM). In certain embodiments, the pharmaceutical excipient comprises α -terpineol; α-tocopherol; α-tocopherol acetate; α-tocopherol ; 1,2,6-hexanetriol; 1,2-dimyristoyl-Sn-glycero-3-( phospho-S-); 1-glycerol; 1,2-dimyristoyl-Sn-glycero-3-; phosphocholine, 1,2-dioleoyl-Sn-glycero-3-phosphocholine; 1,2-di palmitoyl-Sn-glycero-3-(phospho-); Rac-(1-glycerol); 1 ,2-distearoyl-Sn-glycero-3-(phospho-Rac-); 1,2-distear loyl-Sn-glycero-3-phosphocholine; 1-O-tolylbiguanide; 2-ethyl- 1,6-hexanediol; acetic acid; glacial acetic acid; acetic anhydride; acetone; sodium bisulfite acetone lithium; acetylated lanolin alcohol; acetylated monoglyceride; acetylcysteine ; acetyltryptophan, DL-; acrylate copolymer; acrylic acid-isooct yl acrylate copolymer; acrylic adhesive 788; activated carbon; Adcote 72A 103; adipic acid; Aerotex Resin 3730; alanine (injection); aggregation Albumin; Colloidal albumin; Human albumin; Alcohol; Absolute alcohol; Modified Dilute alcohol; Dilute alcohol; Alfadex; Alginic acid; Alkyl ammonium sulfide Sulfonic acid betaine;Sodium alkylarylsulfonate;Allantoin;Allyl α- Ionone; Almond oil; Aluminum acetate; Aluminum chlorohydroxyallantoy hydrate;aluminum hydroxide;hydrated aluminum hydroxide-sucrose;aluminum hydroxide Aluminum Hydroxide Gel F 500; Aluminum Hydroxide Gel F 5000; Mo Aluminum stearate;Aluminum oxide;Aluminum polyester;Aluminum silicate Aluminum;Aluminum starch octenylsuccinate;Aluminum stearate;Salts Basic aluminum acetate;Anhydrous aluminum sulfate;Amerchol C;Amercho l-Cab; Aminomethylpropanol; Ammonia; Ammonia solution; Concentrated ammonia solution ammonium acetate;ammonium hydroxide;ammonium lauryl sulfate;ammonium Nonoxynol-4 sulfate; C-12 to C-15 linear ammonium salt; Primary ammonium salt Alcohol ethoxylate; Ammonium sulfate; Ammonyx; Amphoteric- 2;Amphoteric-9;Anethole;Anhydrous citric acid;Anhydrous dextrose;Anhydrous Lactose;Anhydrous Trisodium Citrate;Anise Oil;Anoxid Sbn;Antifoam;A Intipirin; Apaflurane; Apricot kernel oil Peg-6 ester; Aquaphor; Arginine ;Arlacel;Ascorbic acid;Ascorbyl palmitate;Aspartic acid;Per - Balsam Peru; Barium Sulfate; Beeswax; Synthetic Beeswax; Beheneth -10;Bentonite;Benzalkonium chloride;Benzenesulfonic acid;Benzonitrile chloride Mu; Benzyldodecinium Bromide; Benzoic Acid; Benzyl Alcohol; Benzyl Benzoate; Chloride Benzyl; Beta Dex; Vivapotide; Bismuth Subgallate; Boric Acid; Broclynaato; Butane; Butyl Alcohol; Butyl Ester of Vinyl Methyl Ether / Maleic Acid; Anhydrous Substance Copolymer (125000 Mw); Butyl Stearate; Butylated Hydroxyanisole L; Butylated Hydroxytoluene; Butylene Glycol; Butyl Paraben; Butyric Acid; C20 ~40 Paless - 24; Caffeine; Calcium; Calcium Carbonate; Calcium Chloride; Glu Septate Calcium; Calcium Hydroxide; Calcium Lactate; Calcobutrol (Calc obutrol); Cardiamid Sodium; Trisodium Caloxetate (Caloxe tate Trisodium); Cartelidol Calcium; Canada Balsam; Capri lic Acid / Capric Acid Triglyceride; Caprylic Acid / Capric Acid / Stearic Acid Triglycery d; Captan; Captizol; Caramel; Carbomer 1342; Carbomer 1382; Ca rbomer 934; Carbomer 934p; Carbomer 940; Carbomer 941; Carbom er 980; Carbomer 981; Carbomer Homopolymer B Type (Allyl); Pentaerythri tol (Crosslinked)); Carbomer Homopolymer C Type (Allyl); Pentaerythritol (Cross linked)); Carbon Dioxide; Carboxyvinyl Copolymer; Carboxymethyl Cellulose; Car boxymethyl Cellulose Sodium; Carboxypolymethylene; Carrageenan; Carragee nan Salt; Castor Oil; Japanese Cypress Oil Cellulose; Microcrystalline Cellulose; Cerasynt - S e; Cerasin; Ceteareth - 12; Ceteareth - 15; Ceteareth - 30; Cetearyl A Lecol / Setearyl-20; Cetearyl Ethylhexanoate; Cetes-10; Cetes-2 ; Cetes-20; Cetes-23; Cetostearyl Alcohol; Cetrimonium Chloride; Cetyl Alcohol; Cetyl Ester Wax l; Cetyl Palmitate; Cetylpyridinium Chloride ; Chlorobutanol; Chlorobutanol Hemihydrate I; Anhydrous Chlorobutanol; Chloroc Resol; Chloroxylenol; Cholesterol; Cholest; Cholest-24; Citrate; Citric Acid; Citric Acid Monohydrate; Hydrous Citric Acid; Cocamidopropyl Ether Sulfate; Cocamine Oxide; Cocobetaine; Cocodiethanolamide; Cocomonoethanolamide; Cocoa Butter; Coco-Glycerides; Palm Oil; Hydrogenated Palm Oil; Palm Kernel Oil / Glyceride; Coconut Oil Caprylocaprate; Cola Nitida Seed Extract; Collagen; Colorant Suspending Agent; Corn Oil; Cottonseed Oil; Cream Base; Creatine; Creatinine; Cro Scarmellose Sodium; Crospovidone; Copper Sulfate; Anhydrous Copper Sulfate; Cyclomethicone; Cy clomethicone / Dimethicone Copolyol; Cysteine; Cysteine Hydrochloride; Anhydrous Cysteine Hydrochloride ; D&C Red No.28; D&C Red No.33; D&C Red No.36; D &C Red No.39; D&C Yellow No.10; Dalfram Pridine; Dauber t 1-5 Pestr(Matte)164z; Decyl Methyl Sulfoxide; Dehy dag Wax Sx; Dehydroacetic Acid; Dehymuls E; Sodium Benzoate; Deoxycholic Acid; Dextran; Dextran 40; Dextrin; Dextrose; Dextrose Monohydrate; Dextrose Solution; Diatrizoic Acid; Diazolidinyl Urea; Di chlorobenzyl Alcohol; Dichlorodifluoromethane; Dichlorotetrafluoroethane ; Dichlorobenzyl Alcohol; Dichlorodifluoromethane; Dichlorotetrafluoroethane ; Diethanolamine; Diethyl pyrocarbonate; Diethyl sebacate; Diethylene glycol Monoethyl ether; Diethylhexyl phthalate; Dihydroxyaluminum aminoacetate ; Diisopropanolamine; Diisopropyl adipate; Diisopropyl dilinoleate ; Dimethicone 350; Dimethicone copolyol; Dimethicone Mdx4 - 4210; Dimeth icone medical fluid 360; Dimethyl isosorbide; Dimethyl sulfoxide; Dimethylamino Ethyl methacrylate - butyl; Methacrylate - methyl methacrylate, copolymer; Dimethyldioctadecylammonium bentonite; Dimethylsiloxane / methylvinyl siloxane, copolymer; Dinoseb ammonium salt; Dipalmitoyl phosphatidylgly cerol; Dipropylene glycol; Cocamidodiacetic acid disodium; Sulfosuccinic acid la ureth sodium; Sulfosuccinic acid lauryl disodium; Sulfosalicylic acid disodium um; Disophenin; Divinylbenzene - styrene copolymer; Dmdm hydantoin; Dox osanol; Docusate sodium; Duro - Tak 280 - 2516; Duro - Tak 387 - 2516; Duro - Tak 80 - 1196; Duro - Tak 8 7 - 2070; Duro - Tak 87 - 2194; Duro - Tak 87 - 2287 ; Duro - Tak 87 - 2296; Duro - Tak 87 - 2888; Duro - Tak 87 - 2979; Calcium disodium edetate; Disodium edetate; Anhydrous disodium edetate; Sodium edetate; Egg lecithin; Enzofone; Enzofone; Epilactose; Epitetracycline hydrochloride; Essence Bouquet 92 00; Ethanolamine hydrochloride; Ethyl acetate; Ethyl oleate; Ethyl cellulose; E thylene glycol; Ethylene vinyl acetate copolymer; Ethylenediamine; Ethylenediam ine dihydrochloride; Ethylene-propylene copolymer; Ethylene-vinyl acetate copolymer; Eth ylene-vinyl acetate copolymer; Ethylhexyl hydroxystearate; Ethylparaben ; Eucalyptol; Examethium; Edible fat; Solid fat; Fatty acid ester; Fatty acid p entaerythritol ester; Fatty acid; Citric acid fatty alcohol; Fatty alcohol; Fd &C Blue No.1; Fd&C Green No.3; Fd&C Red No.4; Fd&C Re d No.40; Fd&C Yellow No.10; Fd&C Yellow No.5; Fd&C I ellow No.6; Ferric chloride; Ferric oxide; Flavor 89-186; Flavor 89-259; Flavor Df-119; Flavor Df-1530; Flavor enhancer; Flavor Jujube 827118 ; Flavor Raspberry Pfc-8407; Pharmaceutical flavor Rhodia No.Rf 451 ; Fluorochlorohydrocarbon; Formaldehyde; Formaldehyde; Fractionated palm oil; Spice 3 949-5; Spice 520a; Spice 6.007; Spice 91-122; Spice 9128-Y; S pice 93498g; Spice Matsubar balsam No.5124; Spice Bouquet 10328; Spice Chemoderm 6401-B; Spice Chemoderm 6411; Spice Cr eam No.73457; Spice Cs-28197; Spice Felton 066m; Spice Firmenich 47373; Spice Givaudan Ess 9090 / 1 c; Spice H-6540; Spice Herbal 10396; Spice Nj-1085; Spice P O Fl-147; Spice Pa 52805; Spice Pera Derm D; Spice Rbd-98 19; Spice Shaw Mudge U-7776; Spice Tf 044078; Spice Ung erer Honeysuckle K 2771; Spice Ungerer N5195; Fructose; Gadolinium oxide; Galactose; γ-Cyclodextrin; Gelatin; Crosslinked gelatin; Gelfoam sponge; Gellan gum (low acyl); Gelva 737; Gentisic acid; Ethanolamine gentisate; Sodium gluceptate; Gluceptic acid Sodium dihydrate; Glucono-lactone; Glucuronic acid; Glutamic acid; Glutathione; Glycerin; Glycerol ester of hydrogenated rosin; Glyceryl citrate; Glyceryl isostearate; Glyceryl laurate; Glyceryl monostearate; Glyceryl oleate; Glyceryl oleate / propylene glycol; Glyceryl palmitate; Lysyl -oleate; Glyceryl stearate; Stearyl glyceryl stearate-23 ; Glyceryl stearate / Peg stearate; Glyceryl stearate / Peg-1 00 stearate; Glyceryl stearate / Peg-40 stearate; Stearic acid Glyceryl stearyl-stearamide ethyl; Diethylamine; Glyceryl trioleate; Glycine ; Glycine hydrochloride; Glycol distearate; Glycol stearate; Guanidine hydrochloride; Guar gum; Hair conditioner (18nl95-lm); Heptane; Hetastarch ; Hexylene glycol; High density polyethylene; Histidine; Human albumin micro spheres; Sodium hyaluronate; Hydrocarbon; Plasticized hydrocarbon gel; Hydrochloric acid; Dilute hydrochloric acid; Hydrocortisone; Hydrogel polymer; Hydrogen peroxide; Hydrogenated castor oil; Hydrogenated palm oil; Hard ​Hydrogenated palm / palm kernel oil Peg-6; ester; hydrogenated polybutene 635-690; hydroxide Ion; hydroxyethyl cellulose; hydroxyethylpiperazineethanesulfonic acid; Hydroxymethyl cellulose; hydroxyoctacosanyl hydroxystearate; hyd Roxypropyl cellulose; hydroxypropyl methylcellulose 2906; hydroxy Propyl-β-cyclodextrin; hypromellose 2208(15000Mpa.S); Hypromellose 2910(15000Mpa.S); hypromellose; imidurea; iodine Element; iodoxamic acid; iophentamine hydrochloride; Irish moss extract; isobutane; i Sosetes-20; isoleucine; isooctyl acrylate; isopropyl alcohol; Isopropyl isostearate; isopropyl myristate; isopropyl myristate -Myristyl alcohol; isopropyl palmitate; isopropyl stearate; iso Stearic acid; isostearyl alcohol; isotonic sodium chloride solution; Jelene; ca Orin; Kathon Cg; Kathon Cg II; lactate; lactic acid; lactic acid; milk Acid; lactobionic acid; lactose; lactose monohydrate; hydrous lactose; lanese; lano Lin; lanolin alcohol - mineral oil; lanolin alcohol; anhydrous lanolin; lanolin cholest Terol; lanolin nonionic derivative; ethoxylated lanolin; hydrogenated lanolin; lauryl chloride Lalconium; lauramine oxide; laurdimonium hydrolyzed animal collagen; lauryl sulfate ; laureth-2; laureth-23; laureth-4 T; lauric acid diethanolamide ; lauric acid myristic acid diethanolamide; lauroyl sarcosine; lauryl lactate; Sodium lauryl sulfate; English lavender (Lavandula Angustifol ia) flower top; lecithin; unbleached lecithin; egg lecithin; hydrogenated lecithin; hydrogenated soybean le cithin; soybean lecithin; lemon oil; leucine; levulinic acid; lidofenin; light oil; light oil( 85Ssu); limonene, (+ / -)-; Lipocol Sc-15; lysine; lysine acetate; lysine monohydrate; magnesium aluminum silicate; magnesium aluminum silicate water hydrate; magnesium chloride; magnesium nitrate; magnesium stearate; maleic acid; mannitol; Maprofix; Mebrofenin(Mebrof enin); medical adhesive modified S-15; medical defoamer A-F emulsion; medro sodium acid disodium; medronic acid; meglumine; menthol; metacresol; metaphosphoric acid; methanesulfonic acid; methionine; methyl alcohol; methyl gluceth-10; methyl gluc eth-20; methyl gluceth sesquistearate-20; methyl gluceth sesquistearate course; methyl laurate; methyl pyrrolidone; methyl salicylate; methyl stearate ; methyl boronic acid; methyl cellulose(4000Mpa.S); methyl cellulose; meth ylchloroisothiazolinone; methylene blue; methylisothiazolinone; methyl paraben ; microcrystalline wax; mineral oil; mono and diglycerides; citric acid monostearyl; monothio glycerol; Multisterol extract; myristyl alcohol ; myristyl lactate; myristyl-γ-picolinium chloride; N-(carbamoyl- methoxy Peg-40)-1,2-; distearoyl-cephalin sodium; N,N- dimethylacetamide; niacinamide; nioxime; nitric acid; Peg-2 stearate ; Phenylmercury acetate; Phenylmercury nitrate; Egg phosphatidylglycerol; Phospholipid; Egg Phospholipid; Phospholipon 90g; Phosphoric acid; Pine oil (Pinus sylvestris); Piperazine hexahydrate; Plastibase-50w; Polacrilin ylvestris)); Piperazine hexahydrate; Plastibase-50w; Polacrilin Iontophoresis; Polydronium chloride; Poloxamer -124; Poloxamer 181; Poloxamer 182; Poloxamer 188; Poloxamer -237; Poloxamer 407; Poly(bis(P-carboxyphenoxy)propane; anhydride, Sebacic acid; End-blocked poly(dimethylsiloxane / methylvinylsiloxane / methylhydrogensiloxane) dimethylvinyl, dimethylhydroxy, or trimethyl ; Poly(Dl-lactic acid-Co-glycolic acid); Poly(Dl-lactic acid-Co-glycolic acid); Polyacrylic acid (250000 Mw); Polybutene (1400 Mw); Polycarb fil; Polyester; Polyester polyamine copolymer; Polyester rayon; Poly ethylene glycol 1000; Polyethylene glycol 1450; Polyethylene glycol 1500; Polyethylene glycol 1540; Polyethylene glycol 200; Poly ethylene glycol 300; Polyethylene glycol 300-1600; Polyethylene glycol 3350; Polyethylene glycol 400; Polyethylene glycol 4000; Polyethylene glycol 540; Polyethylene glycol 600; Polyethylene glycol 6000; Polyethylene glycol 8000; Polyethylene glycol 900; Ferric oxide containing high density polyethylene; Black oxide (<1%); Low density polyethylene; Barium sulfate ( 20~24%); Polyethylene T; Polyethylene terephthalate; Polyglyactin; Poly 20~24%); Polyethylene T; Polyethylene terephthalate; Polyglyactin; Poly Glyceryl 3 Oleate; Polyglyceryl 4 Oleate; Polyhydroxyethyl Methacrylate Rate; Polyisobutylene; Polyisobutylene (1100000 Mw); Polyisobutylene (35000 Mw); Polyisobutylene 178 - 236; Polyisobutylene 241 - 2 94; Polyisobutylene 35 - 39; Low Molecular Weight Polyisobutylene; Medium Molecular Weight Polyisobutylene Lene; Polyisobutylene / Polybutene Adhesive; Polylactide; Polyol; Polyoxyethylene -Polyoxypropylene 1800; Polyoxyethylene Alcohol; Polyoxyethylene Fatty Acid Ester; Polyoxyethylene Propylene; Polyoxyl 20 Cetostearyl Ether; Polyoxyl 35 Castor Oil; Polyoxyl 40 Hydrogenated Castor Oil; Stearic Acid Polyoxyl 40; Polyoxyl 400 Stearate; Polyoxyl 6 Palmitostearate and Polyoxyl 32; Polyoxyl Distearate; Polyoxyl Stearic Acid Gly ceryl; Polyoxyl Lanolin; Polyoxyl Palmitate; Polyoxyl Stearate ; Polypropylene; Polypropylene Glycol; Polyquaternium - 10; Polyquaternium -7; Acrylamide / Dadmac; Polysiloxane; Polysorbate 20; Pol ysorbate 40; Polysorbate 60; Polysorbate 65; Polysorbate 80; Pol yurethane; Polyvinyl Acetate; Polyvinyl Alcohol; Polyvinyl Chloride; Polyvinyl Chloride -Polyvinyl Acetate, Copolymer; Polyvinyl Pyridine; Rose Oil; Potassium Carbonate; Potassium Acetate ; Potassium Alum; Potassium Hydrogen Carbonate; Potassium Bisulfite; Potassium Chloride; Cro tonic Acid Potassium; Potassium Hydroxide; Potassium Metabisulfite; Dipotassium Phosphate; Dipotassium Phosphate; Potassium hydrogen; Potassium soap; Potassium sorbate; Povidone acrylate copolymer; Povidone hydrogel iontophoresis; Povidone K1 7; Povidone K25; Povidone K29 / 32; Povidone K30; Povidone K90; Povid one K90f; Povidone / eicosene copolymer; Povidone; Ppg-12 / Smdi cop olymer; Ppg-15 stearyl ether; Ppg-20 methyl glucose ether dist earate; Ppg-26 oleate; Product Wat; Proline; Promulgen D; Promulgen G; Propane; Propellant A-46; Propyl gallate; Propylene carbonate; Propylene glycol; Propylene gly col diacetate; Propylene glycol dicaprylate; Propylene glycol mono laurate; Propylene glycol monopalmitostearate; Propylene glycol pa lmitostearate; Propylene glycol ricinoleate; Propylene glycol / di azolidinyl; Urea / methylparaben / propylparaben; Propylparaben; Pro sulfate tamine; Protein hydrolyzate; Pvm / Ma copolymer; Quaternium-15; Qua ternnium-15 cis type; Quaternium-52; Ra-2397; Ra-3011; Sa ccarin; Sodium saccharin; Anhydrous sodium saccharin; Safflower oil; Sd al cohol 3a; Sd alcohol 40; Sd alcohol 40-2; Sd alcohol 40b; S epineo P 600; Serine; Sesame oil; Shea butter; Silastic medical adhesive agent; Silicone A type; Silica; Silicon; Silicon dioxide; Silicone; Silicone adhesive 4 102; Silicone adhesive 4502; Silicone adhesive Bio-Psa Q7-4201 ; Silicone Adhesive Bio-Psa Q7-4301; Silicone Emulsion; Silic one / Polyester Film Strip; Simethicone; Simethicone Emulsion; Sip on Ls 20np; Soda Ash; Sodium Acetate; Sodium Acetate Anhydrous; Alkyl Sulfuric Sodium; Sodium Ascorbate; Sodium Benzoate; Sodium Bicarbonate; Di Sodium Sulfate; Sodium Bisulfite; Sodium Borate; Sodium Borate Decahydrate; Sodium Carbonate; Sodium Carbonate Decahydrate; Sodium Carbonate Monohydrate; Cetostearyl Sulf ate Sodium; Sodium Chlorate; Sodium Chloride; Sodium Cholesteryl Sulfate; Citr ate Sodium; Sodium Cocoyl Sarcosinate; Sodium Deoxycholate; Sodium Dithion ite; Sodium Dodecylbenzenesulfonate; Sodium Formaldehyde Sulfox ylate; Sodium Gluconate; Sodium Hydroxide; Sodium Hypochlorite; Sodium Iodide; Sodium Lactate; Sodium Lactate, L-; Sodium Laureth-2 Sulfate ; Sodium Laureth-3 Sulfate; Sodium Laureth-5 Sulfate; Sodium Lauroyl Sarcosine Sodium; Sodium Lauryl Sulfate; Sodium Lauryl Sulfacetate; Sodium Metabisulfit e; Sodium Nitrate; Sodium Phosphate; Sodium Phosphate Dihydrate; Sodium Hydrogen Phosphate; Sodium Hydrogen Phosphate Anhydrous; Sodium Hydrogen Phosphate Dihydrate; Sodium Hydrogen Phosphate Dodecahydrate; Sodium Hydrogen Phosphate Heptahydrate; Sodium Dihydrogen Phosphate; Sodium Dihydrogen Phosphate Anhydrous; Sodium Dihydrogen Phosphate Dihydrate; Sodium Dihydrogen Phosphate or Monohydrate; Polyac rylic Acid Sodium (2500000 Mw); Sodium Pyrophosphate; Pyrrolidonecarbo Sodium phosphate; Sodium starch glycolate; Sodium succinate hexahydrate; Sulfuric acid Sodium; Anhydrous sodium sulfate; Sodium sulfate decahydrate; Sodium sulfite; Sulfo Sodium sulfosuccinated undecylenic acid (Sodium Sulfosuccinated Undecyclenic), or monoalkylolamide; Sodium tartrate; Thi Sodium glycolate; Sodium thiomalate; Sodium thiosulfate; Anhydrous thiosulfuric Sodium acid; Sodium trimetaphosphate; Sodium xylene sulfonate; Somay 44; Sorbic acid; Sorbitan; Sorbitan isostearate; Sorbitan monolaurate ; Sorbitan monooleate; Sorbitan monopalmitate; Sorbitan monostearate Sorb Tan; Sesquioleic acid sorbitan; Trioleic acid sorbitan; Tristearic acid sorbitan Tan; Sorbitol; Sorbitol solution; Soybean flour; Soybean oil; Spearmint oil; Spermaceti; Squalane; Stabilized oxy chloro complex; Stannous 2-ethylhexanoate; Stannous chloride; Anhydrous stannous chloride; Stannous fluoride; Stannous tartrate; Starch; Alpha starch 1 500; Corn starch; Stearylconium chloride; Stearylconium hectorite / Propylene carbonate; Stearamide ethyldiethylamine; Steareth-10; Ste areth-100; Steareth-2; Steareth-20; Steareth-21; Steareth- 40; Stearic acid; Stearic acid diethanolamide; Stearoxytrimethylsilane ; Stearyltrimonium hydrolyzed animal; Collagen; Sterile water for stearyl alcohol; Sty rene / isoprene / styrene block copolymer; Saccimer; Succinic acid; Sucralose ; Sucrose; Sucrose Distearate; Sucrose Polyester; Sulfacetamide Sodium; Sulfobutylether β-Cyclodextrin Intramuscular; Sulfur Dioxide; Sulfuric Acid; Sulfurous Acid; Surfactol Qs; Tagatose, D-; Talc; Tall Oil; Tallow Glyceride Lidocaine; Tartaric Acid; Tartaric Acid; Tenox; Tenox-2; Tert-Butyl Alcohol; T ert-Butyl Hydroperoxide; Tert-Butyl Hydroquinone; Tetrakis(2- Methoxyisobutyl Isocyanide) Copper(I); Tetrafluoroborate; Tetrapropyl Orthosilicate Tetrophosmin; Theophylline; Thimerosal; Threonine; Thymol; Tin; Titanium Dioxide; Tocopherol; Tocopherolsolan; Triacetin; Tricaprylin ; Trichloromonofluoromethane; Tridecenes-10; Triethanolamine Lauryl Sulfate ; Trifluoroacetic Acid; Medium-Chain Triglycerides; Trihydroxystearic Acid; Trilanes -4 Phosphate; Trilauryl-4 Phosphate; Trisodium Citrate Dihydrate; Hedta Trisodium ; Triton 720; Triton X-200; Tromethamine; Tromantane ; Tromethamine; Tryptophan; Tyloxapol; Tyrosine; Undecylenic Acid; U nion 76 Amsco-Res 6038; Urea; Valine; Vegetable Oil; Hydrogenated Vegetable Glyceride ; Hydrogenated Vegetable Oil; Versetamide; Viscarin; Viscose / Cotton; Vitamin E ; Emulsifying Wax, Wecobee Fs; White Ceresin Wax; White Wax; Xanthan Gum ; Zinc; Zinc Acetate; Zinc Carbonate; Zinc Chloride; or Zinc Oxide. In certain embodiments , the formulation of the cyclic polynucleotide is combined with lipid nanoparticles (LNP).

