Thermostable ribonuclease r for circular RNA purification

Abstract

The present disclosure provides, among other things, methods for removing linear ribonucleic acid molecules from a sample by contacting the sample with ribonuclease R (RNase R) from a thermophilic organism, for example, Geobacillus stearothermophilus, Fervidobacterium pennivorans or Thermus thermophilus expressed and purified in E.coli. Also provided herein are methods of producing circular RNA and removing uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products using an RNase R expressed or purified from a thermophilic organism. The present disclosure also provides methods of purifying RNase R from a thermophilic organism in E. coll, wherein the method comprises heat treatment at high temperatures, and compositions and kits comprising the same.

Description

[0001] THERMOSTABLE RIBONUCLEASE R FOR CIRCULAR RNA PURIFICATION

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] [1] The present application claims priority to and the benefit of U.S. Provisional Application Number 63 / 659,011, filed on June 12, 2024, the contents of which are incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] [2] Circular RNA is a highly stable closed loop structure that, as compared to linear mRNA molecules, is more stable because its loop structure can resist exonucleolytic degradation. Circular RNA is therefore useful for RNA therapeutics and RNA vaccines.

[0006] [3] Ribonuclease R (RNase R) is a ubiquitous 3' exoribonuclease that can efficiently degrade linear RNAs, including highly structured RNAs. RNase R contains a nuclease as well as having RNA helicase activity that facilitates degradation of duplex RNA. For example, RNase R isolated from Escherichia coli effectively degrades linear RNA, Y- structure RNAs, but not lariat loops or circular RNAs. There is a need for methods to more efficiently and effectively manufacture and purify circular RNAs.

[0007] SUMMARY OF THE INVENTION

[0008] [4] The present disclosure provides, among other things, methods for removing linear ribonucleic acid (RNA) molecules from a sample by contacting the sample with ribonuclease R (RNase R) from a thermophilic organism, such as but not limited to Geobacillus stearothermophilus, Fervidobacterium pennivorans or Thermus thermophilus, expressed and purified in a non-thermophilic bacterium, e.g., E. coli. Also provided herein are methods of producing circular RNA and removing linear RNA molecules (e.g., uncircularized RNA, linear byproducts or linear side products) using an RNase R from a thermophilic organism recombinantly expressed and isolated or purified from E. coli. The present disclosure also provides methods of making RNase R from a thermophilic organism, wherein the method comprises heat treatment at a temperature between 72°C to 79°C, and compositions and kits comprising the same. [5] The present disclosure thus provides an improved RNase R, having beneficial features such as reduced endoribonuclease enzyme contamination (which would undesirably cleave internal to the circular RNA) and high activity and specificity at higher temperatures. Since thermophilic RNase R has activity at higher temperatures, the duration of enzyme reaction is reduced as excess RNase R enzyme can be added in the reaction without risk of endonucleolytic cleavage and degradation of circular RNA, leading to a more efficient process, reducing time and costs for manufacturing and purifying circular RNA. Further, at high temperatures, RNA is denatured, which aids in RNase R digestion of any highly structured regions. Lack of or reduced endoribonuclease contamination also provides the advantage of reducing or obviating the need to titrate RNase enzyme quantities carefully in a reaction, as unintended degradation is substantially reduced or abolished. Larger enzyme concentrations are also tolerated effectively, which reduces reaction time.

[0009] [6] Without wishing to be bound by any particular theory, RNase R from a thermophilic organism can be purified in E. coli to remove endoribonuclease contamination by heating to high temperatures during the isolation and purification, for example, between 72°C to 79°C since RNase R from a thermophilic bacterium is resistant to being heated to high temperatures. In contrast, stability and RNase activity of RNase R from E. coli (also referred to herein as “E. coli RNase R”) is reduced or abolished at high temperatures, for example, of between 72°C to 79°C. Further, in some embodiments, using Size Exclusion Chromatography provides an advantage in that lower molecular weight species were found to have high RNase R activity and specificity compared with high molecular weight species that exhibit endonucleolytic activity in addition to specific / exonucleolytic RNase R activity.

[0010] [7] During circular RNA manufacturing, RNase R from a thermophilic organism can also efficiently remove linear or uncircularized RNA at a high temperature used for circular RNA preparation reaction (e.g., 50 °C to 65 °C). This provides an advantage in using thermophilic RNase R having a wider thermal range over using E. coli RNase R, which functions between 37 °C-50 °C (e.g., optimally at 37°C).

[0011] [8] In some aspects, provided herein is a method for removing linear ribonucleic acid (RNA) molecules from a sample comprising contacting the sample comprising the linear RNA molecules with a recombinant Ribonuclease R (RNase R) from a thermophilic organism, thereby removing or reducing the linear RNA molecules in the sample.

[0012] [9] In some embodiments, the thermophilic organism is a thermophilic bacterium.

[0010] In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus.

[0013]

[0011] In some embodiments, the sample is a RNA sample.

[0014]

[0012] In some embodiments, the RNA sample comprises a circular RNA or a lariat RNA preparation sample.

[0015]

[0013] In some embodiments, at least over 85% of linear RNA molecules are removed from the sample.

[0016]

[0014] In some embodiments, at least over 90% of linear RNA molecules are removed from the sample.

[0017]

[0015] In some embodiments, at least over 95% of linear RNA molecules are removed from the sample.

[0018]

[0016] In some embodiments, at least over 99% of linear RNA molecules are removed from the sample.

[0019]

[0017] In some embodiments, the method is carried out at a temperature of 50°C to 65°C.

[0020]

[0018] In some embodiments, the sample is treated with the RNase R for less than 2 hours.

[0021]

[0019] In some embodiments, the sample is treated for about 1 hour.

[0022]

[0020] In some embodiments, the sample is treated for about 10 minutes, about 20 minutes or about 30 minutes.

[0023]

[0021] In some embodiments, the sample is treated with RNase R in the presence of about 0.1 mM -1 mM magnesium chloride (MgCh).

[0024]

[0022] In some embodiments, the sample is treated with RNase R in the presence of 0.2 mM -20 mM Tris buffer.

[0025]

[0023] In some embodiments, the sample is treated with RNase R in the presence of 100 mM to 1000 mM potassium chloride (KC1), sodium chloride (NaCl), or lithium chloride (LiCl).

[0026]

[0024] In some embodiments, the sample is treated with RNase R at a pH of between 6.5-8.

[0025] In some embodiments, the sample is treated with RNase R in the presence of 20 mM Tris-HCl at pH 8.0, 100 mM KC1, and 0.1 mM MgCk

[0027]

[0026] In some aspects, provided herein is a method for producing circular RNA comprising circularizing linear RNA molecules and removing the uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products using an RNase R isolated or purified from a thermophilic organism.

[0028]

[0027] In some embodiments, the thermophilic organism is a thermophilic bacterium.

[0029]

[0028] In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus.

[0030]

[0029] In some embodiments, the uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products are present in a circular RNA preparation.

[0031]

[0030] In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of between 50 °C to 65 °C.

[0032]

[0031] In some aspects, provided herein is a method of producing circular RNA, the method comprising:

[0033] (i) transcribing a nucleic acid molecule comprising a sequence of interest to produce a linear RNA molecule,

[0034] (ii) circularizing the linear RNA molecule, thereby producing a circular RNA molecule, and

[0035] (iii) removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products using an RNase R from a thermophilic organism.

[0036]

[0032] In some embodiments, the thermophilic organism is a thermophilic bacterium.

[0037]

[0033] In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus.

[0038]

[0034] In some embodiments, the RNase R is at a concentration of 0.1 - 10 pg / pl.

[0039]

[0035] In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of between 7.0 and 8.0.

[0040]

[0036] In some embodiments, the buffer comprises a pH of about 7.5.

[0037] In some embodiments, the buffer comprises 20 mM Tris-HCl at pH 8.0, 100 mM KC1, and 0.1 mM MgCl2

[0041]

[0038] In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in the absence of adenosine triphosphate (ATP).

[0042]

[0039] In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in less than two hours.

[0043]

[0040] In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in one hour.

[0044]

[0041] In some embodiments, provided herein is a purified circular RNA prepared according to the methods of the present invention.

[0045]

[0042] In some embodiments, provided herein is a composition comprising the purified circular RNA.

[0046]

[0043] In some aspects, provided herein is a method of purifying RNase R from a thermophilic organism recombinantly produced in E. coli, wherein the method comprises heat treatment at between 72°C to 79°C.