[0160] In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising a disaccharide such as sucrose, lactose , or trehalose. In certain embodiments , the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising sucrose . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising a polysaccharide . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising a surfactant such as glycerol or polysorbate 80 . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising α-tocopherol . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising phosphocholine . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising an alcohol . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising isopropyl alcohol . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising lanolin alcohol . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising human albumin . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising aluminum hydroxide gel F 500 . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising aspartic acid . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising barium sulfate . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising asparagine . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising aluminum hydroxide gel F 500 . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising aspartic acid . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising barium sulfate . In certain embodiments, the formulation of the cyclic polynucleotide is subsequently combined with a pharmaceutical excipient comprising asparagine They are then combined. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing benzoic acid. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing calcium. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing calcium chloride. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing carboxymethyl cellulose. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing citric acid. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing ethylene glycol. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing ferric chloride. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing a hydrocarbon gel. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing magnesium chloride. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing niacinamide. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing polyethylene glycol. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing potassium chloride. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing propylene glycol. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing propylene glycol. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing propylene glycol. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing propylene glycol. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing propylene glycol. In certain embodiments, the formulation of the cyclic polynucleotide is then combined with a pharmaceutical excipient containing propylene glycol. It is subsequently combined with a pharmaceutical excipient containing a call. In certain embodiments, the cyclic poly The formulation of ribonucleotides is subsequently combined with a pharmaceutical excipient containing sodium carbonate. In certain embodiments, the formulation of cyclic polyribonucleotides is a pharmaceutical subsequently combined with an excipient containing sodium chloride. In certain embodiments, the cyclic polyribonucleotide formulation is subsequently combined with a pharmaceutical excipient containing sodium lactate. In certain embodiments the formulation of cyclic polyribonucleotides is subsequently combined with a pharmaceutical excipient containing zinc acetate.

[0161] In certain embodiments, the amount of impurities (e.g., cellular proteins, cellular nucleic acids, enzymes, reagent components, gel components, or chromatography materials, protein contamination, or endotoxin contamination) is measured to determine whether a pharmaceutical composition, pharmaceutical drug, or pharmaceutical product meets a reference standard.

[0162] For example, the reference standard for the amount of DNA present in a formulation is the presence of zero DNA molecules, substantially free of DNA molecules, or the presence of DNA at 1 pg / ml, 10 pg / ml, 0. 1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 n g / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 n g / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, or 500 ng / ml, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL, or the presence of DNA at 100,000 μg / mL or less.

[0163] For example, the reference standard for the amount of protein contamination present in a formulation is cyclic polyribo​ 0.1 ng, 1 ng, 5 ng, 10 ng, per milligram (mg) of nucleotide molecules, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or the presence of protein contamination of less than 500 ng of protein contamination.

[0164] In certain embodiments, the amount of endotoxin present in a pharmaceutical composition, pharmaceutical drug, or pharmaceutical product is 20 EU / kg (weight), 10 EU / kg, 5 EU / kg, 1 EU / kg, or less than a predetermined threshold, e.g., the formulation contains endotoxin at a level below the limit of detection by a specified method. In certain embodiments, the reference standard is a pharmaceutical release specification.

[0165] In certain embodiments, a pharmaceutical composition, pharmaceutical drug, or pharmaceutical product is a sterile pharmaceutical product or is substantially free of microorganisms (e.g., supports the growth of less than 100 viable microorganisms when tested in a sterile state). In certain embodiments, a pharmaceutical composition, pharmaceutical drug, or pharmaceutical product meets the standards of USP <71> and / or USP <85>. In certain embodiments, a pharmaceutical composition, pharmaceutical drug, or pharmaceutical product is further labeled and shipped for pharmaceutical use. In certain embodiments, a pharmaceutical composition, pharmaceutical drug, or pharmaceutical product contains a bioburden of 100 CFU / 100 ml, 50 CFU / 100 ml, 40 CFU / 100 ml, 30 CFU / 100 ml, 200 CFU / 100 ml, 10 CFU / 100 ml, or less than 10 CFU / 100 ml prior to sterilization.

[0166] In certain embodiments, a pharmaceutical composition, a medicament, or a pharmaceutical product comprises a concentration of circular polynucleotide molecules of at least 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL, 100 mg / mL, or 500 mg / mL.

[0167] In certain embodiments, the pharmaceutical composition, medicament, or pharmaceutical product may further be purified using techniques known in the art for removing impurities, such as column chromatography or pH / vial inactivation.

[0168] Circularization In one embodiment, the linear circular polynucleotide can be circularized or concatemerized. In certain embodiments, the linear circular polynucleotide can be circularized in vitro prior to formulation and / or delivery. In certain embodiments, the linear circular polynucleotide can be circularized intracellularly.

[0169] Extracellular circularization In certain embodiments, the linear circular polynucleotide is circularized or concatemerized using chemical methods to form a circular polynucleotide. In certain chemical methods, the 5' and 3' ends of the nucleic acid (e.g., the linear circular polynucleotide) contain chemically reactive groups that can form a new covalent bond between the 5' and 3' ends of the molecule when brought close to each other. The 5' end may contain an NHS ester reactive group and the 3' end may contain a 3' - may contain an amino-terminal nucleotide and, in an organic solvent, the 3' end of a linear RNA molecule the 3'-amino-terminal nucleotide at the 3' end undergoes a nucleophilic attack on the 5'-NHS-ester moiety to form a new 5'- / 3'-amide bond

[0170] In one embodiment, a DNA or RNA ligase is used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule ( e.g., a linear circular polynucleotide) to the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) to form a new phosphodiester bond. In an example reaction, the linear circular polynucleotide is incubated with 1 to 10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) for 1 hour at 37°C according to the manufacturer's instructions. The ligation reaction can occur in the presence of a linear nucleic acid that can base pair with both the 5'- and 3'-regions flanking the enzymatic ligation reaction to assist the enzymatic ligation reaction. In one embodiment, the ligation is a splint ligation. For example, a splint ligase such as RNA ligase 2 can be used for splint ligation. In splint ligation, a single-stranded polynucleotide (the splint), such as single-stranded DNA, is designed to hybridize to both ends of a linear polynucleotide such that the two ends can be juxtaposed upon hybridization with the single-stranded splint. Thus, RNA ligase 2 can catalyze the ligation of the two juxtaposed ends of the linear polynucleotide to produce a covalently linked circular polynucleotide linked circular polynucleotide

[0171] ​​​​​​​​In one embodiment, the DNA or RNA ligase can be used for the synthesis of circular polynucleotides. As a non-limiting example, the ligase can be a circ ligase or a circular ligase. In one embodiment, either the 5'-end or the 3'-end of the linear circular polynucleotide can be encoded with a ligase ribozyme sequence such that during in vitro transcription, the resulting linear circular polynucleotide can ligate the 5'-end of the linear circular polynucleotide to the 3'-end of the linear circular polynucleotide and contains an active ribozyme sequence.

[0172] The ligase ribozyme can be derived from group I introns, hepatitis delta virus, hairpin ribozymes, or can be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction can be carried out at a temperature of 0 to 37°C for 1 to 24 hours.

[0173] In one embodiment, the linear circular polynucleotide can be circularized or concatenated by using at least one non-nucleic acid moiety. In one aspect, at least one non-nucleic acid moiety can react with a region or feature near the 5'-end and / or near the 3'-end of the linear circular polynucleotide to circularize or concatenate the linear circular polynucleotide. In another aspect, at least one non-nucleic acid moiety can be located at or linked to or near the 5'-end and / or 3'-end of the linear circular polynucleotide. The non-nucleic acid moieties considered can be of the same or different types. As non-limiting examples, the non-nucleic acid moieties can be hydrophobic bonds, ionic bonds, biodegradable bonds, and / or cleavable bonds. ​​​​​​​​​​​​​​​can be a linkage such as. As another non-limiting example, the non-nucleic acid moiety is at the ligation moiety there is. As yet another non-limiting example, the non-nucleic acid moiety can be an oligonucleotide or peptide moiety such as an aptamer described herein or a non-nucleic acid linker.

[0174] In one embodiment, the linear circular polyribonucleotide is at, in the vicinity of, or such 5' and 3' ends at the 5' and 3' ends of the linear circular polyribonucleotide is cyclized or concatemerized by a non-nucleic acid moiety that causes an attractive force between atoms or between molecular surfaces linked thereto. As a non-limiting example, one or more linear circular polyribonucleotides can be cyclized or concatemerized by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, agnostic bonds( agnostic bond), dipole bonds, conjugation, hyperconjugation, and antibonding. In one embodiment, the linear circular polyribonucleotide can include ribozyme RNA sequences in the vicinity of the 5' end and in the vicinity of the 3' end. The ribozyme RNA sequence can covalently bind to a peptide when the sequence is exposed to the rest of the ribozyme

[0175] In one embodiment, the linear circular polyribonucleotide can include ribozyme RNA sequences near the 5' end and near the 3' end The ribozyme RNA sequence can covalently bind to a peptide when the sequence is exposed to the rest of the ribozyme In one aspect, peptides covalently bound to the ribozyme RNA sequences near the 5' and 3' ends bind to each other to cyclize or concatemerize the linear circular polyribonucleotide. In another aspect, peptides covalently bound to the ribozyme RNA near the 5' and 3' ends are limited to cyclize or concatemerize the linear circular polyribonucleotide. In another aspect, peptides covalently bound to the ribozyme RNA near the 5' and 3' ends are limited to cyclize or concatemerize the linear circular polyribonucleotide. In another aspect, peptides covalently bound to the ribozyme RNA near the 5' and 3' ends are limited ​​Although not limited thereto, various methods known in the art such as protein ligation After subjecting the linear primary construct or linear mRNA to ligation using, the resulting constructs can be circularized or concatemerized. Non-limiting examples of ribozymes or methods for incorporating and / or covalently binding peptides for use with the linear primary constructs or linear RNAs of the present invention are listed in U.S. Patent Application Publication No. 20030082768, the entire contents of which are incorporated herein by reference. In certain embodiments, the circularization efficiency of the circularization methods provided herein is at least

[0176] In certain embodiments, the linear circular polynucleotide can comprise a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase (apyrase). Alternatively, conversion of the 5' triphosphate of the linear circular polynucleotide to a 5' monophosphate can be accomplished by a two-step reaction comprising: (a) contacting the 5' nucleotide of the linear circular polynucleotide with a phosphatase (e.g., Antarctic phosphatase, shrimp alkaline phosphatase, or calf intestinal phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., polynucleotide kinase) that adds one phosphate. In certain embodiments, the circularization efficiency of the circularization methods provided herein is at least In certain embodiments, the linear circular polynucleotide can comprise a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase In certain embodiments, the linear circular polynucleotide can comprise a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase In certain embodiments, the linear circular polynucleotide can comprise a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase In certain embodiments, the linear circular polynucleotide can comprise a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase In certain embodiments, the linear circular polynucleotide can comprise a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase In certain embodiments, the linear circular polynucleotide can comprise a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase In certain embodiments, the linear circular polynucleotide can comprise a 5' triphosphate of a nucleic acid that is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase

[0177] In certain embodiments, the circularization efficiency of the circularization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least At least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In certain embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%.

[0178] Splicing element In certain embodiments, the circular polynucleotide comprises at least one splicing element. In the circular polynucleotides provided herein, the splicing element can be a complete splicing element that can mediate splicing of the circular polynucleotide. Alternatively, the splicing element can also be a residual splicing element from a completed splicing event. For example, in some cases, the splicing element of a linear polynucleotide can mediate a splicing event that results in circularization of the linear polynucleotide, such that the resulting circular polynucleotide contains a residual splicing element from such a splicing-mediated circularization event. In some cases, the residual splicing element is unable to mediate splicing. In other cases, the residual splicing element can still mediate splicing under certain circumstances. In certain embodiments, the splicing element is adjacent to at least one expression sequence. In certain embodiments, the circular polynucleotide comprises a splicing element adjacent to each expression sequence. In certain embodiments, the splicing element is on one or both sides of each expression sequence and the expression product, e.g., a peptide and / or a poly Resulting in the separation of the peptide.

[0179] In certain embodiments, the circular polynucleotide, when replicated, is spliced to contain internal splicing elements whose ends are joined to each other. Some examples are splicing site sequences and short inverted repeats (30- 40 nt), such as AluSq2, AluJr, and AluSz, inverted sequences in adjacent introns, Alu elements in adjacent introns, and sequences that are 200 bp before (upstream) or after (downstream) a back-splice site having an adjacent exon, motifs found in cis-sequence elements proximal to back-splice events (su ptable4-enriched motifs), such as small introns (<100 nt) having motifs. In certain embodiments, the circular polynucleotide contains at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repetitive nucleotide sequence may contain repetitive sequences from the Alu family of introns. In certain embodiments, ribosome-binding proteins associated with splicing may regulate circular polynucleotide biosynthesis (e.g., Muscleblind and Quaking (QKI) splicing factors).

[0180] In certain embodiments, the circular polynucleotide contains a canonical splice site adjacent to the head-to-tail junction of the circular polynucleotide.

[0181] In certain embodiments, the circular polynucleotide contains two 3-nucleotide bulges. containing a bulge-helix-bulge motif comprising adjacent 4-base pair stems is obtained. Cleavage occurs in the bulge region, generating characteristic fragments having a terminal 5'-hydroxyl group and a 2',3'-cyclic phosphate. Cyclization proceeds by nucleophilic attack of the 5'-OH group of the same molecule on the 2',3'-cyclic phosphate to form a 3',5'-phospho diester bridge.

[0182] In certain embodiments, the cyclic polynucleotide can include a multimeric repeat RNA sequence having an HPR element. The HPR contains a 2',3'-cyclic phosphate and a 5'-OH terminus and the HPR element self-processes the 5' and 3' termini of the linear cyclic polynucleotide, thereby ligating the termini to each other.

[0183] In certain embodiments, the cyclic polynucleotide can include a sequence that mediates self-ligation In one embodiment, the cyclic polynucleotide can include an HDV sequence (e.g., an HDV replication domain conserved sequence,

Chemical Formula

[0184] Other circularization methods In certain embodiments, the linear circular polynucleotide may contain complementary sequences that include repetitive or non-repetitive nucleic acid sequences within individual introns or across adjacent introns. The repetitive nucleic acid sequences are sequences present within segments of the circular polynucleotide. In certain embodiments, the circular polynucleotide contains repetitive nucleic acid sequences. In certain embodiments, the repetitive nucleotide sequence includes a polyCA or polyUG sequence. In certain embodiments, the circular polynucleotide contains at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the circular polynucleotide, and the hybridized segments form an internal double strand. In certain embodiments, repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate circular polynucleotides hybridize to produce a single circularized polynucleotide, and the hybridized segments form an internal double strand. In certain embodiments, the complementary sequences are found at the 5' and 3' ends of the linear circular polynucleotide. In certain embodiments, the complementary sequences contain about 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, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100

[0185] In certain embodiments, chemical methods of cyclization can be used to generate cyclic polynucleotides. Such methods include, but are not limited to, click chemistry (e.g., methods based on alkynes and azides, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal - imine cross - linking, base modification, and any combination thereof. In certain embodiments, enzymatic methods of cyclization can be used to generate cyclic polynucleotides. In certain embodiments, ligation enzymes, such as DNA or RNA ligases, can be used with a cyclic polynuclease or a complementary template, a complementary strand of the cyclic polynuclease, or to generate a cyclic polynuclease. The cyclization of cyclic polynucleotides can be performed by methods known in the art, such as those described in Petkovic and Muller, "RNA circularization strategies in vivo and in vitro" from Nucleic Acids Res, 2015, 43(4):2454 - 2465, and Muller and Appel, "In vitro circularization of RNA" from RNA Biol, 2017, 14(8):1018 - 1027. In certain embodiments, the cyclic polynucleotide encodes a peptide or polypeptide.

[0186] In certain embodiments, ligation enzymes, such as DNA or RNA ligases, can be used with a cyclic polynuclease or a complementary template, a complementary strand of the cyclic polynuclease, or to generate a cyclic polynuclease.

[0187] The cyclization of cyclic polynucleotides can be performed by methods known in the art, such as those described in Petkovic and Muller, "RNA circularization strategies in vivo and in vitro" from Nucleic Acids Res, 2015, 43(4):2454 - 2465, and Muller and Appel, "In vitro circularization of RNA" from RNA Biol, 2017, 14(8):1018 - 1027.