[0047]

[0044] In some embodiments, the method includes size exclusion chromatography.

[0048]

[0045] In some embodiments, the RNase R from a thermophilic organism has reduced endoribonuclease as compared to E. coli RNase R.

[0049]

[0046] In some embodiments, endoribonuclease activity is 0.1%, 1%, or 10% as compared to E.coli RNase R.

[0050]

[0047] In some embodiments, the thermophilic organism is a thermophilic bacterium.

[0051]

[0048] In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans and / or Thermus thermophilus.

[0052]

[0049] In some aspects, provided herein is a kit comprising a recombinant RNase R from a thermophilic bacterium, wherein the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans and / or Thermus thermophilus, and at least one buffer.

[0050] In some embodiments, the buffer comprises 20 mM Tris-HCl at pH 8.0, 100 mM KC1, and 0.1 mM MgCl2

[0053]

[0051] In some embodiments, the kit comprises RNase R isolated from Geobacillus stearothermophilus .

[0054]

[0052] In this application, the use of “or” means “and / or” unless stated otherwise. As used in this disclosure, the term “comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps. As used in this application, the terms “about” and “approximately” are used as equivalents. Both terms are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.

[0055]

[0053] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, and are not limiting. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057]

[0054] The drawings are for illustration purposes only, and are not limiting.

[0058]

[0055] FIG. 1A shows a schematic of the RNase R purification process. FIG. IB shows gel images of overexpressed RNase R from exemplary mesophilic, piezophilic, psychrophilic and thermophilic organisms, and depicts subsequent purification and analytical steps. FIG. 1C shows gel images of nickel-nitrilotriacetic acid (Ni-NTA) purification of RNase R purified from Shewanella piezotolerans, Psychrobacter arcticus, Geobacillus stearothermophilus, and Escherichia coli.

[0059]

[0056] FIG. 2A shows gel images of linear and circular forms of RNA upon treatment with RNase R purified from Shewanella piezotolerans (SpRNR), Psychrobacter arcticus (PaRNR), Geobacillus stearothermophilus (GsRNR), and Escherichia coli (EcRNR). RNase R activity is denoted by digestion of linear RNA and disappearance or reduction of the linear RNA band, while for circular RNA, the band was intact.

[0057] FIG. 2B shows that exemplary RNase R from Bacillus subtilis and Bacillus [multispecies] was active, but exemplary RNase R purified from Synechocystis and Shewanella was not active, as seen from the presence of linear RNA.

[0060]

[0058] FIG. 3 and FIG. 4 showed gel images depicting that RNase R activity in RNase R purified from heat-treated soluble fractions of G. stearothermophilus was intact and comparable to fractions that were not heat-treated. FIG. 3 and FIG. 4 also showed that at very high temperatures, greater than about 79 °C, GsRNase R activity was inactivated. Heat treatment at 72 °C or 79 °C reduced endoribonuclease activity. Overnight incubation at 50 °C resulted in loss of RNA stability.

[0061]

[0059] FIG. 5A shows nickel-NTA purification of GsRNase R. FIG. 5B showed that subsequent purification using size exclusion chromatography (SEM) further reduced endoribonuclease contamination.

[0062]

[0060] FIG. 6A shows gel images that G. stearothermophilus RNase R retained activity up to 65 °C, as shown by the digestion of the upper linear RNA bands and retention of lower circular RNA bands. FIG. 6B shows gel images of G. stearothermophilus RNase R activity tested at 37 °C, 50 °C, 60 °C and 65 °C for 10 minutes, 30 minutes and 60 minutes, using IX and 10X concentrations of RNase R. In FIG. 6B, the gel images showed that RNase R digestion with a 10-fold higher concentration for 10 minutes was sufficient at temperatures of 50 °C and higher. As shown by the arrows in the gel images, when linear RNA and intron bands are not visible, this reflects that the linear RNA and introns had degraded. When the circular RNA band is visible, this reflects that the circular RNA had not degraded.

[0063] DEFINITIONS

[0064]

[0061] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0065]

[0062] Approximately or about'. As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0066]

[0063] Biologically active'. As used herein, the term “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent, e.g., circular RNA, that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.

[0067]

[0064] Delivery. As used herein, “delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, sequence, cargo, or payload. “Delivery” encompasses both local and systemic delivery. For example, delivery of RNA encompasses situations in which an RNA is delivered to a target tissue, and the protein encoded by the RNA is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”); and situations in which RNA is delivered to a target tissue, and the encoded protein is expressed and secreted into a patient’s circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery). In some embodiments, delivery is pulmonary delivery, e.g., comprising nebulization.

[0068]

[0065] DNA Nuclease: As used herein, “DNA nuclease” is an enzyme that catalyzes the cleavage of phosphodiester bonds. DNA nucleases play a role in DNA replication and various DNA repair processes, base excision repair, nucleotide excision repair, mismatch repair, and double strand break repair. Depending on whether a 5' or 3' end is required for substrate recognition and whether cleavage products are single or oligo nucleotides, DNA nucleases are classified as exonucleases and endonucleases. For example, self-cleaving ribozymes cleave RNAs endonucleolytically, while exonucleases cleave one nucleotide at a time from one end, either from 5' to 3' or from 3' to 52 Some nucleases have both exonuclease and endonuclease activities, for example, Flap endonuclease 1 (FEN1) has 5' to 3' exonuclease activity in addition to endonuclease activity, and Mrel, has both endonuclease and 3' to 5' exonuclease activities.

[0066] DNase I: As used herein, “DNase I” or “deoxyribonuclease I” refers to enzymes that cleave single or double-stranded DNA and require divalent metal ions to hydrolyze DNA yielding 3 -hydroxyl and 5 -phosphorylated products.

[0069]

[0067] Encode'. As used herein, the term “encode” or “encoding” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule. For example, DNA encodes RNA, and RNA encodes a polypeptide or protein.

[0070]

[0068] Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing (including self-splicing in the case of circular RNA), editing, 5' cap formation, and / or 3' end processing) (RNA expression); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein (protein expression).

[0071]

[0069] Functional'. As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which the biological molecule is characterized.

[0072]

[0070] Homology. As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules). In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two polynucleotide sequences. In accordance with the disclosure, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For example, in some embodiments, for polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids.

[0071] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;

[0073] Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to, those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0074]

[0072] Improve, increase, or reduce'. As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment or method described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment or method described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[0075]

[0073] Intron: As used herein, the term “intron” means in general a non-coding sequence of a DNA, or an RNA transcript. The term “intron” includes any sequence within a gene that is transcribed but removed by RNA splicing during maturation of RNA transcripts from pre-mRNA transcripts. Following transcription, new, immature strands of messenger RNA, called pre-mRNA, may contain, among other sequences, both introns and exons (exons are any DNA or RNA sequence encoding for protein). The pre-mRNA molecule goes through a modification process called splicing during which noncoding introns are cut out, and only coding exons remain. Splicing produces a mature messenger RNA molecule that is then translated into a protein. As used herein, “intron” also refers to any sequence within a precursor linear RNA that is spliced out during circularization to form circular RNA, such as through self-splicing of the intron (e.g., Group I or Group II introns in the linear mRNA that results in circular RNA. Self-splicing intron refers to introns that act as ribozymes to autocatalytically splice introns out from precursor RNA in the absence of any added protein or RNA. “Intron,” as used herein, can also refer to intron fragments.

[0076]

[0074] In Vitro'. As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0077]

[0075] In Vivo'. As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cellbased systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0076] Isolated'. As used herein, the term “isolated” refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by a person or under the direction of a person. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and / or entities should not include excipients (e.g., buffer, solvent, water, etc.).

[0078]

[0077] Linear RNA byproduct'. As used herein, the term “linear RNA byproduct” refers to a partial linear RNA product that is not desired but results from molecular fragments of nucleotides and / or reagents that are not incorporated into the circular RNA.

[0079]

[0078] Linear RNA side product: As used herein, the term “linear RNA side product” refers to a product that is formed from a competitive process that results in generation of linear RNA instead of circular RNA and could be reduced or suppressed by optimization of reaction conditions for production of circular RNA.

[0080]

[0079] Messenger RNA ( mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. Messenger RNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5’ to 3’ direction unless otherwise indicated.

[0080] Modified'. As used herein “modified” or, as appropriate, “modification” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. With respect to nucleic acid molecules (e.g., DNA and RNA), the modifications are A, G, C, U or T nucleotides. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moieties. With respect to polypeptides, the term “modification” refers to a modification as compared to the canonical set of 20 amino acids.

[0081]

[0081] miRNA sponge or microRNA sponge: As used herein, the term “miRNA sponge” or “microRNA sponge” refers to a circular polynucleotide comprising a singlestranded non-coding polynucleotide with repeat copies of at least one specific microRNA (miRNA) binding site to hold microRNA molecules of interest. The miRNA sponge acts as an artificial microRNA inhibitor that would decrease the cellular level of the microRNA of interest when expressed in a cell.

[0082]

[0082] miRNA Response Element (MRE): As used herein, the term “miRNA response element (MRE)” refers to a target site (i.e., a short nucleic acid fragment) that binds to a miRNA. In some embodiments, the circular polynucleotide comprise two or more MREs. The number of MREs in the circular polynucleotide is variable and relates to the length of the circular polynucleotide. As non-limiting examples, the circular polynucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more MREs. The multiple MREs may have the same nucleic acid sequences and bind to the same miRNA; or alternatively, the MREs have different nucleic acid sequences and bind to different miRNAs, such as 2, 3, 4, 5, or more different miRNAs.

[0083]

[0083] Nucleoside: As described herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or a pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). Five primary / canonical nucleobases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) are the fundamental units of nucleic acid molecules, in which adenine and guanine, referred to as purine bases, have a fused-ring skeletal structure derived of purine, while uracil and thymine, derived of pyrimidine, are referred to pyrimidine bases.

[0084]

[0084] Nucleotide: As described herein, “nucleotide” is defined as a nucleoside including a phosphate group or other backbone linkage (intemucleoside linkage).

[0085] Nucleic acid: As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double- stranded DNA and / or cDNA.

[0085] Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so- called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences that encode proteins and / or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5’ to 3’ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5’-N-phosphoramidite linkages). In some embodiments, the present invention is specifically directed to “unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery. In some embodiments, the nucleotides T and U are used interchangeably in sequence descriptions.

[0086]

[0086] Preadenylation'. As used herein, “preadenylation” or “preadenylated” refers to adenylation prior to ligation. In some embodiments, the 5' end is preadenylated. In some embodiments, 5' adenylated RNA (5' AppRNA) removes the need to dephosphorylate RNA prior to ligation and prevents undesirable ligation products. In some embodiments, a truncated KQ ligase ligates 3' end of ssRNA to 5' adenylated ssDNA or 5' adenylated ssRNA.

[0087]

[0087] Pharmaceutical composition'. As used herein, the term “pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

[0088]

[0088] RNA Integrity: As used herein, the term “RNA integrity” generally refers to the quality of RNA. In some embodiments, RNA integrity refers to the percentage of mRNA that is not degraded after a purification process (e.g., after the purification process described herein). mRNA integrity may be determined using methods particularly described herein, such as TAE Agarose gel electrophoresis or by SDS-PAGE with silver staining, or by methods well known in the art, for example, by RNA agarose gel electrophoresis (e.g., Ausubel et al., John Wiley & Sons, Inc., 1997, Current Protocols in Molecular Biology).

[0089]

[0089] Substantially. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0090]

[0090] Transcription'. As used herein, the term “transcription” refers to the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template.

[0091]

[0091] Translation'. As used herein, the term “translation” refers to the formation of a polypeptide molecule by a ribosome based upon an RNA template.

[0092] Treat and Prevent'. As used herein, the terms “treat” or “prevent” as well as words stemming therefrom do not necessarily imply 100% or complete treatment or prevention. Rather there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Also, “prevention” can encompass delaying the onset of the disease, symptom or condition thereof.

[0092]

[0093] Therapeutically effective amount'. As used herein, the term “therapeutically effective amount” means an amount effective, at dosages, frequency of administration and for duration of time necessary to achieve the desired results such that one or more symptoms or biomarkers is improved after treatment.

[0093] DETAILED DESCRIPTION

[0094]

[0094] Ribonuclease (RNase) is a type of enzyme that cleaves RNA sequences.

[0095] RNase enzymes have several applications in, for example, DNA isolation, mapping single base mutations in DNA or RNA, removal of RNA from protein preparations, and cDNA synthesis. Ribonuclease R (RNase R) is an exoribonuclease that belongs to the RNase II superfamily. RNase R, in particular, has applications in enrichment and purification of circular RNAs including, for example, enriching endogenous circular RNAs from total RNA preparations or circular RNAs generated from circularizing linear RNAs in vitro. The present invention provides, among other things, methods that result in a pure and efficient RNase R activity that is not contaminated with endoribonucleases.

[0096]

[0095] The present disclosure provides, in part, methods for removing linear ribonucleic acid (RNA) molecules from a sample by contacting the sample comprising linear RNA molecules with a recombinant ribonuclease R (RNase R) from a thermophilic organism, including as non-limiting examples, Geobacillus sterothermophilus, Fervidobacterium pennivorans or Thermus thermophilus, thereby removing or reducing the linear RNA molecules in the sample. In some embodiments, the linear RNA is mRNA.

[0097]

[0096] The present disclosure, also provides, in part, methods of producing circular RNA comprising circularizing linear RNA molecules and removing the uncircularized linear RNA molecules using an RNase R isolated or purified from a thermophilic organism.

[0098]

[0097] Also provided herein is a method of producing circular RNA, the method comprising: (i) transcribing a nucleic acid molecule comprising a sequence of interest to produce a linear RNA molecule,

[0099] (ii) circularizing the linear RNA molecule, thereby producing a circular RNA molecule, and

[0100] (iii) removing uncircularized linear RNA molecules using an RNase R from a thermophilic organism.

[0101]

[0098] Among other things, provided herein are methods of purifying RNase R from a thermophilic organism recombinantly produced in E.coli, wherein the method comprises heat treatment at between 72°C to 79°C, and compositions and kits comprising the same.

[0102]

[0099] In some aspects, provided herein is a kit comprising a recombinant RNase R from a thermophilic bacterium, wherein the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans and / or Thermus thermophilus, and at least one buffer.

[0103]

[0100] RNase R from a thermophilic organism isolated in E.coli has higher purity, e.g., due to lower endoribonuclease contamination and / or activity, than RNase R isolated from Escherichia coli (RNase R isolated from E. coli is also described herein as “E. coli RNase R”), which is not a thermophilic organism. Without wishing to be bound by any particular theory, reduced endoribonuclease contamination is due to a heating step during the isolation and purification to high temperatures, for example, between 72°C to 79°C, and by selecting RNase R of lower molecular weight as purified by size exclusion chromatography having higher activity. For example, 80 to 90 kDa for GsRNase R, which has a molecular weight of 86 kDa, has no endoribonuclease contamination and has high RNase R activity. Further, during circular RNA manufacturing, RNase R from a thermophilic organism recombinantly produced in E. coli can also efficiently remove linear or uncircularized RNA at a high temperature used for circular RNA preparation reaction (e.g., 50 °C to 65 °C). This purification process using RNase R from thermophilic organism provides an advantage over using E. coli RNase R, which functions optimally at a relatively lower temperature of 37 °C, and at that lower temperature requires longer reaction times. In addition, while current approaches in manufacturing circular RNA and purifying circular RNA using E.coli RNase R require the additional purification step be carried out at a reduced temperature, the present invention provides, among other things, a pure and efficient thermostable RNase R, which can be used at a single temperature for both manufacturing circular RNA as well as removing uncircularized RNA. Ribonuclease (RNase)

[0104]

[0101] Ribonucleases (RNases) are enzymes that digest RNA, and they are either exoribonucleases or endoribonucleases based on their mode of action. Exoribonucleases digest RNA molecules from either a 3' or 5' terminus, releasing nucleotide residues.

[0105] Exoribonucleases may act processively, z.e., by releasing residues continuously while moving along a substrate, or distributively, by dissociating and rebinding after one or a few catalytic events. Digestion may be complete or may stop due to the presence of secondary structure in the RNA.

[0106]

[0102] In bacteria, known exoribonucleases all initiate degradation at the 3' terminus of the RNA and release 5' mononucleotides. RNase R is an example of such a ribonuclease.