[0188] In certain embodiments, the cyclic polynucleotide encodes a peptide or polypeptide. It includes at least one expression array to be induced. Such peptides include, but are not limited to, small peptides, peptidomimetics (e.g., peptoids), amino acids and amino acid analogs. The peptide can be linear or branched. Such a peptide has a molecular weight of less than about 5,000 grams per mole, less than about 2,000 grams per mole of molecular weight, less than about 1,000 grams per mole of molecular weight, less than about 500 grams per mole of molecular weight, as well as salts, esters and other pharmaceutically acceptable forms of such compounds. Such peptides include, but are not limited to, neurotransmitters, hormones , drugs, toxins, virus or microbial particles, synthetic molecules and their agonists or ant agonists.

[0189] The polypeptide can be linear or branched. The polypeptide has about 5 to about 40,0 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, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids , about 1,000 to about 2,500 amino acids or any range of lengths therebetween. In certain embodiments, the polypeptide has less than about 40,000 amino acids, less than about 35,00 0 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, 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 less than amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, about 2,0 00 less than amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, about 9 00 less than amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or having a length below that, and may be useful.

[0190] Peptides or polypeptides expressed by the expression sequences in the cyclic polynucleotides of the present invention include, as non-limiting examples, those described in

[0149] ,

[0150] , and

[0152] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is incorporated herein by reference in its entirety. and those described in

[0152] .

[0191] In certain embodiments, the cyclic polynucleotide includes an expression sequence encoding a protein, such as a therapeutic protein. In certain embodiments, the therapeutic proteins that can be expressed from the cyclic polynucleotides disclosed herein have antioxidant activity, binding, cation receptor activity, catalytic activity, molecular carrier activity, molecular function regulator, molecular transducer activity, nutrient storage activity, protein tag, structural molecular activity, toxin activity, transcriptional regulatory activity, translational regulatory activity or transport activity. Some examples of therapeutic proteins include, but are not limited to, enzyme replacement proteins, replacement proteins, protein vaccination, antigens (e.g., tumor antigens, viruses, bacteria), hormones, cytokines, antibodies, immunotherapy (e.g., cancer), reprogramming / differentiation conversion factors, transcription factors, chimeric antigen receptors, transposases or nucleases ​​Claresase, immune effector (e.g., affecting susceptibility to immune response / signal), regulated death effector protein (e.g., inducer of apoptosis or necrosis), tumor non-lytic inhibitor (e.g., inhibitor of cancer protein), epigenetic modifier, epigenetic enzyme, transcription factor, DNA or protein modification enzyme, DNA insert, efflux pump inhibitor, nuclear receptor activator or inhibitor, proteasome inhibitor, competitive inhibitor for enzyme, protein synthesis effector or inhibitor, nuclease, protein fragment or domain, ligand or receptor, and CRISPR system or its components are included. or inducer of necrosis), tumor non-lytic inhibitor (e.g., inhibitor of cancer protein), epigenetic modifier, epigenetic enzyme, transcription factor, DNA or protein modification enzyme, DNA insert, efflux pump inhibitor, nuclear receptor activator or inhibitor, proteasome inhibitor, competitive inhibitor for enzyme, protein synthesis effector or inhibitor, nuclease, protein fragment or domain, ligand or receptor, and CRISPR system or its components are included. or inducer of necrosis), tumor non-lytic inhibitor (e.g., inhibitor of cancer protein), epigenetic modifier, epigenetic enzyme, transcription factor, DNA or protein modification enzyme, DNA insert, efflux pump inhibitor, nuclear receptor activator or inhibitor, proteasome inhibitor, competitive inhibitor for enzyme, protein synthesis effector or inhibitor, nuclease, protein fragment or domain, ligand or receptor, and CRISPR system or its components are included. or inducer of necrosis), tumor non-lytic inhibitor (e.g., inhibitor of cancer protein), epigenetic modifier, epigenetic enzyme, transcription factor, DNA or protein modification enzyme, DNA insert, efflux pump inhibitor, nuclear receptor activator or inhibitor, proteasome inhibitor, competitive inhibitor for enzyme, protein synthesis effector or inhibitor, nuclease, protein fragment or domain, ligand or receptor, and CRISPR system or its components are included. or inducer of necrosis), tumor non-lytic inhibitor (e.g., inhibitor of cancer protein), epigenetic modifier, epigenetic enzyme, transcription factor, DNA or protein modification enzyme, DNA insert, efflux pump inhibitor, nuclear receptor activator or inhibitor, proteasome inhibitor, competitive inhibitor for enzyme, protein synthesis effector or inhibitor, nuclease, protein fragment or domain, ligand or receptor, and CRISPR system or its components are included. or inducer of necrosis), tumor non-lytic inhibitor (e.g., inhibitor of cancer protein), epigenetic modifier, epigenetic enzyme, transcription factor, DNA or protein modification enzyme, DNA insert, efflux pump inhibitor, nuclear receptor activator or inhibitor, proteasome inhibitor, competitive inhibitor for enzyme, protein synthesis effector or inhibitor, nuclease, protein fragment or domain, ligand or receptor, and CRISPR system or its components are included. or inducer of necrosis), tumor non-lytic inhibitor (e.g., inhibitor of cancer protein), epigenetic modifier, epigenetic enzyme, transcription factor, DNA or protein modification enzyme, DNA insert, efflux pump inhibitor, nuclear receptor activator or inhibitor, proteasome inhibitor, competitive inhibitor for enzyme, protein synthesis effector or inhibitor, nuclease, protein fragment or domain, ligand or receptor, and CRISPR system or its components are included. or inducer of necrosis), tumor non-lytic inhibitor (e.g., inhibitor of cancer protein), epigenetic modifier, epigenetic enzyme, transcription factor, DNA or protein modification enzyme, DNA insert, efflux pump inhibitor, nuclear receptor activator or inhibitor, proteasome inhibitor, competitive inhibitor for enzyme, protein synthesis effector or inhibitor, nuclease, protein fragment or domain, ligand or receptor, and CRISPR system or its components are included.

[0192] In certain embodiments, exemplary proteins that can be expressed from the cyclic polynucleotides disclosed herein include intracellular or cytoplasmic proteins. In certain embodiments, exemplary proteins that can be expressed from the cyclic polynucleotides disclosed herein include intracellular or cytoplasmic proteins. In certain embodiments, the cyclic polynucleotide expresses a reporter molecule, e.g., NanoLuc® luciferase (nLuc). In certain embodiments In certain embodiments, the cyclic polynucleotide expresses a reporter molecule, e.g., NanoLuc® luciferase (nLuc). In certain embodiments In certain embodiments, exemplary proteins that can be expressed from the cyclic polynucleotides disclosed herein include secreted proteins, e.g., secreted enzymes. In certain cases, the cyclic poly In certain embodiments, exemplary proteins that can be expressed from the cyclic polynucleotides disclosed herein include secreted proteins, e.g., secreted enzymes. In certain cases, the cyclic poly ribonucleotide expresses a secreted protein that may have a short half-life with therapeutic effect in the blood, or is a protein with an intracellular localization signal or a protein with a secretion signal peptide ribonucleotide expresses a secreted protein that may have a short half-life with therapeutic effect in the blood, or is a protein with an intracellular localization signal or a protein with a secretion signal peptide In certain embodiments, the cyclic polynucleotide expresses Gaussia luciferase (gLuc). In certain cases, the cyclic polyribonucleotide In certain embodiments, the cyclic polynucleotide expresses Gaussia luciferase (gLuc). In certain cases, the cyclic polyribonucleotide ribonucleotide is a non-human protein, e.g., a fluorescent protein, an energy transfer acceptor, or Fl expresses a protein tag such as ag, Myc or His. In certain embodiments , an exemplary protein that can be expressed from the cyclic polynucleotide is GFP . In certain embodiments, the cyclic polynucleotide is a tagged protein, e.g . a fusion protein or a modified protein containing a protein tag, e.g., a chitin-binding t protein (CBP), maltose-binding protein (MBP), Fc tag, glutathione- S-transferase (GST), SNAP-tag, tandem protein A(ZZ) tag, Halo-tag, AviTag (GLNDIFEAQKIEWHE), calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL); p olyglutamate tag (EEEEEE); E-tag (GAPVPYPDPLEPR); FL AG-tag (DYKDDDDK), HA-tag (YPYDVPDYA); His-tag ( e.g., HHHHHH); Myc-tag (EQKLISEEDL); NE-tag (TKE NPRSNQEESYDDNES); S-tag (KETAAAKFERQHMDS); S BP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQG QREP); Softag 1 (SLAELLNAGLGGS); Softag 3 (T QDPSRVG); Spot-tag (PDRVRAVSHWSS); Strep-tag ( Strep-tag II: WSHPQFEK); TC tag (CCPGCC); Ty tag (E VHTNQDPLD); V5 tag (GKPIPNPLLGLDST); VSV-tag (Y TDIEMNRLGK); or an Xpress tag (DLYDDDDK).

[0193] In certain embodiments, the cyclic polynucleotide expresses an antigen-binding protein, such as, an antibody, such as an antibody fragment or a portion thereof. In certain embodiments, the cyclic po lyribonucleotide-expressed antibody can be of any isotype, such as IgA, IgD, IgE, IgG, Ig M. In certain embodiments, the cyclic polynucleotide expresses a portion of an antibody, such as a light chain, a heavy chain, an Fc fragment, a CDR (complementary determining region), an Fv fragment, or a Fab fragment, or a further portion thereof. In certain embodiments, the cyclic polynucleotide expresses one or more portions of an antibody. For example, the cyclic polynucleotide can include two or more expression sequences, which each express a portion of the antibody, and the collection of which can constitute an antibody. In certain cases, the cyclic polynucleotide includes one expression sequence encoding the heavy chain of an antibody and another expression sequence encoding the light chain of the antibody. In certain cases, when the cyclic polynucleotide is expressed intracellularly or in a cell-free environment, the light and heavy chains can be subjected to appropriate modifications, folding, or other post-translational modifications that form a functional antibody.

[0194] Regulatory elements In certain embodiments, the cyclic polynucleotide includes a regulatory element, such as a sequence that regulates the expression of an expression sequence within the cyclic polyribo nucleotide.

[0195] The regulatory element can include a sequence located adjacent to the expression sequence encoding the expression product. The regulatory element can be operably linked to the adjacent sequence. The regulatory element can increase the amount of the product expressed as compared to the amount of the product expressed in the absence of the regulatory element. Further, one The regulatory element increases the amount of product expressed for a plurality of expression arrays joined in series to obtain. Thus, one regulatory element can promote the expression of one or more expression arrays . A plurality of regulatory elements are well known to those skilled in the art.

[0196] The regulatory elements provided herein may include alternative translation arrays. As used herein , the term "alternative translation array" refers to a circular polynucleotide, e.g., a nucleic acid sequence that selectively initiates or activates the translation of an expression array in a particular riboswitch aptazyme . In certain embodiments, the regulatory element is a translation modulator . A translation modulator can regulate the translation of an expression array in a circular polynucleotide. The translation modulator can be a translation enhancer or suppressor. In certain embodiments , the translation initiation sequence can function as a regulatory element. Nucleotides adjacent to a codon that initiates translation, such as, but not limited to, an initiation codon or another initiation codon, are known to affect the translation efficiency, length, and / or structure of circular polynucleotides. (See, e.g., Matsuda and Mauro, PLoS ONE, 2010 5:11, which is hereby incorporated by reference in its entirety). Changing the translation initiation position, translation efficiency, length, and / or structure of a circular polynucleotide can be achieved by masking any of the nucleotides adjacent to the codon that initiates translation . (See, e.g., Matsuda and Mauro, PLoS ONE, 2010 5:11, which is hereby incorporated by reference in its entirety). In one embodiment, the masking agent masks or hides the codon to reduce the likelihood of translation initiation at the masked initiation codon or another initiation codon .

[0197] In one embodiment, the masking agent masks or hides the codon to reduce the likelihood of translation initiation at the masked initiation codon or another initiation codon in order to reduce the likelihood of translation initiation at the masked initiation codon or another initiation codon It can be used near the start codon or another start codon. In another embodiment, masking agents can be used to mask the start codon of circular polynucleotides in order to increase the likelihood that translation will start from another start codon.

[0198] The regulatory elements provided herein can include any of the regulatory elements described in paragraphs

[0156] to

[0161] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is incorporated herein by reference in its entirety.

[0199] Translation initiation sequence In certain embodiments, the circular polynucleotide can encode a polypeptide and include a translation initiation sequence, such as a start codon. In certain embodiments, the translation initiation sequence includes a Kozak sequence or a Shine-Dalgarno sequence. In certain embodiments, the circular polynucleotide includes a translation initiation sequence, such as a Kozak sequence, adjacent to the expression sequence. In certain embodiments, the translation initiation sequence is a non-coding start codon. In certain embodiments, the translation initiation sequence, such as a Kozak sequence, is present on one or both sides of each expression sequence and results in the separation of the expression products. In certain embodiments, the circular polynucleotide includes at least one translation initiation sequence adjacent to the expression sequence. In certain embodiments, the translation initiation sequence confers conformational flexibility to the circular polynucleotide. In certain embodiments, the translation initiation sequence is within a substantially single-stranded region of the circular polynucleotide.

[0200] The circular polynucleotide can include, but is not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, ​​​​​​At least 9, at least 10, at least 11, at least 12, at least 13, At least 14, at least 15, at least 16, at least 17, at least 18, At least 19, at least 20, at least 25, at least 30, at least 35, Two or more start codons such as at least 40, at least 50, at least 60 or more than 60 may be included. The translation may start at the first start codon or may start downstream of the first start codon. In certain embodiments, the cyclic polynucleotide may start with a codon that is not the first start codon, for example AUG. The translation of the cyclic polynucleotide may start from another translation start sequence such as that described in

[0164] of International Patent Publication No. WO 2019 / 118919 A1, which is hereby incorporated by reference in its entirety. In certain embodiments, the translation is initiated by treatment with Rocaglates of eukaryotic initiation factor 4A (eIF4A) (translation is inhibited by blocking 43S scanning, leading to early upstream translation initiation and reduced protein expression from transcripts having a RocA-eIF4A target sequence; see, for example, www.nature.com / articles / nature17978).

[0201] In certain embodiments, the cyclic polynucleotides described herein include an internal ribosome entry site (IRES) element. Suitable IRES elements for inclusion in the cyclic polynucleotides are those described in International Patent Publication No. WO 2 which is hereby incorporated by reference in its entirety. International Patent Publication No. WO 2019 / 118919 A1 pamphlet which is hereby incorporated by reference in its entirety.

[0202] In certain embodiments, the translation is initiated by treatment with Rocaglates of eukaryotic initiation factor 4A ( eIF4A) (translation is inhibited by blocking 43S scanning, leading to early upstream translation initiation and reduced protein expression from transcripts having a RocA-eIF4A target sequence; see, for example, www.nature.com / articles / nature17978). See, for example, www.nature.com / articles / nature17978.

[0203] IRES In certain embodiments, the cyclic polynucleotides described herein include an internal ribosome entry site (IRES) element. Suitable IRES elements for inclusion in the cyclic polynucleotides are those described in International Patent Publication No. WO 2 which is hereby incorporated by reference in its entirety. as described in

[0166] to

[0167] of Pamphlet No. 019118919A1 and contains an RNA sequence capable of binding to a eukaryotic ribosome, such as those described above.

[0204] In certain embodiments, the circular polynucleotide comprises at least one (e.g., 2, 3, 4, 5, or more) IRESs adjacent to at least one expression sequence. In certain embodiments, the IRES is adjacent to both sides of at least one (e.g., 2, 3, 4, 5, or more) expression sequences. In certain embodiments, the circular polynucleotide is present on one side or both sides of each expression sequence and contains one or more IRES sequences that result in the separation of the resulting peptide and / or polypeptide.

[0205] Termination element In certain embodiments, the circular polynucleotide comprises one or more expression sequences, and each expression sequence may or may not have a termination element. In certain embodiments the circular polynucleotide comprises one or more expression sequences, and the expression sequences lack a termination element such that the circular polynucleotide is translated continuously. Excluding the termination element can result in rolling circle-type translation or continuous expression of the expression product, such as a peptide or polypeptide, because there is no ribosome stalling or dropout. In such embodiments, rolling circle-type translation expresses a continuous expression product through each expression sequence. In certain other embodiments, the termination element of the expression sequence may be part of a stager element. In certain embodiments, one or more Rolling circle type translation or expression of an expression array such as 5 is performed. In such a case, When the ribosome encounters a termination element, such as a stop codon, and terminates translation, the expression product can fall off the ribosome. In certain embodiments, translation is terminated while at least one subunit of the ribosome, for example, remains in contact with the circular polynucleotide.

[0206] In certain embodiments, the circular polynucleotide contains a termination element at the end of one or more expression arrays. In certain embodiments, one or more expression arrays contain two or more consecutive termination elements. In such embodiments, translation is terminated and rolling circle type translation is terminated. In certain embodiments, the ribosome completely detaches from the circular polynucleotide. In certain such embodiments, the generation of subsequent ( for example, second, third, fourth, fifth, etc.) expression arrays in the circular polynucleotide may require the ribosome to re-engage the circular polynucleotide before the start of translation. Generally, termination elements include in-frame nucleotide triplets that indicate the termination of translation, such as UAA, UGA, UAG. In certain embodiments, one or more termination elements in the circular polynucleotide are, without limitation, off-frame or -1 and +1 shifted reading frame (e.g., hidden stop) termination elements that can terminate translation. Frame shift termination elements include nucleotide triplets, TAA, TAG, and TGA, that appear in the second and third reading frames of the expression array. Frame shift termination elements can be important in preventing misreading of mRNA, which is often harmful to the cell. ​​​

[0207] Stagger element In certain embodiments, the circular polynucleotide comprises at least one stagger element adjacent to the expression sequence. In certain embodiments, the circular polynucleotide comprises a stagger element adjacent to each expression sequence. In certain embodiments, the stagger element is present on one or both sides of each expression sequence to effect the separation of the expression product, such as a peptide and / or polypeptide. In certain embodiments, the stagger element is part of one or more expression sequences. In certain embodiments, the circular polynucleotide comprises one or more expression sequences, each of the one or more expression sequences being separated from subsequent expression sequences by a stagger element in the circular polynucleotide. In certain embodiments, the stagger element prevents the production of a single polypeptide from (a) two translations of a single expression sequence or (b) one or more translations of two or more expression sequences. In certain embodiments, the stagger element is a sequence separated from one or more expression sequences. In certain embodiments, the stagger element comprises a part of the expression sequence of one or more expression sequences. In certain embodiments, the circular polynucleotide comprises a stagger element. The stagger element may be included to induce stalling of the ribosome during translation to avoid the production of consecutive expression products, such as peptides or polypeptides, while maintaining rolling circle type translation. In certain embodiments, the stagger element is at at least one 3' end of one or more expression sequences. The stagger element may be configured to stall the ribosome during rolling circle type translation of the circular polynucleotide. Examples of the stagger element include, but are not limited to sequences separated from one or more expression sequences. In certain embodiments, the stagger element comprises a part of the expression sequence of one or more expression sequences.

[0208] In certain embodiments, the circular polynucleotide comprises a stagger element. The stagger element may be included to induce stalling of the ribosome during translation to avoid the production of consecutive expression products, such as peptides or polypeptides, while maintaining rolling circle type translation. In certain embodiments, the stagger element is at at least one 3' end of one or more expression sequences. The stagger element may be configured to stall the ribosome during rolling circle type translation of the circular polynucleotide. Examples of the stagger element include, but are not limited to sequences separated from one or more expression sequences. In certain embodiments, the stagger element comprises a part of the expression sequence of one or more expression sequences. In certain embodiments, the stagger element is at at least one 3' end of one or more expression sequences. The stagger element may be configured to stall the ribosome during rolling circle type translation of the circular polynucleotide. Examples of the stagger element include, but are not limited to sequences separated from one or more expression sequences. Examples of the stagger element include, but are not limited to but includes 2A-like or CHYSEL (cis-acting hydrolase element) sequences . In certain embodiments, the stager element is X1X2X3EX5NPGP (where X1 is absent or is G or H, X2 is absent or is D or G , X3 is D, or V, or I, or S, or M, and X5 is any amino acid ). In certain embodiments, the sequence encodes a sequence having a C-terminal consensus sequence of ) followed by a non-conserved sequence of amino acids having a strong alpha helix tendency, followed by a consensus sequence -D(V / I)ExNPG P (where x = any amino acid). Some non-limiting examples of stager elements include GDVESNPGP, GDIEENPGP , VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDI ETNPGP, GDVENPGP, GDVEENPGP, GDVEQNPGP, IESN PGP, GDIELNPGP, HDIETNPGP, HDVETNPGP, HDVEMN PGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP and DSEFN PGP.

[0209] In certain embodiments, the stager elements described herein cleave the expression product, such as between the G and P of the consensus sequences described herein. As one non-limiting example , the cyclic polynucleotide includes at least one stager element to cleave the expression product. In certain embodiments, the cyclic polynucleotide includes a stager element adjacent to at least one expression sequence. In certain embodiments, the cyclic polynucleotide includes a stager element after each expression sequence. In certain embodiments, the cyclic polynucleotide includes a stager element after each expression sequence. In certain embodiments, the cyclic polynucleotide includes a stager element after each expression sequence. In certain embodiments, the cyclic polynucleotide The staggered elements are present on one or both sides of each expression sequence. This results in the translation of individual peptides and / or polypeptides from each expressed sequence.

[0210] In one embodiment, the stagger element comprises one or more ribosome-specific nucleotides that induce pausing of the ribosome during translation. The non-natural nucleotides include the above modified nucleotides or non-natural nucleotides. Nucleic Acid (PNA), Morpholino and Locked Nucleic Acid (LNA) and Glycol Nucleic Acid (GNA Examples such as these include threose nucleic acid (TNA) and threose nucleic acid (TNA). Exemplary modifications include sugar, nucleobase, nuclease, and nucleotide modifications. for interosidic bonds (e.g., phosphate / phosphodiester bonds / phospho Any modifications (to the diester backbone) and those that can induce ribosome pausing during translation Some of the exemplary modifications provided herein may be included in the present invention. These are described elsewhere in the Specification.

[0211] In some embodiments, the stagger element is present in the circular polyribonucleotide in another form. For example, in certain exemplary circular polyribonucleotides, the stagger element is The termination element of the first expression sequence in the polyribonucleotide sequence and the expression sequence following the first expression sequence It contains a nucleotide spacer sequence that separates the termination element from the first translation initiation sequence. In one example, the first staggered element of the first expression sequence is It is located upstream (5' end side) of the first translation initiation sequence of the expression following the first expression sequence. The first expression sequence and the expression sequence following the first expression sequence are circular polyribonucleoside. They are two separate expression arrays in the chid. Between the first stager element and the first translation initiation array The distance may enable continuous translation of the first expression array and its subsequent expression arrays. In a certain embodiment, the first stager element includes a termination element and separates the expression product of the first expression array from the expression product of its subsequent expression array, thereby forming separate expression products. In certain cases, a circular polynucleotide containing a first stager element upstream of the first translation initiation array of a subsequent array in the circular polynucleotide is continuously translated, while a circular polynucleotide containing a stager element of a second expression array upstream of the second translation initiation array of an expression array following the second expression array is not continuously translated. In certain cases there is only one expression array in the circular polynucleotide, and the first expression array and its subsequent expression array are the same expression array. In an exemplary circular polynucleotide, the stager element includes a nucleotide spacer array that separates the first termination element of the first expression array in the circular polynucleotide and the termination element from the downstream translation initiation array. In such an example, the first stager element is upstream (5'-end side) of the first translation initiation array of the first expression array in the circular polynucleotide. In certain cases, the distance between the first stager element and the first translation initiation array enables continuous translation of the first expression array and any subsequent expression arrays. In a certain embodiment, the first stager element separates the expression product of a certain round of the first expression array from the expression product of the next round of the first expression array, thereby forming separate expression products. In certain cases, a circular polynucleotide containing a first stager element upstream of the first translation initiation array of the first expression array in the circular polynucleotide The do is continuously translated, while the second expression sequence in the corresponding circular polynucleotide The corresponding circular polynucleotide containing the stager element upstream of the second translation start sequence of is not continuously translated. In certain cases, the distance between the second stager element and the second translation start sequence is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater in the corresponding circular polynucleotide than the distance between the first stager element and the first translation start in the corresponding circular polynucleotide. In certain cases, the distance between the first stager element and the first translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt t, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 1 5 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 3 5 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 7 5 nt or more. In certain embodiments, the distance between the second stager element and the second translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or more greater than the distance between the first stager element and the first translation start. In certain embodiments, the circular polynucleotide comprises two or more expression sequences. In certain embodiments, the circular polynucleotide encodes a regulatory nucleic acid, such as an internal

[0212] Regulatory nucleic acid In certain embodiments, the circular polynucleotide encodes a regulatory nucleic acid, for example, internal It contains one or more expression sequences that regulate the expression of sex genes and / or foreign genes. In one embodiment, the expression sequence of the circular polynucleotide provided herein is, but not limited to, tRNA, lncRNA, miRNA, rRNA, snRNA, maclRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, sca RNA, Y RNA, hnRNA, etc., and may contain sequences that are antisense to regulatory nucleic acids such as non-coding RNAs.

[0213] In one embodiment, the regulatory nucleic acid targets a gene such as a host gene. The regulatory nucleic acid is any of the regulatory nucleic acids described in International Patent Publication No. WO 2019 / 1189 19A1, paragraphs

[0177] and

[0181] to

[0189] , which are hereby incorporated by reference in their entirety.

[0214] In certain embodiments, the circular polynucleotide comprises a guide RNA (gRNA). In certain embodiments, the circular polynucleotide comprises or encodes a guide RNA. The gRNA short-chain synthetic RNA consists of a "scaffold" sequence necessary to bind to the incomplete effector portion and a user-defined approximately 20-nucleotide targeting sequence for the genomic target. In fact, the guide RNA sequence generally has a length of 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and is designed to be complementary to the targeted nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for use in the design of effective guide RNAs. Gene editing mimics the natural cr RNA-tracrRNA complex and tracrRNA (which binds to the nuclease ​ and at least one crRNA (for guiding the nuclease to the sequence targeted for editing), a chimeric " single-guide RNA" ("sgRNA"), which is a modified (synthetic) single RNA molecule containing both has also been performed using chemically modified sgRNA. Chemically modified sgRNA has also been demonstrated to be effective for genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991.

[0215] The gRNA can recognize a specific DNA sequence (e.g., a sequence adjacent to or within a gene's promoter, enhancer, silencer or repressor).

[0216] In one embodiment, the gRNA is used as part of a CRISPR system for gene editing. For gene editing, the circular polynucleotide can be designed to include one or more guide RNA sequences corresponding to the desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. At least about 16 or 17 nucleotides of the gRNA sequence are required by Cas9 for DNA cleavage to occur; for Cpf1, at least about 16 nucleotides of the gRNA sequence are required to achieve detectable DNA cleavage.

[0217] The circular polynucleotide can regulate the expression of RNA encoded by a gene. Since multiple genes can share a certain degree of sequence homology with each other, in certain embodiments Thus, the cyclic polynucleotide can be designed to target a class of genes having sufficient sequence homology. In certain embodiments, the cyclic polynucleotide can contain sequences that are shared among different gene targets or have complementarity to sequences that are specific to a particular gene target. In certain embodiments, the cyclic polynucleotide can be designed to target conserved regions of RNA sequences that are homologous among several genes, thereby targeting several genes in a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In certain embodiments, the cyclic polynucleotide can be designed to target sequences specific to a particular RNA sequence of a single gene.

[0218] In certain embodiments, the expression sequence has a length of less than 5000 bp (e.g., about 5000 bp, 4000 bp, 3000 bp, 2000 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 50 bp, 40 bp, 30 bp, 20 bp, 10 bp or less). In certain embodiments, the expression sequence independently or in addition has a length of more than 10 bp (e.g., at least about 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb or more). 5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3 kb, 3.1 kb, 3. 2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 4 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, 5 kb, 10 kb, 20 kb, or longer than that).

[0219] In certain embodiments, the expression array is one or more of the features described herein, e.g., 1 or more sequences encoding one or more peptides or proteins, one or more regulatory elements, one or more reg ulatory nucleic acids, e.g., one or more non-coding RNAs, other expression arrays, and any combination thereof including.