[0107]

[0103] Endoribonucleases are enzymes that cleave internal to the RNA, releasing RNA fragments of various sizes. Endoribonucleases may be highly specific, cleaving at only one or a few sites per RNA molecule, or nonspecific, cleaving throughout the RNA chain and leading to extensive degradation. Depending on the reaction mechanism of the RNase, cleavage can be on either side of the phosphodiester linkage. Endoribonuclease enzyme or activity in the RNase R of the present disclosure is undesirable owing to its ability to cleave circular RNA.

[0108] Ribonuclease R (RNase R)

[0109]

[0104] Ribonuclease R (RNase R) is a 3' to 5' exoribonuclease, belonging to the RNase II superfamily. It is highly effective in degrading RNA from the 3’ end, which is chemically unprotected. RNase R degrades linear RNAs, as well as double stranded RNAs with 3' overhangs of seven nucleotides or longer than seven nucleotides.

[0110]

[0105] RNase R cannot digest covalently closed circular RNA, lariat RNA, or double stranded RNA with short 3' overhangs that are less than seven nucleotides, and this property is used in enrichment and purification of circular RNA, lariat RNA or double- stranded RNA species with less than seven nucleotide overhangs. RNase R is highly conserved and degrades a variety of RNA species, including messenger RNA, non-coding RNA, transfer RNA and ribosomal RNA. In E. coli, RNase R is a 92 kD protein comprising two cold shock domains, an RNase catalytic domain, an SI domain and a basic domain. It comprises both a nuclease and a helicase activity, although RNase R from some thermophilic organisms may not have an intact helicase activity.

[0106] RNase R has several applications, such as in isolation and purification of circular RNA manufactured in vitro, enrichment and isolation or purification of endogenous circular RNA, e.g., enrichment and isolation of circular RNA arising from back-splicing or lariat RNAs produced by traditional splicing. RNase R degrades and removes linear RNAs from cellular or RNA extracts, allowing for enrichment, purification or identification of circular RNA species, including exonic circular RNAs or intronic lariat sequences through RNA sequencing, discovery or manipulation of alternative splicing events, as well as production of circular RNAs in vitro.

[0111]

[0107] The present disclosure provides, among other things, methods for linear RNA degradation, for example, in circular RNA manufacturing, as well as methods for expression and purification of RNase R from thermophilic species in E. coli, and compositions and kits comprising the same.

[0112] Organisms for RNase R isolation and purification

[0113]

[0108] Apart from E.coli, several RNase R from mesophilic, piezophilic, psychrophilic and thermophilic species are known that share amino acid sequence identity with E. coli.

[0114]

[0109] Mesophiles are organisms that have optimum growth conditions that require a moderate temperature from 20 °C to 45 °C (68 °F to 113 °F), for example, 37 °C (about

[0115] 99 °F). Mesophiles include bacteria, archaea, and fungi. Most bacteria and some archaea have RNase R homologs, for example, halophilic archaea H. volcanii RNase.

[0116]

[0110] Piezophiles are organisms that live under elevated hydrostatic pressure. While piezotolerant organisms only tolerate high pressures, piezophiles grow better under high pressures, and strict or obligate piezophiles require pressures above atmospheric pressure for growth.

[0117]

[0111] Organisms that prefer extreme environments are known as extremophiles. Extremophiles that prefer cold environments are termed psychrophilic, those preferring warmer temperatures are termed thermophilic or thermotropic and those thriving in extremely hot environments are referred to as hyperthermophilic.

[0118]

[0112] Thermophiles are organisms that grow at above 40 °C, and that have optimal growth temperatures between 50 °C and 55 °C (Gleeson et al., 2013). Obligate thermophiles have an absolute requirement of growth above 40 °C and facultative thermophiles, e.g., Anoxybacillus flavithermus and some strains of G. stearothermophilus, grow at 37 °C (Eijlander et al., 2019).

[0119]

[0113] Sequence identity of various mesophiles, piezophiles, psychrophiles and thermophiles is shown in Table 1 as compared to E. coli RNase R.

[0120] Table 1. RNase R homologs of RNase R from E.coli and percent identity to RNase R from E. coli

[0121]

[0114] The present disclosure, provides, among other things, RNase R from thermophilic organisms, for example, Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus, among others, recombinantly expressed and purified from E. coli for a variety of applications, for example, removal of linear RNA during production of circular RNA. Other applications include affinity RNA purification, generating cDNA, intronic screening of cDNA libraries, as well as RNA isolation of splicing intermediates, lariats and tRNA.

[0122]

[0115] The present disclosure provides alternative RNase R from thermophilic organisms with improved features and properties relative to E. coli RNase R, including high specificity, greater purity (e.g., less endoribonuclease contamination), and activity at higher reaction temperatures and shorter times during manufacturing of circular RNA.

[0123] Circular RNA

[0124]

[0116] As used herein, the term “circular RNA” or “circRNA” refers to an RNA that forms a circular structure through covalent or non-covalent bonds. The terms “circRNA” or “circular polyribonucleotide” or “circular RNA” are used interchangeably herein. In some embodiments, circRNAs are covalently closed, single stranded RNA molecules. A circular RNA can be produced by back- splicing of a linear precursor RNA, by chemical ligation and / or enzymatic ligation. Circular RNAs (circRNAs) can be endogenous or synthetic. Synthetically created and exogenously delivered circRNAs can be synthesized in vitro using self-splicing permuted introns (e.g., self-splicing Group I or Group II intron) from in vitro transcribed constructs. Unlike linear RNAs, circular RNAs are more resistant to the degradation by exonuclease and have a longer half-life than their corresponding linear counterparts. A circular RNA can be formed from an mRNA precursor that encodes a polypeptide of interest (e.g., an immunogen, a therapeutic polypeptide, including, e.g., a chimeric antigen receptor or a T cell receptor).

[0125]

[0117] Endogenous and synthetic circular RNAs (circRNAs) are closed loop singlestranded RNAs. Unlike linear RNAs which interact with various cellular proteins through their 5' and 3' ends, circular RNAs lack this capacity and are more stable and conserved than linear RNA. Circular RNAs have a variety of protein-coding and non-coding functions, for example, among other things, they function as miRNA sponges to regulate miRNA expression and mRNA expression, transcriptional regulation by binding RNA binding proteins, protein translocation between cytosol and nucleus, protein-protein interactions, and enhancing protein translation. In some embodiments, circular RNAs comprise one or more RNA sequences encoding one or more polypeptides. In other embodiments, circular RNAs comprises one or more non-coding RNA sequences. In some embodiments, circular RNAs comprises one or more coding sequences and non-coding sequences.

[0118] In some aspects, since circRNAs contain miRNA binding sites, they can function as miRNA sponges to regulate miRNA expression, eliminating miRNA-mediated repression of linear RNA transcripts and promoting mRNA expression. For example, circ- ITCH binds to miR-17 and miR-224 to act as a miRNA sponge, inhibiting bladder cancer through regulation of p21 and PTEN 31, circCCDC9 binds to miR-6792-3p inhibiting the development of gastric cancer through regulation of CAVE In some embodiments, circular RNA binds to miRNA promoting development of cancer, for example, circSDHC binds to miR-127-3p to CDKN3 / E2F1 to promote the development of kidney cancer and circ-TFRC combined with miR-107 promotes the development of bladder cancer.

[0126]

[0119] In some aspects, circular RNAs can regulate transcription by binding RNA- binding proteins (RBPs), e.g., circRNA formed by the insulin gene binds an RNA binding protein, TDP-43 and regulates insulin secretion, circPABPN 1 binds an RNA binding protein, HuR, thereby reducing binding of HuR to PABPN 1 mRNA, and inhibiting translation of PABPN1.

[0127]

[0120] In some aspects, circular RNAs form three dimensional structures by interacting with other proteins and regulate signaling. For example, circFoxo3, p53 and MDM2 forms a complex and promotes p53 ubiquitination and degradation, while a complex of circFoxo3, Foxo3 and MDM2 protects Foxo3 from degradation.

[0128]

[0121] In some aspects, circular RNAs function in translocating proteins, for example, circFoxo3 can bind multiple proteins such as ID-1, E2F1, HIFla and FAK and promote their retention in cytoplasm. In contrast, circAmotll binds c-myc and facilitates its translocation to the nucleus, improving stability and target binding. CircAmotll binds and promotes STAT3 translocation to the nucleus.