[0220] Translation efficiency In certain embodiments, the translation of the circular polynucleotides provided herein efficiency is higher than that of a reference, e.g., a linear equivalent, a linear expression array, or a linear circular polynucleotide. In certain embodiments, the circular polynucleotides provided herein are at least about 5%, 10%, 15%, 20%, 25%, 30%, 3 5%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8 5%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250% 300%, 350%, 400%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2000%, 5000%, 10000%, 100000% or more higher translation efficiency than that of the reference. In certain embodiments, the circular polynucleotide is ​、has a translation efficiency that is 10% higher than that of its linear equivalent. In certain embodiments, the circular poly ribonucleotide has a translation efficiency that is 300% higher than that of its linear equivalent.

[0221] In certain embodiments, the circular polyribonucleotide produces a stoichiometric ratio of expression products Rolling circle type translation continuously produces expression products at substantially equivalent ratios. In certain embodiments, the circular polyribonucleotide has a stoichiometric translation efficiency such that the expression products are produced at substantially equivalent ratios. In certain embodiments, the circular polyribo nucleotide has a stoichiometric translation efficiency for products from multiple expression products, e.g., 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12 or more expression sequences.

[0222] Rolling circle type translation In certain embodiments, once translation of the circular polyribonucleotide is initiated, the ribosome bound to the circular polyribo nucleotide does not dissociate from the circular polyribonucleotide until at least one round of translation of the circular polyribonucleotide is completed. In certain embodiments the circular polyribonucleotides described herein have the ability for rolling circle type translation In certain embodiments, during rolling circle type translation, once translation of the circular polyribonucleotide is initiated, the ribosome bound to the circular polyribo nucleotide does not dissociate from the circular polyribo nucleotide until at least 2, at least 3, at least 4, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least 9 rounds, at least 1 0 rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds, at least At least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 500 times, at least 1000 times, at least 1500 times, at least 200 0 times, at least 5000 times, at least 10000 times, at least 10 5 times or at least 10 6 times of translation is completed and does not dissociate from the circular polynucleotide.

[0223] In certain embodiments, rolling circle type translation of the circular polynucleotide results in the production of polypeptide products (``continuous '' expression products) translated from two or more translations of the circular polynucleotide. In certain embodiments, the circular polynucleotide comprises a stager element, and rolling circle type translation of the circular polynucleotide results in the production of polypeptide products ( ``discrete'' expression products) generated from one or less than one translation of the circular polynucleotide. In certain embodiments, the circular polynucleotide is configured such that at least 10%, 20%, 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of all polypeptides (mol / mol) generated during rolling circle type translation of the circular polynucleotide are discrete polypeptides. In certain embodiments, the amount ratio of discrete products across all polypeptides is tested in an in vitro translation system. In certain embodiments at least 96%, at least 97%, at least 98%, at least 99% or 10 0% are discrete polypeptides. In certain embodiments, the amount ratio of discrete products across all polypeptides is tested in an in vitro translation system. In certain embodiments across all polypeptides is tested in an in vitro translation system. In certain embodiments In this case, the in vitro translation system used for the quantitative ratio test contains rabbit reticulocyte lysate. In certain embodiments, the quantitative ratio is tested in an in vivo translation system such as eukaryotic or prokaryotic cells, cultured cells or cells in an organism.

[0224] Untranslated region In certain embodiments, the cyclic polynucleotide contains an untranslated region (UTR). The UTR of the genomic region containing the gene can be transcribed but not translated. In certain embodiments the UTR can be included upstream of the translation initiation sequence of the expression sequence described herein. In certain embodiments, the UTR can be included downstream of the expression sequence described herein. In some cases, one UTR for a first expression sequence is the same as, continuous with, or overlapping with another UTR for a second expression sequence. In certain embodiments the intron is a human intron. In certain embodiments, the intron is a full-length human intron, such as ZKSCAN1.

[0225] In certain embodiments, the cyclic polynucleotide contains a UTR having one or more stretches of adenosine and uridine embedded therein. These AU-rich signatures can increase the turnover rate of the expression product.

[0226] Introduction, removal or modification of UTR AU-rich elements (AREs) can be useful for modulating the stability or immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response) of the cyclic polynucleotide. When modifying a particular cyclic polynucleotide, one or more copies of ARE can be introduced into the cyclic polynucleotide and the copy of ARE is expressed ​​​​​It can regulate the translation and / or generation of products. Similarly, AREs are identified, removed or modified in circular polynucleotides to regulate intracellular stability and thus can affect the resulting protein translation and generation. identified, removed or modified in the circular polynucleotides to regulate intracellular stability and thus the resulting protein translation and generation can be affected.

[0227] It should be understood that any UTR from any gene can be incorporated into each adjacent region of the circular polynucleotide. Exemplary UTRs that can be used in the circular polynucleotides provided herein are those described in

[0200] -

[0201] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is hereby incorporated by reference in its entirety. It should be understood that any UTR from any gene can be incorporated into each adjacent region of the circular polynucleotide. Exemplary UTRs that can be used in the circular polynucleotides provided herein are those described in

[0200] -

[0201] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is hereby incorporated by reference in its entirety. It should be understood that any UTR from any gene can be incorporated into each adjacent region of the circular polynucleotide. Exemplary UTRs that can be used in the circular polynucleotides provided herein are those described in

[0200] -

[0201] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is hereby incorporated by reference in its entirety. It should be understood that any UTR from any gene can be incorporated into each adjacent region of the circular polynucleotide. Exemplary UTRs that can be used in the circular polynucleotides provided herein are those described in

[0200] -

[0201] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is hereby incorporated by reference in its entirety. to

[0201] .

[0228] Poly - A sequence In certain embodiments, the circular polynucleotide can include a poly - A sequence. In certain embodiments, the length of the poly - A sequence exceeds 10 nucleotides. In one embodiment, the poly - A sequence exceeds 15 nucleotides in length (e.g., at least about 10, 1 5, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 10 0, 120, 140, 160, 180, 200, 250, 300, 350, 400, 45 0, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,90 0, 2,000, 2,500 and 3,000 nucleotides or more). In certain embodiments, the poly - A sequence is designed according to the description of the poly - A sequence in International Patent Publication No. WO 2019 / 118919 A1, which is hereby incorporated by reference in its entirety, from

[0202] to

[0204] . In certain embodiments, the poly - A sequence is designed according to the description of the poly - A sequence in International Patent Publication No. WO 2019 / 118919 A1, which is hereby incorporated by reference in its entirety, from

[0202] to

[0204] .

[0229] In certain embodiments, the circular polynucleotide contains polyA, lacks polyA, or has a modified polyA for modulating one or more properties of the circular polynucleotide. In certain embodiments, the circular polynucleotide lacking polyA or having a modified polyA improves one or more functional properties, such as immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response), half-life, expression efficiency, etc.

[0230] RNA Binding In certain embodiments, the circular polynucleotide contains one or more RNA binding sites. MicroRNA (or miRNA) is a short non-coding RNA that binds to the 3’UTR of a nucleic acid molecule and downregulates gene expression by decreasing nucleic acid molecule stability or inhibiting translation. The circular polynucleotide may contain one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNA, such as those taught in U.S. Patent Application Publication No. 2005 / 0261218, U.S. Patent Application Publication No. 2005 / 0059005, and paragraphs

[0207] to

[0215] of International Patent Publication No. WO 2019 / 118919A1, the entire contents of which are incorporated herein by reference.

[0231] Protein Binding In certain embodiments, the circular polynucleotide contains one or more protein binding sites that enable a protein, such as a ribosome, to bind to an internal site in the RNA sequence. Protein binding sites, such as ribosome binding sites, in the circular polynucleotide ​​​ By operating in the host, the cyclic polynucleotide can be from components of the host's immune system By masking the cyclic polynucleotide, the host's immune system can be avoided or It may have reduced detection by the host's immune system and may have regulated degradation or regulated translation be able to.

[0232] In certain embodiments, the cyclic polynucleotide is, for example, an immune response, such as CT Includes at least one immune protein binding site to avoid the L (cytotoxic T lymphocyte) response. In certain embodiments, the immune protein binding site binds to the immune protein and is a nucleotide sequence that helps to mask the cyclic polynucleotide as being exogenous. In certain embodiments, the immune protein binding site binds to the immune protein and is a nucleotide sequence that helps to hide the cyclic polynucleotide as being exogenous or foreign. and is a nucleotide sequence that helps to hide the cyclic polynucleotide as being exogenous or foreign. and is a nucleotide sequence that helps to hide the cyclic polynucleotide as being exogenous or foreign. is a nucleotide sequence.

[0233] The conventional mechanism of ribosome engagement with linear RNA involves ribosomes that bind to the capped 5' end of the RNA. From the 5' end, as soon as the ribosome moves to the start codon, the first peptide bond is formed immediately. According to the present invention, internal initiation (i.e., cap-independent) of translation of the cyclic polynucleotide does not require a free end or a capped end. Rather, the ribosome binds to an uncapped internal site, whereby the ribosome begins polypeptide elongation at the start codon. In certain embodiments, the cyclic polynucleotide includes one or more RNA sequences, such as a ribosome binding site, for example, a start codon. begins polypeptide elongation at the start codon. In certain embodiments, the cyclic polynucleotide includes one or more RNA sequences, such as a ribosome binding site, for example, a start codon. begins polypeptide elongation at the start codon. In certain embodiments, the cyclic polynucleotide includes one or more RNA sequences, such as a ribosome binding site, for example, a start codon. begins polypeptide elongation at the start codon. In certain embodiments, the cyclic polynucleotide includes one or more RNA sequences, such as a ribosome binding site, for example, a start codon. begins polypeptide elongation at the start codon. In certain embodiments, the cyclic polynucleotide includes one or more RNA sequences, such as a ribosome binding site, for example, a start codon. begins polypeptide elongation at the start codon. In certain embodiments, the cyclic polynucleotide includes one or more RNA sequences, such as a ribosome binding site, for example, a start codon. A sequences.

[0234] Natural 5’UTRs have features that play a role in translation initiation. They are generally known to be involved in the process by which ribosomes initiate translation of many genes and have signatures such as the Kozak sequence. The Kozak sequence has the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". 5’UTRs are also known to be involved in secondary structures that participate in elongation factor binding. They have signatures such as the Kozak sequence, which is generally known to be involved in the process by which ribosomes initiate translation of many genes. The Kozak sequence has the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". 5’UTRs are also known to be involved in secondary structures that participate in elongation factor binding.

[0235] In certain embodiments, the circular polynucleotide encodes a protein-binding sequence that binds to a protein. In certain embodiments, the protein-binding sequence targets or confines the circular polynucleotide to a specific target. In certain embodiments, the protein-binding sequence specifically binds to the arginine-rich region of a protein.

[0236] In certain embodiments, protein-binding sites include, but are not limited to, ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, C APRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CST F2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DG CR8, EIF3A, EIF4A3, EIF4G2, ELAVL1, ELAVL3, FA M120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR 2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, H NRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2 ​​​BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, L IN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1 , MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2 , POLR2A, PRPF8, PTBP1, RBFOX2, RBM10, RBM22, R BM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1, TNRC 6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF 1, WDR33, XRN2, YBX1, YTHDC1, YTHDF1, YTHDF2, Y WHAG, ZC3H7B, PDK1, AKT1 and any other protein that binds to RNA such as a binding site to a protein.

[0237] Encryptogen When described herein, the cyclic polynucleotide comprises an encryptogen to reduce, avoid or evade the innate immune response of the cell. In one embodiment, when delivered to a cell, a reduced immune response from the host is provided by a cyclic polynucleotide that results in a reduced immune response compared to the response elicited by a reference compound, such as a cyclic polynucleotide described or a linear polynucleotide corresponding to a cyclic polynucleotide lacking an encryptogen. In certain embodiments, the cyclic polynucleotide has lower immunogenicity (e.g., an immune or inflammatory response than an equivalent lacking an encryptogen. having a lower level of one or more markers.

[0238] In certain embodiments, the cryptogen improves stability. Evidence is increasing regarding the regulatory role played by UTRs with respect to the stability of nucleic acid molecules and translation The regulatory features of UTRs can be included in the cryptogen to improve the stability of circular polynucleotides. can be included in the cryptogen.

[0239] In certain embodiments, the 5' or 3' UTR can constitute the cryptogen in a circular polynucleotide. For example, removal or modification of the UTR AU-rich element (ARE) can be useful for modulating the stability or immunogenicity of the circular polynucleotide (e.g., modulating the level of one or more markers of an immune or inflammatory response).

[0240] In certain embodiments, removal of modification of an AU-rich element (ARE) in an expression sequence, such as a translatable region, can be useful for modulating the stability or immunogenicity of the circular polynucleotide (e.g., modulating the level of one or more markers of an immune or inflammatory response).

[0241] In certain embodiments, the cryptogen includes a miRNA binding site or a binding site to any other non-coding RNA. For example, incorporation of the miR-142 site into the circular polynucleotides described herein not only modulates expression in hematopoietic cells, but can also reduce or eliminate the immune response against the protein encoded in the circular polynucleotide.

[0242] In certain embodiments, the cryptogen is a protein, such as an immune protein that is R ​​​​​It contains one or more protein binding sites that enable binding to the NA sequence. Protein By engineering the binding site into the cyclic polynucleotide, the cyclic polynucleo tide can mask the cyclic polynucleotide from components of the host immune system, thereby avoiding or having reduced detection by the host immune system, and may have regulated degradation or regulated translation. In certain embodiments, the cyclic polynucleotide contains, for example, at least one immune protein binding site to avoid an immune response, such as a CTL response. In certain embodiments, the immune protein binding site binds to an immune protein and serves to mask the cyclic polynucleotide as being exogenous.

[0243] In certain embodiments, the cryptogen contains one or more modified nucleotides. Ex emplary modifications include any modification to the sugar, nucleobase, internucleo side linkage (e.g., to the linking phosphate / phosphodiester bond / phosphodiester backbone) and any combination thereof that can prevent or reduce the immune response to the cyclic polynucleotide. Some of the exemplary modifications provided

[0244] herein are described in detail below. In certain embodiments, the cyclic polynucleotide contains one or more of the modifications described elsewhere herein to reduce the immune response from the host as compared to the response elicited by a reference compound, such as a It has been shown that, for example, Yu, Z. et al. ( 2015) RNA editing by ADAR1 marks dsRNA as “self”. See Cell Res. 25, 1283-1284, which is incorporated herein by reference in its entirety.