[0129]

[0122] In some aspects, circular RNAs comprising modifications, for example, m6A modifications, or IRES (modified and engineered IRESs) can be translated. CircRNAs containing multiple ORFs without stop codons can be translated by rolling circle translation. For example, circMbl3 and circ-ZNF609 can be translated into proteins, CircAXINl encodes AXIN 1 protein and promotes gastric cancer.

[0130]

[0123] In some aspects, the circular RNA to be purified may be of any length, e.g., between 100 base pair (bp) to 10 kilobase (kb) in length. In some embodiments, the circular RNA is between 100 bp to 500 bp in length. In some embodiments, the circular RNA is between 500 bp to 10 kb in length.

[0124] The present disclosure provides, among other things, a method of making circular RNA and removing uncircularized or linear RNA. Synthetic circular RNA is more stable than linear RNA since the lack of 5' and 3' ends protect circular RNA from exonuclease degradation. Further, circular RNA can be translated for sustained periods both in vitro and in vivo. Compared to linear RNA, circular RNAs are more stable and so are less likely to be degraded during storage or transportation at temperatures higher than ultra-low temperatures, such as -80 °C or -20 °C. Accordingly, circular RNA is valuable for various therapeutic uses, including as circular RNA vaccines and as therapeutics for various diseases / conditions where increased stability of the RNA increases efficacy of the treatment. The present disclosure provides a much-needed method for manufacturing circular RNA with increased purity and decreased linear RNA contamination for use in circular RNA therapeutics and vaccines.

[0131] Linear RNA

[0132]

[0125] Linear RNA is used as a precursor to generate circularized RNA. After circularization, the remaining linear RNA is a contaminant of circular RNA preparations. To purify circular RNA, RNase R from thermophilic organisms of the present disclosure is used to remove linear RNAs.

[0133]

[0126] In some embodiments, the linear RNA is chemically synthesized. In some embodiments, RNA synthesis is carried out in synthesizer machines using nucleotide triphosphate derivatives known as phosphoramidites, which are building blocks of linear oligonucleotides. Nucleoside phosphoramidites use inert substituents to protect reactive moieties such as hydroxyl and amino groups from undesirable reactions and promote phosphodiester bond formation leading to greater homogenous yields. Once synthesis is complete, these groups are removed to generate RNA oligonucleotides of high purity. However, these are typically small RNAs, of up to 70-80 nucleotides in length, beyond which the method is associated with low yields and high costs.

[0134]

[0127] In some embodiments, the linear RNA is in vitro transcribed RNA. An in vitro transcription (IVT) reaction typically comprises a double- stranded DNA (dsDNA) template, ribonucleotide triphosphates, and a DNA-dependent RNA polymerase. In some embodiments, the DNA-dependent RNA polymerase is derived from bacteriophage. In some embodiments, the DNA-dependent RNA polymerase is a T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase, or variants thereof.

[0128] The DNA template contains a promoter sequence to which the polymerase binds and catalyzes downstream transcription. In some embodiments, the promoter is about 20 nucleotides to 40 nucleotides long. In some embodiments, the DNA template is a doublestranded PCR product. In some embodiments, the DNA template is a linearized plasmid containing a promoter upstream of the DNA sequence to be transcribed. Nucleotide triphosphates used during in vitro transcription (IVT) result in a single-stranded RNA containing guanine nucleotide triphosphate (GTP) at its 5' end. Since enzymatic intramolecular ligation of RNA requires a 5' monophosphate, an excess molar ratio of GMP to GTP is used during the IVT reaction.

[0135]

[0129] In vitro transcription may be carried out in the presence of GMP, for example, in some embodiments, there is 5-fold excess GMP as compared to GTP.

[0136]

[0130] If GTP alone was used to prime transcription, monophosphorylation is achieved by treatment of the RNA with a phosphatase followed by a kinase, or by treatment with a pyrophosphatase. For example, RppH converts triphosphate to monophosphate.

[0137]

[0131] Enzymatic ligation of a monophosphorylated RNA is achieved using a DNA ligase or an RNA ligase. Exemplary ligases include T4 DNA ligase, T4 RNA ligase 1, and T4 RNA ligase 2. A DNA splint, or, in some embodiments, an RNA splint, is used to bring the terminal ends of the RNA together. Compared to chemical synthesis, enzymatic synthesis allows for the generation of much larger linear RNAs (kb in length).

[0138]

[0132] In some embodiments, the linear RNA is messenger RNA. In some embodiments, the linear RNA encodes one or more polypeptide(s), comprises one or more modified nucleotides and / or does not comprise an intron.

[0139] RNase R degradation of linear RNA

[0140]

[0133] In some aspects, provided herein are methods for removing linear ribonucleic acid (RNA) molecules from a sample comprising contacting the sample comprising the linear RNA molecules with a Ribonuclease R (RNase R) from a thermophilic organism expressed and isolated and / or purified in E. coli, thereby removing or reducing the linear RNA molecules in the sample.

[0141]

[0134] In some embodiments, the thermophilic organism is a thermophilic bacterium.

[0142]

[0135] In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus. In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus. In some embodiments, the thermophilic bacterium is Fervidobacterium pennivorans . In some embodiments, the thermophilic bacterium is Thermus thermophilus.

[0143]

[0136] In some embodiments, the sample is a RNA sample.

[0144]

[0137] In some embodiments, the RNA sample comprises a circular RNA or a lariat RNA preparation sample.

[0145]

[0138] In some embodiments, at least over 85% of linear RNA molecules are removed from the sample. In some embodiments, at least over 90% of linear RNA molecules are removed from the sample. In some embodiments, at least over 95% of linear RNA molecules are removed from the sample. In some embodiments, at least over 99% of linear RNA molecules are removed from the sample. In some embodiments, 100% of linear RNA molecules are removed from the sample.

[0146]

[0139] In some embodiments, the method is carried out at a temperature of 50 °C to 65 °C. In some embodiments, the method is carried out at a temperature of 50 °C. In some embodiments, the method is carried out at a temperature of 51 °C. In some embodiments, the method is carried out at a temperature of 52 °C. In some embodiments, the method is carried out at a temperature of 53 °C. In some embodiments, the method is carried out at a temperature of 54 °C. In some embodiments, the method is carried out at a temperature of 55 °C. In some embodiments, the method is carried out at a temperature of 56 °C. In some embodiments, the method is carried out at a temperature of 57 °C. In some embodiments, the method is carried out at a temperature of 58 °C. In some embodiments, the method is carried out at a temperature of 59 °C. In some embodiments, the method is carried out at a temperature of 60 °C. In some embodiments, the method is carried out at a temperature of 61 °C. In some embodiments, the method is carried out at a temperature of 62 °C. In some embodiments, the method is carried out at a temperature of 63 °C. In some embodiments, the method is carried out at a temperature of 64 °C. In some embodiments, the method is carried out at a temperature of 65 °C.

[0147]

[0140] In some embodiments, the sample is treated with the RNase R for less than 2 hours. In some embodiments, the sample is treated for about 1 hour and 45 minutes. In some embodiment, the same is treated for about 1 hour and 30 minutes. In some embodiment, the same is treated for about 1 hour and 15 minutes. In some embodiments, the sample is treated for about 1 hour.

[0141] In some embodiments, the sample is treated for about 10 minutes, about 20 minutes or about 30 minutes. In some embodiments, the sample is treated for about 10 minutes. In some embodiments, the sample is treated for about 20 minutes. In some embodiments, the sample is treated for about 30 minutes.

[0148]

[0142] In some embodiments, the sample is treated with RNase R in the presence of about 0.1 mM to 1 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.1 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.2 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.3 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.4 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.5 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.6 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.7 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.8 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 0.9 mM MgCh. In some embodiments, the sample is treated with RNase R in the presence of about 1 mM MgCh.

[0149]

[0143] In some embodiments, the sample is treated with RNase R in the presence of 0.2 mM to 20 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 0.2 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 0.3 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 0.4 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 0.5 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 0.6 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 0.7 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 0.8 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 0.9 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 1 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 2 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 3 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 4 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 5 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 6 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 7 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 8 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 9 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 10 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 11 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 12 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 13 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 14 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 15 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 16 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 17 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 18 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 19 mM Tris buffer. In some embodiments, the sample is treated with RNase R in the presence of 20 mM Tris buffer.

[0150]

[0144] In some embodiments, the sample is treated with RNase R in the presence of 100 mM to 1000 mM KC1, NaCl, or LiCl.

[0151]

[0145] In some embodiments, the sample is treated with RNase R in the presence of

[0152] 100 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0153] 200 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0154] 300 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0155] 400 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0156] 500 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0157] 600 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0158] 700 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0159] 800 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0160] 900 mM KC1. In some embodiments, the sample is treated with RNase R in the presence of

[0161] 1000 mM KC1.

[0162]

[0146] In some embodiments, the sample is treated with RNase R in the presence of

[0163] 100 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of

[0164] 200 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of

[0165] 300 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of 400 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of

[0166] 500 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of

[0167] 600 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of

[0168] 700 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of

[0169] 800 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of

[0170] 900 mM NaCl. In some embodiments, the sample is treated with RNase R in the presence of

[0171] 1000 mM NaCl.

[0172]

[0147] In some embodiments, the sample is treated with RNase R in the presence of 100 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 200 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 300 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 400 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 500 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 600 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 700 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 800 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 900 mM LiCl. In some embodiments, the sample is treated with RNase R in the presence of 1000 mM LiCl.

[0173]

[0148] In some embodiments, the sample is treated with RNase R at a pH of between 6.5 to 8. In some embodiments, the sample is treated with RNase R at a pH of 6.5. In some embodiments, the sample is treated with RNase R at a pH of 7. In some embodiments, the sample is treated with RNase R at a pH of 7.5. In some embodiments, the sample is treated with RNase R at a pH of 8.

[0174]

[0149] In some embodiments, the sample is treated with RNase R in the presence of 20 mM Tris-HCl at pH 8.0, 100 mM KC1, and 0.1 mM MgCl2.

[0175]

[0150] Various other aspects of the invention are further described below.

[0176] Production of Circular RNA

[0177]

[0151] In some aspects, provided herein is a method for producing circular RNA comprising circularizing linear RNA molecules using one of various methods, and then removing the uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products using an RNase R from a thermophilic organism expressed and isolated and / or purified in E. coli.

[0152] Methods used to circularize linear RNA molecules include any methods known in for generating circular RNA, including but not limited to, T4 RNA / DNA ligase ligation, splint ligation, ribozyme or intron-based methods, including using self-splicing introns and permuted intron-exon (PIE) constructs.

[0178]

[0153] For example, ligases used for circularization are either T4 DNA ligase or T4 RNA ligases 1 and 2. Circular RNAs can be produced by direct ligation of 5' and 3' ends of linear RNAs, as intermediates in RNA processing reactions, or by “backsplicing,” wherein a downstream 5' splice site (splice donor) is joined to an upstream 3' splice site (splice acceptor).

[0179]

[0154] For example, a method of making circular RNA comprises: (i) contacting a linear RNA comprising a 5' end and a 3' end with a RNA ligase and a DNA splint comprising a region of homology to the 5' end of the linear RNA and a region of homology to the 3' end of the linear RNA, wherein the DNA splint binds the linear RNA and the ligase joins the 5' end and the 3' end; and (ii) removing the DNA splint; thereby generating circular RNA.

[0180]

[0155] Further, a method of making circular RNA comprises contacting two linear RNA molecules, each comprising a 5' end and a 3' end with a RNA ligase, and two DNA splints, wherein the first DNA splint comprises two regions of homology to the 3' end of the first linear RNA and to the 5' end of the second linear RNA, wherein the second DNA splint comprises two regions of homology to the 3' end of the second linear RNA and to the 5' end of the first linear RNA, such that the two DNA splints bind the linear RNA molecules and the ligase joins the ends of the linear RNA molecules; and (ii) removing the DNA splints; thereby generating a circular RNA.

[0181]

[0156] RNase R from thermophilic organisms of the present disclosure specifically removes linear RNA from circular RNA species, having low endoribonuclease enzyme and high specificity as well as activity at high temperatures during circular RNA manufacturing.

[0182]

[0157] In some embodiments, the thermophilic organism is a thermophilic bacterium.

[0183]

[0158] In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus. In some embodiments, the thermophilic bacterium is Geobacillus sterothermophilus . In some embodiments, the thermophilic bacterium is Fervidobacterium pennivorans . In some embodiments, the thermophilic bacterium is Thermus thermophilus.

[0159] In some embodiments, the uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products are present in a circular RNA preparation.

[0184]

[0160] In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of between 15°C to 65 °C, and every temperature in between those ranges. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 15 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 20 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 25 °C.

[0185]

[0161] In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 30 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 35 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 37 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA is performed at a temperature of 40 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 45 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 50 °C.

[0186]

[0162] In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of between 50°C-65 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 50 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 55 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 60 °C. In some embodiments, the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts or linear RNA side products is performed at a temperature of 65 °C.

[0187]

[0163] In some aspects, provided herein is a method of producing circular RNA, the method comprising: (i) transcribing a nucleic acid molecule comprising a sequence of interest to produce a linear RNA molecule, (ii) circularizing the linear RNA molecule, thereby producing a circular RNA molecule, and (iii) removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products using an RNase R from a thermophilic organism.

[0188]

[0164] In some embodiments, the thermophilic organism is a thermophilic bacterium.

[0189]

[0165] In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus. In some embodiments, the thermophilic bacterium is Geobacillus sterothermophilus. In some embodiments, the thermophilic bacterium is Fervidobacterium pennivorans . In some embodiments, the thermophilic bacterium is Thermus thermophilus.

[0190]

[0166] In some embodiments, the RNase R is at a concentration of 0.1 pg / pl to 10 pg / pl. In some embodiments, the RNase R is at a concentration of 0.1 pg / pl. In some embodiments, the RNase R is at a concentration of 0.2 pg / pl. In some embodiments, the RNase R is at a concentration of 0.3 pg / pl. In some embodiments, the RNase R is at a concentration of 0.4 pg / pl. In some embodiments, the RNase R is at a concentration of 0.5 pg / pl. In some embodiments, the RNase R is at a concentration of 0.6 pg / pl. In some embodiments, the RNase R is at a concentration of 0.7 pg / pl. In some embodiments, the RNase R is at a concentration of 0.8 pg / pl. In some embodiments, the RNase R is at a concentration of 0.9 pg / pl. In some embodiments, the RNase R is at a concentration of 1 pg / pl. In some embodiments, the RNase R is at a concentration of 2 pg / pl. In some embodiments, the RNase R is at a concentration of 3 pg / pl. In some embodiments, the RNase R is at a concentration of 4 pg / pl. In some embodiments, the RNase R is at a concentration of 5 pg / pl. In some embodiments, the RNase R is at a concentration of 6 pg / pl. In some embodiments, the RNase R is at a concentration of 7 pg / pl. In some embodiments, the RNase R is at a concentration of 8 pg / pl. In some embodiments, the RNase R is at a concentration of 9 pg / pl. In some embodiments, the RNase R is at a concentration of 10 pg / pl.

[0167] In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of between 7.0 and 8.0. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.0. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.1. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.2. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.3. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.4. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.5. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.6. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.7. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.8. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 7.9. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products occurs in a buffer comprising a pH of 8.0.

[0191]

[0168] In some embodiments, the buffer comprises a pH of about 7.5.

[0192]

[0169] In some embodiments, the buffer comprises 20 mM Tris-HCl at pH 8.0, 100 mM KC1 and 0.1 mM MgCh

[0193]

[0170] In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in the absence of adenosine triphosphate (ATP).

[0171] In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in less than two hours. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in one hour and 45 minutes. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in one hour and 30 minutes. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in one hour and 15 minutes. In some embodiments, the step of removing uncircularized linear RNA molecules, linear RNA byproducts or linear RNA side products is carried out in one hour.

[0194]

[0172] In some embodiments, provided herein is a purified circular RNA prepared according to the methods of the present disclosure.

[0195]

[0173] In some embodiments, provided herein is a composition comprising the purified circular RNA of the present disclosure.

[0196]

[0174] In some embodiments, the RNase R is at a concentration of between 0.1 pg / pl to 10 pg / pl. In some embodiments, the RNase R is at a concentration of 0.1 pg / pl. In some embodiments, the RNase R is at a concentration of 0.2 pg / pl. In some embodiments, the RNase R is at a concentration of 0.3 pg / pl. In some embodiments, the RNase R is at a concentration of 0.4 pg / pl. In some embodiments, the RNase R is at a concentration of 0.5 pg / pl. In some embodiments, the RNase R is at a concentration of 0.6 pg / pl. In some embodiments, the RNase R is at a concentration of 0.7 pg / pl. In some embodiments, the RNase R is at a concentration of 0.9 pg / pl. In some embodiments, the RNase R is at a concentration of 1.0 pg / pl.