[0245] In certain embodiments, the circular polynucleotide comprises an RNA sequence that can be processed into one or more expression sequences for shRNA or an RNA sequence for siRNA, and the shRNA or si RNA targets RIG-I and reduces the expression of RIG-I. RIG-I can sense exogenous circular RNA and effect the degradation of exogenous circular RNA. Thus, a circular polynucleotide having a sequence for RIG-1 targeting shRNA, siRNA or any other regulatory nucleic acid can reduce immunity against the circular polynucleotide, e.g., host cell immunity thereby.

[0246] In certain embodiments, the circular polynucleotide lacks sequences, elements or structures that assist the circular polynucleotide in reducing, avoiding or evading the innate immune response of the cell. In certain such embodiments, the circular polynucleotide may lack poly sequences, 5' ends, 3' ends, phosphate groups, hydroxyl groups or any combination thereof.

[0247] Riboswitches In certain embodiments, the circular polynucleotide comprises one or more riboswitches.

[0248] Riboswitches are typically thought to be part of the circular polynucleotide that can directly bind to small target molecules, and binding of the target affects RNA translation, expression product stability, and ​ and affect activity (Tucker B J, Breaker R R (2005), Curr Opin Struct Biol 15(3):342-8). Thus , cyclic polynucleotides containing riboswitches are directly involved in regulating their own activity in response to the presence or absence of their target molecules. In certain embodiments, the riboswitch has a region of affinity, such as an aptamer for a separate molecule. Thus, in the broader context of the present invention, any aptamer contained within a non-coding nucleic acid can be used for the isolation of molecules from the bulk volume. Reporting downstream of events due to "(ribo)switch" activity can be particularly advantageous. In certain embodiments, riboswitches can affect gene expression including, but not limited to, transcription termination, inhibition of translation initiation, mRNA self-cleavage and alteration of the splicing pathway in eukaryotes. Riboswitches can function to control gene expression by the binding or removal of a trigger molecule. Thus, a cyclic polynucleotide containing a riboswitch is subjected to conditions that activate, inactivate or block the riboswitch to change expression. Expression can be changed, for example, as a result of termination or blockage of transcription of ribosomes that bind to RNA. Binding of a trigger molecule or an analog thereof can decrease or prevent the expression of an RNA molecule, or promote or increase the expression of an RNA molecule,

[0249] depending on the nature of the riboswitch. Some examples of riboswitches are described herein. In certain embodiments, riboswitches include, but are not limited to, cyclic di-GMP riboswitches, FMN ribos witches, and the like. By subjecting a cyclic polynucleotide containing a riboswitch to conditions that activate, inactivate or block the riboswitch, expression is changed. Expression can be changed, for example, as a result of termination or blockage of transcription of ribosomes that bind to RNA. Binding of a trigger molecule or an analog thereof can decrease or prevent the expression of an RNA molecule, or promote or increase the expression of an RNA molecule, depending on the nature of the riboswitch. Some examples of riboswitches are described herein.

[0250] In certain embodiments, the riboswitch is a cyclic di-GMP riboswitch, FMN ribos Ichi (also called RFN element), glmS riboswitch, glutamine riboswitch, glycine riboswitch, lysine riboswitch (also called L-box), PreQ1 riboswitches (e.g., PreQ1-l riboswitch and PreQ1-ll riboswitch ), purine riboswitch, SAH riboswitch, SAM riboswitch, SAM-SAH ri boswitch, tetrahydrofolate riboswitch, theophylline-binding riboswitch, thymine pi rophosphate-binding riboswitch, T. tengcongensis (T.tengcongensi s) glmS catalytic riboswitch, TPP riboswitch (also called THI-box) , Moco riboswitch, or adenine-sensing add-A riboswitch, which are each incorporated herein by reference in their entirety from International Patent Publication No. International Publication No. 20191 18919A1, pages

[0235] to

[0252] .

[0251] Aptazymes In certain embodiments, the cyclic polynucleotide comprises an aptazyme. An aptazyme is a switch for conditional expression where the aptamer region is used as an allosteric control element and is bound to a region of catalytic RNA (the "ribozyme" described below). In certain embodiments, the aptazyme is active in cell-type specific translation. In certain embodiments the aptazyme is active in cell-state specific translation, e.g., in virus-infected cells or in the presence of viral nucleic acids or viral proteins.

[0252] A ribozyme (derived from a ribonucleic acid enzyme, also called an RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Some non-limiting examples of ribozymes include ha hammerhead ribozymes, VL ribozymes, leadzymes, hairpin ribozymes, and other ribozymes described in

[0254] to

[0259] of International Patent Publication No. WO201911819A1 pamphlet, which is incorporated herein by reference in its entirety, may be mentioned.

[0253] Replication element Circular polynucleotides may encode sequences and / or motifs useful for replication. Replication of circular polynucleotides can be carried out by generating complementary circular polynucleotides. In certain embodiments, the circular polynucleotide contains a motif for initiating transcription, where the transcription is driven by either an endogenous cellular machinery (DNA- dependent RNA polymerase) or an RNA-dependent RNA polymerase encoded by the circular polynucleotide. The products of rolling circle type transcription events can be cleaved by ribozymes to generate either unit- length complementary or propagated circular polynucleotides. The ribozyme can be encoded by an RNA sequence in the circular polynucleotide, its complement, or trans. In certain embodiments, the encoded ribozyme may contain a sequence or motif that regulates (inhibits or promotes) the activity of the ribozyme to control circular RNA propagation. In certain embodiments, the unit-length sequence can be ligated into a Those described in

[0280] to

[0282] of Pamphlet No. 019118919A1 may be mentioned.

[0254] In certain embodiments, the cyclic polynucleotide is, for example, substantially resistant to degradation by exonucleases.

[0255] In certain embodiments, the cyclic polynucleotide replicates intracellularly. In certain embodiments, the cyclic polynucleotide replicates intracellularly at a rate of about 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 75%, 75% - 80%, 80% - 85%, 85% - 90%, 90% - 95%, 95% - 99% or any percentage in between. In certain embodiments, the cyclic polynucleotide is replicated intracellularly and sent to daughter cells. In certain embodiments, the cell sends at least one cyclic polynucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90% , 95% or 99%. In certain embodiments, cells undergoing meiosis send cyclic polynucleotides to daughter cells with an efficiency of at least 25%, 50%, 6 0%, 70%, 80%, 85%, 90%, 95% or 99%. In certain embodiments, cells undergoing mitosis send cyclic polynucleotides to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% .

[0256] In certain embodiments, the cyclic polynucleotide replicates within a host cell. In one embodiment, the cyclic polynucleotide replicates within mammalian cells, such as human cells.​​​​​ is possible.

[0257] In an embodiment, the circular polynucleotide replicates in the host cell, but the circular poly ribonucleotide is not integrated into the host genome by, for example, the host chromosome. In certain embodiments, the circular polynucleotide has, for example, a very low recombination frequency due to, for example, the host chromosome. In certain embodiments, the circular polynucleotide has, for example, a recombination frequency of, for example, about 1.0 cM / Mb, 0.9 cM / Mb, 0.8 cM / Mb, 0.7 cM / Mb, 0.6 cM / Mb, 0.5 cM / Mb, 0.4 cM / Mb, 0.3 cM / Mb, 0.2 cM / Mb, less than 0.1 cM / Mb or below that due to, for example, the host chromosome.

[0258] scaffold sequence In certain embodiments, the circular polynucleotide molecule comprises one or more scaffold sequences. The scaffold sequence can be an aptamer sequence. In certain embodiments of each of the aspects listed above, the circular polynucleotide molecule has a sequence encoding an endogenous or native circular polynucleotide sequence.

[0259] In certain embodiments, the circular RNA binds to one or more targets. In certain embodiments, the circular RNA is a circular aptamer. In one embodiment, the circular RNA comprises one or more binding sites that bind to one or more targets. In one embodiment, the circular RNA comprises an aptamer sequence. In one embodiment, the circular RNA binds to both DNA and protein targets and, for example, mediates Protein complexes combine to mediate, for example, post-translational modifications or signal transduction. In another embodiment, the circular RNA binds to two or more different targets such as proteins and, for example, transports these proteins to the cytoplasm or mediates the degradation of one or more of the targets.

[0260] In certain embodiments, the circular RNA binds to at least one of DNA, RNA, and proteins, thereby regulating cellular processes (e.g., changing protein expression, regulating gene expression, regulating cell signaling, etc.). In certain embodiments, the synthetic circular RNA contains at least one portion of a target or selected DNA, RNA, or protein, e.g., a binding site for interaction with a binding portion, thereby competing with binding to endogenous counterparts.

[0261] In certain embodiments, the circular RNA forms a complex that regulates cellular processes (e.g., changing protein expression, regulating gene expression, regulating cell signaling, etc.). In certain embodiments, the circular RNA sensitizes cells to a cytotoxic agent (e.g., a chemotherapeutic agent) by binding to a target (e.g., a transcription factor), thereby reducing cell survival. For example, by sensitizing cells to a cytotoxic agent, cell survival is reduced after delivery of the cytotoxic agent and the circular RNA. In certain embodiments, the reduced cell survival is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage therebetween.

[0262] In certain embodiments, the complex persists in the cell for at least 5 days after delivery of the is detectable over time. In certain embodiments, the complex is the delivery of circular RNA to cells and is detectable for 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 15 days, or 16 days.

[0263] In one embodiment, the synthetic circular RNA binds and / or sequesters miRNAs. In another embodiment, the synthetic circular RNA binds and / or sequesters proteins. In another embodiment the synthetic circular RNA binds and / or sequesters mRNAs. In another embodiment the synthetic circular RNA binds and / or sequesters ribosomes. In another embodiment the synthetic circular RNA binds and / or sequesters circular RNAs. In another embodiment the synthetic circular RNA binds and / or sequesters long non-coding RNAs (lncRNAs) or any other non-coding RNAs, such as miRNAs, tRNAs, rRNAs, snoRNAs, ncRNAs siRNAs, long non-coding RNAs, shRNAs. In addition to the binding and / or sequestering site, the circular RNA may include a degradation element, which results in the degradation of the bound and / or sequestered RNA and / or protein.

[0264] In one embodiment, the circular RNA includes an lncRNA or the sequence of an lncRNA, e.g., the circular RNA includes the sequence of a native, non-circular lncRNA or a fragment thereof. In one embodiment, the lncRNA or the sequence of the lncRNA is circularized with or without a spacer sequence to form the synthetic circular RNA.

[0265] In one embodiment, the circular RNA has ribozyme activity. In one embodiment Circular RNAs can act as ribozymes and be used to cleave pathogenic or endogenous RNAs, DNAs, small molecules, or proteins. In one embodiment, the circular RNA has enzymatic activity . In one embodiment, the synthetic circular RNA can specifically recognize and cleave RNA (e.g., viral RNA) . In another embodiment, the circular RNA can specifically recognize and cleave a protein . In another embodiment, the circular RNA can specifically recognize and degrade small molecules.

[0266] In one embodiment, the circular RNA is an immolating or self-cleaving or cleavable circular RNA. In one embodiment, the circular RNA can be used to deliver RNA, such as miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, long non-coding RNA, shRNA. In one embodiment, the synthetic circular RNA is composed of a microRNA separated by (1) a self-cleavable element (e.g., hammerhead type, splicing element), (2) a cleavage mobilization site (e.g., ADAR), (3) a degradable linker (e.g., glycerol), (4) a chemical linker, and / or (5) a spacer sequence . In another embodiment, the synthetic circular RNA is composed of an siRNA separated by (1) a self-cleavable element (e.g., hammerhead type, splicing element), (2) a cleavage mobilization site (e.g., ADAR), (3) a degradable linker (e.g., glycerol), (4) a chemical linker, and / or (5) a spacer sequence . . .

[0267] In one embodiment, the circular RNA is a transcription / replication-capable circular RNA. This circular R NA can encode any type of RNA. In one embodiment, the synthetic circular RNA has antisense miRNAs and transcriptional elements. In one embodiment, after transcription, linear functional miRNAs are generated from the circular RNA. In one embodiment, the circular RNA is a non-translatable circular polynucleotide.

[0268] In one embodiment, the circular RNA has one or more of the above attributes in combination with a translation element.

[0269] Other sequences In certain embodiments, the circular polynucleotide further comprises another nucleic acid sequence. In certain embodiments, the circular polynucleotide can include other sequences including DNA, RNA, or artificial nucleic acids. Other sequences include, but are not limited to, genomic DNA, cDNA, or sequences encoding tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecules. In one embodiment, the circular polynucleotide includes siRNA to target different loci of the same gene expression product as the circular polynucleotide. In one embodiment, the circular polynucleotide includes siRNA to target a different gene expression product than the circular polynucleotide.

[0270] In certain embodiments, the circular polynucleotide lacks a 5'-UTR. In certain embodiments, the circular polynucleotide lacks a 3'-UTR. In certain embodiments, the circular polynucleotide lacks a poly-A sequence. In certain embodiments, the circular polynucleotide lacks a termination element. In certain embodiments, ​​​​​​​In addition, the circular polynucleotide lacks an internal ribosome entry site. In certain embodiments the circular polynucleotide lacks susceptibility to degradation by exonucleases In certain embodiments, the lack of susceptibility to degradation of the circular polynucleotide means that the circular polynucleotide is not degraded by exonucleases, or is degraded only in the presence of exonucleases to an extent equivalent or similar to that in the absence of exonucleases. In certain embodiments, the circular polynucleotide lacks susceptibility to degradation by exonucleases. In certain embodiments, the circular polynucleotide has reduced degradation when exposed to exonucleases. In certain embodiments, the circular polynucleotide lacks binding to cap-binding proteins. In certain embodiments, the circular polynucleotide lacks a 5' cap In certain embodiments, the circular polynucleotide lacks a 5'-UTR and has the ability to express protein from one or more of its expression sequences. In certain embodiments, the circular polynucleotide lacks a 3'-UTR and has the ability to express protein from one or more of its expression sequences. In certain embodiments, the circular polynucleotide lacks a poly-A sequence and has the ability to express protein from one or more of its expression sequences. In certain embodiments, the circular polynucleotide lacks a termination element and has the ability to express protein from one or more of its expression sequences. In certain embodiments, the circular .