[0197]

[0175] In some embodiments, the RNase R is at a concentration of 1 pg / pl. In some embodiments, the RNase R is at a concentration of 2 pg / pl. In some embodiments, the RNase R is at a concentration of 3 pg / pl. In some embodiments, the RNase R is at a concentration of 4 pg / pl. In some embodiments, the RNase R is at a concentration of 5 pg / pl. In some embodiments, the RNase R is at a concentration of 6 pg / pl. In some embodiments, the RNase R is at a concentration of 7 pg / pl. In some embodiments, the RNase R is at a concentration of 8 pg / pl. In some embodiments, the RNase R is at a concentration of 9 pg / pl. In some embodiments, the RNase R is at a concentration of 10 pg / pl.

[0198] Expression and Purification of RNase Rfrom thermophilic organisms in E. coli

[0176] In a standard or conventional purification method for purification of RNase R from E.coli and other non-thermophilic organisms, RNase R is synthetically generated or cloned with a purification tag, and expressed recombinantly in E. coli. After cell lysis, it is then isolated using standard affinity purification methods, e.g., Nickel-nitrilotriacetic acid (Ni-NTA), Strep-Tactin®, etc.

[0199]

[0177] In the present disclosure, RNase R from thermophilic organisms is synthetically generated or cloned with a purification tag, and expressed recombinantly in E. coli. In the present disclosure, after cells are centrifuged and sonicated, the cells are subject to heat treatment at high temperatures owing to the resistance of thermophilic RNase R to high temperatures. Contaminating enzymes from .S’, coli, including endoribonucleases are degraded by this heat treatment. In some embodiments, cells are heated from about 72 C to 79 °C. In some embodiments, cells are heated to about 72 °C. After cell lysis, RNase R is then isolated using standard affinity purification methods, e.g., Nickel-nitrilotriacetic acid (Ni- NTA), Strep-Tactin®, etc.

[0200]

[0178] For example, in some aspects, RNase R from thermophilic organisms is purified as described in the schematic shown in FIG. 1A.

[0201]

[0179] Briefly, proteins can be purified under native, denaturing, or hybrid conditions using, for example, Ni-NTA Agarose purification. Tagged recombinant thermophilic RNase R, for example, with a 6xHis tag (a HHHHHH tag), is purified with high affinity and selectivity in a Ni-NTA column. Ni-NTA agarose is a high-binding, high-capacity nickel charged Immobilized Metal Affinity Chromatography (IMAC) resin that purifies recombinant proteins with the polyhistidine (6XHis) sequence. Proteins (e.g. RNase R) bound to the resin is then eluted with either low pH buffer or by competition with imidazole or histidine.

[0202]

[0180] In some embodiments, Strep-Tactin® columns are also used in the purification process. Strep-Tactin® specifically interacts with commercially available Strep-tag® II as well as Twin-Strep-tag® via an engineered biotin binding pocket with affinity in the pM range for Strep-tag®II and nM range for Twin-Strep-tag® and high specificity. Elution of RNase R is performed by the addition of desthiobiotin, which is a specific competitor that releases tagged Strep-Tactin® from the engineered biotin binding pocket without altering properties of the RNase R. Further, desthiobiotin can optionally be removed via dialysis or gel chromatography. After elution with desthiobiotin, Strep-Tactin® resins can also be regenerated with HABA (2-[4'-hydroxy-benzeneazo]benzoic acid), which is a compound that displaces desthiobiotin from the binding pocket and changes to a red color once it binds to the binding site, indicating the activity status of the resin.

[0203]

[0181] Conventional purification methods using E. coli RNase R uses Ni-NTA and / or Strep-Tactin® columns (e.g., FIG. 2A shows RNase R elution from Ni-NTA and Strep- Tactin® columns). In contrast, in some embodiments of the current disclosure, the methods of purifying using thermophilic RNase R also include a heat treatment prior to an affinity column, e.g., Ni-NTA column.

[0204]

[0182] In some aspects, provided herein is a method of purifying RNase R from a thermophilic organism in E. coli, wherein the method comprises heat treatment at between 72 °C to 79 °C. In some embodiments, the method comprises heat treatment at 72 °C. In some embodiments, the method comprises heat treatment at 73 °C. In some embodiments, the method comprises heat treatment at 74 °C. In some embodiments, the method comprises heat treatment at 75 °C. In some embodiments, the method comprises heat treatment at 76 °C. In some embodiments, the method comprises heat treatment at 77 °C. In some embodiments, the method comprises heat treatment at 78 °C. In some embodiments, the method comprises heat treatment at 79 °C.

[0205]

[0183] In some embodiments, the method comprises heat treatment for 10 minutes. In some embodiments, the method comprises heat treatment for 20 minutes. In some embodiments, the method comprises heat treatment for 30 minutes. In some embodiments, the method comprises heat treatment for 40 minutes. In some embodiments, the method comprises heat treatment for 50 minutes. In some embodiments, the method comprises heat treatment for 60 minutes.

[0206]

[0184] In some embodiments, the method includes a step of using size exclusion chromatography. Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix. Accordingly, low molecular weight (LMW) species have more pores that are accessible to them and therefore spend more time inside the pores relative to high molecular weight species. Therefore, smaller molecules elute last and larger molecules elute first in Size Exclusion Chromatography. In some embodiments, SEC is used after Ni-NTA purification. In some embodiments, SEC is used in lieu of Strep- Tactin® purification.

[0207]

[0185] In some embodiments, the lower molecular weight RNase R species as purified by SEC have no endoribonuclease contamination and high RNase R activity. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of between 60 to 100 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of between 60 to 90 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of between 80 to 100 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of between 80 to 90 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 80 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 81 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 82 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 83 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 84 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 85 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 86 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 87 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 88 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 89 kDa. In some embodiments, GsRNase R having no endoribonuclease contamination and high activity has a molecular weight of about 90 kDa.

[0208]

[0186] In some embodiments, the RNase R from a thermophilic organism has reduced endoribonuclease as compared to E. coli RNase R. In some embodiments, the RNase R from a thermophilic organism has greater purity as compared to E. coli RNase R. In some embodiments, the RNase R from a thermophilic organism has greater specificity as compared to E. coli RNase R.

[0187] In some embodiments, endoribonuclease activity is 0.1%, 1%, or 10% as compared to E.coli RNase R. In some embodiments, endoribonuclease activity is 0.1% as compared to E.coli RNase R. In some embodiments, endoribonuclease activity is 1% as compared to E.coli RNase R. In some embodiments, endoribonuclease activity is 10% as compared to E.coli RNase R.

[0209]

[0188] In some embodiments, the RNase R from a thermophilic organism is a thermophilic bacterium.

[0210]

[0189] In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans and / or Thermus thermophilus. In some embodiments, the thermophilic bacterium is Geobacillus stearothermophilus. In some embodiments, the thermophilic bacterium is Fervidobacterium pennivorans . In some embodiments, the thermophilic bacterium is Thermus thermophilus.

[0211] Circular RNA Compositions F brmulated for Delivery

[0212]

[0190] In some embodiments, the circular RNA is formulated for delivery to a subject. In some embodiments, the circular RNA is formulated in lipid nanoparticles for delivery to a subject. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.

[0213]

[0191] In some embodiments, the circular RNA described herein is administered locally to a diseased site. In some embodiments, the circular RNA composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a silastic membrane, or a fiber.

[0214]

[0192] In other embodiments, the circular RNA composition described herein is delivered in a controlled release system. In one embodiment, a pump can be used (See, e.g., Langer, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71: 105.) Other controlled release systems are discussed, for example, in Langer, supra.

[0215]

[0193] In some embodiments, the circular RNA composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, the circular RNA composition in a RNA delivery system for administration by injection are solutions in sterile isotonic use as solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the circular RNA composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0216]

[0194] A circular RNA composition for systemic administration can be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in “stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al, Gene Ther. 1999, 6: 1438-47). Positively charged lipids such as N-[l-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl- amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g. , U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.