[0271] polynucleotide lacks an internal ribosome entry site and has the ability to express protein from one or more of its expression sequences. It has the ability to express proteins from a column. In certain embodiments, the circular polynucleotide lacks a cap and has the ability to express proteins from one or more of its expression sequences. In certain embodiments, the circular polynucleotide lacks a 5'-UTR, 3'-UTR, and IRES and has the ability to express proteins from one or more of its expression sequences. In certain embodiments, the circular polynucleotide lacks a 5'-UTR, 3'-UTR, and IRES and has the ability to express proteins from one or more of its expression sequences. In certain embodiments, the circular polynucleotide comprises one or more of the following sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding a foreign gene, a sequence encoding a therapeutic agent, a regulatory element (e.g., a translation modulator, e.g., a translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids targeting endogenous genes (siRNA, lncRNAs, shRNA), and a sequence encoding a therapeutic mRNA or protein. Other sequences can have a length of about 2 to about 10,000 nt, about 2 to about 5,000 nt, about 10 to about 100 nt, about 50 to about 150 nt, about 100 to about 200 nt, about 150 to about 250 nt, about 200

[0272] to about 300 nt, about 250 to about 350 nt, about 300 to about 500 nt, about 10 to about 100 0 nt, about 50 to about 1,000 nt, about 100 to about 1,000 nt, about 1,000 to about 2,000 nt, about 2,000 to about 3,000 nt, about 3,000 to about 4,000 nt, about 4,000 to about 5,000 nt, or any range in between. As a result of its circularization, the circular polynucleotide can include several features that distinguish it from linear RNA. For example, the circular polynucleotide, compared to linear RNA, can have a length of any range between about 2 and about 10,000 nt, about 2 and about 5,000 nt, about 10 and about 100 nt, about 50 and about 150 nt, about 100 and about 200 nt, about 150 and about 250 nt, about 200 and about 300 nt, about 250 and about 350 nt, about 300 and about 500 nt, about 10 and about 1000 nt, about 100 and about 1000 nt, about 1000 and about 2000 nt, about 2000 and about 3000 nt, about 3000 and about 4000 nt, about 4000 and about 5000 nt.

[0273] As a result of its circularization, the circular polynucleotide can include several features that distinguish it from linear RNA. For example, the circular polynucleotide, compared to linear RNA, can include several features that distinguish it from linear RNA. For example, the circular polynucleotide, compared to linear RNA, Moreover, it is less sensitive to degradation by exonuclease. Therefore, circular polyribonucleotides are more stable than linear RNA, especially when incubated in the presence of exonuclease. The increased stability of circular polyribonucleotides compared to linear RNA makes circular polyribonucleotides more useful as cell transformation reagents for producing polypeptides and more easily and longer storable than linear RNA. The stability of circular polyribonucleotides treated with exonuclease can be tested using standard methods in the art (e.g., by gel electrophoresis) to determine whether RNA degradation has occurred. Moreover, circular polyribonucleotides are more stable than linear RNA, especially when incubated in the presence of exonuclease. The increased stability of circular polyribonucleotides compared to linear RNA makes circular polyribonucleotides more useful as cell transformation reagents for producing polypeptides and more easily and longer storable than linear RNA. The stability of circular polyribonucleotides treated with exonuclease can be tested using standard methods in the art (e.g., by gel electrophoresis) to determine whether RNA degradation has occurred. Moreover, it is less sensitive to degradation by exonuclease. Therefore, circular polyribonucleotides are more stable than linear RNA, especially when incubated in the presence of exonuclease. The increased stability of circular polyribonucleotides compared to linear RNA makes circular polyribonucleotides more useful as cell transformation reagents for producing polypeptides and more easily and longer storable than linear RNA. The stability of circular polyribonucleotides treated with exonuclease can be tested using standard methods in the art (e.g., by gel electrophoresis) to determine whether RNA degradation has occurred. Moreover, it is less sensitive to degradation by exonuclease. Therefore, circular polyribonucleotides are more stable than linear RNA, especially when incubated in the presence of exonuclease.

[0274] Furthermore, unlike linear RNA, circular polyribonucleotides are less sensitive to dephosphorylation when incubated with phosphatases such as calf intestinal phosphatase. Furthermore, unlike linear RNA, circular polyribonucleotides are less sensitive to dephosphorylation when incubated with phosphatases such as calf intestinal phosphatase. Furthermore, unlike linear RNA, circular polyribonucleotides are less sensitive to dephosphorylation when incubated with phosphatases such as calf intestinal phosphatase.

[0275] Nucleotide spacer sequence In certain embodiments, the circular polyribonucleotide comprises a spacer sequence.

[0276] In certain embodiments, the circular polyribonucleotide comprises at least one spacer sequence. In certain embodiments, the circular polyribonucleotide comprises 1, 2, 3, 4, 5, 6, 7 or more spacer sequences.

[0277] In certain embodiments, the circular polyribonucleotide comprises one or more spacer sequences constructed according to the description in

[0295] -

[0302] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is incorporated herein by reference in its entirety. In certain embodiments, the circular polyribonucleotide comprises one or more spacer sequences constructed according to the description in

[0295] -

[0302] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is incorporated herein by reference in its entirety.

[0278] Non-nucleic acid linker The cyclic polynucleotides described herein may also include non-nucleic acid linkers. In a certain embodiment, the cyclic polynucleotides described herein have a non-nucleic acid linker between one or more of the sequences or elements described herein. In one embodiment, one or more of the sequences or elements described herein are linked using a linker. The non-nucleic acid linker can be a chemical bond, such as one or more covalent or non-covalent bonds. In a certain embodiment, the non-nucleic acid linker is a peptide or protein linker. Such a linker can be 2 to 30 amino acids or more. The linker includes flexible, rigid or cleavable linkers such as those described in

[0304] to

[0307] of the pamphlet of International Patent Publication No. WO 2019 / 118919 A1, which is incorporated herein by reference in its entirety.

[0279] Stability / Half-life In certain embodiments, the cyclic polynucleotide formulations provided herein have a longer half-life than a control, such as a linear polynucleotide having the same nucleotide sequence but not being cyclic (e.g., a linear equivalent). In certain embodiments, the cyclic polynucleotide is resistant to degradation, such as exonuclease. In certain embodiments, the cyclic polynucleotide is resistant to self-degradation. In certain embodiments, the cyclic polynucleotide lacks an enzyme cleavage site, such as a Dicer cleavage site. In certain embodiments, the cyclic polynucleotide is compared to a control, such as ​At least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 140%, at least about 150%, at least about 160 %, at least about 180%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000% or at least about 10000% longer half-life.

[0280] In certain embodiments, the circular polynucleotide persists intracellularly during cell division. In certain embodiments, the circular polynucleotide persists in daughter cells after mitosis. In certain embodiments, the circular polynucleotide is replicated intracellularly and sent to daughter cells. In certain embodiments, the circular polynucleotide comprises a replication element that mediates self-replication of the circular polynucleotide. In certain embodiments, the replication element mediates transcription of the circular polynucleotide into a linear polynucleotide (linear complementarity) that is complementary to the circular polynucleotide. In certain embodiments, the linear complementary polynucleotide can be circularized in vivo intracellularly to a complementary circular polynucleotide. In certain embodiments, the complementary polynucleotide can further self-replicate into another circular polynucleotide having the same or a similar nucleotide sequence as the starting circular polynucleotide. One exemplary self-replication element is the HDV replication domain. ​ (as described by Beeharry et al, Virol, 2014, 450 - 451:1 65 - 173). In certain embodiments, the cell is at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% efficient at delivering at least one circular polynucleotide to daughter cells. In certain embodiments, cells undergoing meiosis deliver circular polynucleotides to daughter cells with at least 25%, 50%, 60%, 70%, 80% 85%, 90%, 95% or 99% efficiency. In certain embodiments, cells undergoing mitosis deliver circular polynucleotides to daughter cells with at least 25%, 50%, 60% 70%, 80%, 85%, 90%, 95% or 99% efficiency.

[0281] Modifications Circular polynucleotides can include one or more substitutions, insertions and / or additions to, deletions and covalent modifications to a reference sequence, particularly where the parent polynucleotide is encompassed within the scope of the present invention.

[0282] In certain embodiments, circular polynucleotides include one or more post - transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly - A sequences, methylation, acetylation, phosphorylation, methylation of lysine and arginine residues, nitrosylation of thiol and tyrosine residues, etc.). The one or more post - transcriptional modifications can be any post - transcriptional modification such as any of the over 100 different nucleoside modifications identified in RNA (Rozenski, J, Crain, P, and McCloskey, J .(1999). The RNA Modification Database: 19 .(1999).The RNA Modification Database:19​ 99 update. Nucl Acids Res 27:196 - 197). In one embodiment, the first isolated nucleic acid comprises messenger RNA (mRNA). In one embodiment, the mRNA comprises at least one nucleoside selected from the group consisting of, for example, those described in [

[0311] ] of International Patent Publication No. WO 2019 / 118919 A1, which is incorporated herein by reference in its entirety.

[0283] Cyclic polynucleotides can include any useful modifications to sugars, nucleobases, or internucleoside linkages (e.g., to the linking phosphate / phosphodiester bond / phosphodiester backbone). One or more atoms of the pyrimidine nucleobase can optionally be replaced with an amino group that is optionally substituted, a thiol group that is optionally substituted, an alkyl group that is optionally substituted (e.g., methyl or ethyl), or a halo group (e.g., chloro or fluoro) or can be substituted. In certain embodiments, the modification (e.g., one or more modificat...

Claims

1. 1. A method of making a pharmaceutical product, comprising: a) providing a plurality of linear polyribonucleotide molecules; b) circularizing said plurality of linear polyribonucleotide molecules to provide a preparation of circular polyribonucleotide molecules comprising one or more expression sequences encoding an expression product; c) assessing the amount of circular polyribonucleotide molecules remaining in the formulation; d) treating said preparation of circular polyribonucleotide molecules as a pharmaceutical if said preparation of circular polyribonucleotide molecules meets a reference criterion of at least 91% (w / w) circular polyribonucleotide molecules relative to total ribonucleotide molecules in said preparation of circular polyribonucleotide molecules. The method includes:

2. The method of claim 1 , wherein the circularization step is carried out by splint ligation.

3. The method of claim 1 , wherein the circularization step is carried out by self-splicing.

4. 10. The method of claim 1, wherein the processing step comprises combining the preparation of circular polyribonucleotide molecules with a pharmaceutical excipient.

5. 10. The method of claim 1, wherein the reference standard further comprises at least 2 μg / mL of circular polyribonucleotide molecules in the formulation.

6. 2. The method of claim 1, wherein the reference standard further comprises no more than 5% (w / w) linear polyribonucleotide molecules relative to the total ribonucleotide molecules in the formulation.

7. The method of claim 1, wherein the reference standard further comprises 5% (w / w) or less of nicked molecules relative to the total ribonucleotide molecules in the preparation.

8. The reference standard is characterized in that the formulation is (i) being substantially free of DNA molecules; and / or (ii) containing less than 10 ng / ml of DNA molecules; 2. The method of claim 1, further comprising the amount of DNA molecules present in the formulation:

9. 2. The method of claim 1, wherein the reference standard further comprises an amount of protein contamination of less than 5 ng per milligram (mg) of circular polyribonucleotide molecule.

10. 10. The method of claim 1, wherein the amount of circular polyribonucleotide molecules in the formulation is measured by microscopy, spectrophotometric methods, fluorescence spectroscopy, denaturing urea polyacrylamide gel electrophoresis imaging, UV-Vis spectrophotometry, RNA electrophoresis, RNAse H analysis, UV spectrophotometric or fluorescence detectors, light scattering techniques, surface plasmon resonance (SPR) with or without the use of separation methods including HPLC, HPLC, chip or gel-based electrophoresis with or without pre- or post-separation derivatization methods, the use of detection methods using silver or dye stains or radioactive decay for detection of linear polyribonucleotide molecules, or by microscopy, visual methods or methods using a spectrophotometer.

11. The pharmaceutical product is (i) contains less than 10 EU / kg endotoxin or is endotoxin-free as measured by the Limulus amoebocyte lysate test; (ii) containing a bioburden of less than 100 CFU / 100 ml or less than 10 CFU / 100 ml prior to sterilization; (iii) is a sterile pharmaceutical product or drug; (iv) supports the growth of less than 100 viable microorganisms when tested under sterile conditions; (v) Meets USP <71> standards; (vi) Meets USP <85> standards; (vii) comprises an A260 / A280 absorbance ratio of about 1.6 to 2.3 as measured by a spectrophotometer; or (viii) being pyrogen-free; The method of claim 1.

12. The circular polyribonucleotide molecule is (i) one or more expressed sequences and a stagger element at the 3' end of at least one expressed sequence; (ii) one or more expression sequences encoding an expression product, which may be a therapeutic expression product, such as a therapeutic protein or a therapeutic nucleic acid; or (iii) a scaffold sequence, which may be an aptamer sequence; The method of claim 1 , comprising:

13. A pharmaceutical formulation of a circular polyribonucleotide molecule, said circular polyribonucleotide molecule comprising one or more expression sequences encoding an expression product, and wherein at least 91% (w / w) of the total ribonucleotide molecules in said pharmaceutical formulation are circular polyribonucleotide molecules.

14. The pharmaceutical formulation comprises: (i) contains less than 10 EU / kg endotoxin or is endotoxin-free as measured by the Limulus amoebocyte lysate test; (ii) containing a bioburden of less than 100 CFU / 100 ml or less than 10 CFU / 100 ml prior to sterilization; (iii) a sterile pharmaceutical preparation, wherein said pharmaceutical preparation may support the growth of less than 100 viable microorganisms when tested under sterile conditions; (iv) meets USP <71> standards; (v) Meets USP <85> standards; (vi) is an intermediate pharmaceutical formulation of a final drug product; (vii) is a final pharmaceutical product for administration to a subject; (viii) comprising a concentration of circular polyribonucleotide molecules of at least 0.1 ng / mL; (ix) containing less than 1 ng / ml of deoxyribonucleotide molecules; (x) containing less than 0.1 ng of protein contamination per milligram (mg) of said circular polyribonucleotide molecule; (xi) comprising an A260 / A280 absorbance ratio of about 1.6 to 2.3 as measured by a spectrophotometer; (xii) is substantially free of process-related impurities selected from cellular proteins, cellular deoxyribonucleic acid, enzymes, reagent components, gel components, or chromatographic materials; and / or (xiii) having a reduced level of one or more markers of an immune or inflammatory response after purification compared to before purification; 14. The pharmaceutical formulation of claim 13.

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