[0195] In some embodiments, the circular RNA composition is a pharmaceutical composition that can be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[0217]

[0196] Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent e.g., sterile used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0218]

[0197] In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and can have a sterile access port. For example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture can further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0219] Kits

[0220]

[0198] In one aspect, provided herein is a kit comprising a recombinant RNase R from a thermophilic bacterium, wherein the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans and / or Thermus thermophilus, and at least one buffer.

[0221]

[0199] In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer has a pH from about 6.5 to about 8.

[0222]

[0200] In some embodiments, the buffer comprises between about 0.1 mM to 1 mM MgCh. In some embodiments, the buffer comprises 0.1 mM MgCh. In some embodiments, the buffer comprises 0.2 mM MgCh. In some embodiments, the buffer comprises 0.3 mM MgCh. In some embodiments, the buffer comprises 0.4 mM MgCh. In some embodiments, the buffer comprises 0.5 mM MgCh. In some embodiments, the buffer comprises 0.6 mM MgCh. In some embodiments, the buffer comprises 0.7 mM MgCh. In some embodiments, the buffer comprises 0.8 mM MgCh. In some embodiments, the buffer comprises 0.9 mM MgCh. In some embodiments, the buffer comprises 1.0 mM MgCh.

[0223]

[0201] In some embodiments, the buffer comprises between about 0.2 mM to 20 mM Tris buffer. In some embodiments, the buffer comprises about 0.2 mM Tris buffer. In some embodiments, the buffer comprises about 0.3 mM Tris buffer. In some embodiments, the buffer comprises about 0.4 mM Tris buffer. In some embodiments, the buffer comprises about 0.5 mM Tris buffer. In some embodiments, the buffer comprises about 0.6 mM Tris buffer. In some embodiments, the buffer comprises about 0.7 mM Tris buffer. In some embodiments, the buffer comprises about 0.8 mM Tris buffer. In some embodiments, the buffer comprises about 0.9 mM Tris buffer. In some embodiments, the buffer comprises between about 1 mM Tris buffer.

[0224]

[0202] In some embodiments, the buffer comprises between about 100 mM to 1000 mM KC1, NaCl or LiCl.

[0225]

[0203] In some embodiments, the buffer comprises 100 mM KC1. In some embodiments, the buffer comprises 200 mM KC1. In some embodiments, the buffer comprises 300 mM KC1. In some embodiments, the buffer comprises 400 mM KC1. In some embodiments, the buffer comprises 500 mM KC1. In some embodiments, the buffer comprises 600 mM KC1. In some embodiments, the buffer comprises 700 mM KC1. In some embodiments, the buffer comprises 800 mM KC1. In some embodiments, the buffer comprises 900 mM KC1. In some embodiments, the buffer comprises 1000 mM KC1.

[0226]

[0204] In some embodiments, the buffer comprises 100 mM NaCl. In some embodiments, the buffer comprises 200 mM NaCl. In some embodiments, the buffer comprises 300 mM NaCl. In some embodiments, the buffer comprises 400 mM NaCl. In some embodiments, the buffer comprises 500 mM NaCl. In some embodiments, the buffer comprises 600 mM NaCl. In some embodiments, the buffer comprises 700 mM NaCl. In some embodiments, the buffer comprises 800 mM NaCl. In some embodiments, the buffer comprises 900 mM NaCl. In some embodiments, the buffer comprises 1000 mM NaCl.

[0227]

[0205] In some embodiments, the buffer comprises 100 mM LiCl. In some embodiments, the buffer comprises 200 mM LiCl. In some embodiments, the buffer comprises 300 mM LiCl. In some embodiments, the buffer comprises 400 mM LiCl. In some embodiments, the buffer comprises 500 mM LiCl. In some embodiments, the buffer comprises 600 mM LiCl. In some embodiments, the buffer comprises 700 mM LiCl. In some embodiments, the buffer comprises 800 mM LiCl. In some embodiments, the buffer comprises 900 mM LiCl. In some embodiments, the buffer comprises 1000 mM LiCl.

[0228]

[0206] In some embodiments, the buffer comprises 20 mM Tris-HCl at pH 8.0, 100 mM KC1 and 0.1 mM MgCL

[0229]

[0207] In some embodiments, the kit comprises RNase R from Geobacillus stearothermophilus .

[0230]

[0208] In some embodiments, the kit comprises RNase R from F ervidobacterium pennivorans .

[0231]

[0209] In some embodiments, the kit comprises RNase R from Thermus thermophilus.

[0232] Delivery of RN A Therapeutics

[0233]

[0211] In some embodiments, RNA based drug delivery is carried out through lipid nanoparticle (LNP) delivery. In some embodiments, delivery is carried out through virus-like particle (VLP) delivery. In either forms of delivery, circular RNA is formulated in LNPs or VLPs.

[0234]

[0212] In some embodiments, an RNA is formulated in lipid nanoparticles (LNPs). LNP components are selected based on the desired target, cargo (e.g., circRNA molecules), size, and / or other desired feature. LNP components include, for example, ionizable lipids, helper lipids, sterols, and / or PEG-lipids. The relative amounts, or molar ratios, of ionizable lipid, helper lipid, cholesterol, and PEG-lipid are optimized for a given target or administration route. In some embodiments, the LNPs do not contain a targeting ligand. In some embodiments, the LNPs contain a targeting ligand. In some embodiments, LNPs are used to target specific cells using endogenous or exogenous ligands by encapsulating circular RNA by methods known in the art. In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof that is conjugated to the surface of the engineered nanoparticle, such as a lipid nanoparticle. In some embodiments, the conjugation is via enzymatic or chemical methods. Endocytosis of LNPs destabilizes the endosomal membrane and release circular RNAs into the target cell cytoplasm.

[0235]

[0210] In some embodiments, VLPs are used to deliver RNA. Virus-like particles (VLPs) are protein complexes similar to native virus particles but that do not contain the viral genome so they cannot replicate but can mimic viral antigenicity without being pathogenic. VLPs consist of one or more capsid proteins in multiple copies and can occur naturally (e.g., poliovirus empty capsids outside of the cell), or be recombinantly produced by expressing the proteins required for VLP production. Some VLPs self-assemble from a single type of coat protein (e.g., LI of human papillomavirus), some VLPs require several structural proteins (e.g., bluetongue virus VLPs), or a combination of structural and non- structural proteins (e.g., poliovirus VLPs). Their repetitive surface structure and size of 20-200 nm make VLPs highly immunogenic, efficient at presenting foreign antigens, such as RNAs that can be loaded on the surface and capable of inducing a strong humoral and cellular immune response.

[0236]

[0211] In some embodiments, selective endogenous encapsidation for cellular delivery (SEND) is used which relies on a PEG 10 protein derived from a human retrovirus with the ability to package RNA and transport it in virus-like particles (Segel, M. et al. Science 373, 882-889 (2021).

[0237]

[0212] In some embodiments, gold nanoparticles (AuNPs) are used to deliver circular RNAs due to their high stability, purity, and easy surface modification.

[0213] In some embodiments, engineered exosomes are used to delivery circular RNAs. Exosomes are tiny vesicles smaller than 50 nm secreted by mature reticulocytes, that are associated with transferrin receptors and function in antigen presentation during the regulation of immune cells. In some embodiments, engineered exosomes act as cargo carriers and deliver small hydrophilic or lipophilic molecules, including some therapeutic drugs to cells, participating in the regulation of many major diseases. Exosomes can improve bioavailability of some drugs when taken orally, reducing the total dose required for administration, and minimizing side effects.

[0238]

[0214] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0239] EXAMPLES

[0240]

[0215] While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.

[0241] Example 1. Method of expressing and purifying recombinant RNase R derived from mesophilic and thermophilic organisms in Escherichia coli.

[0242]

[0216] This example illustrates methods of expressing and purifying recombinant RNase R from mesophilic and thermophilic organisms in E. coli.

[0243]

[0217] FIG. 1A shows a schematic of the RNase R purification process. Briefly, recombinant E. coli harboring constructs encoding RNase R from exemplary thermophilic organisms was grown in culture flasks and subsequently cells were spun down and sonicated. The supernatant was heated at 72 °C for 10 minutes. The heat treatment resulted in the denaturation and precipitation of a majority of E. coli proteins, while the RNase R from exemplary thermophilic organisms remained unaffected. Afterwards, centrifugation removed the denatured proteins. The supernatant, which contained the RNase R, was subjected to Ni- NTA purification and size-exclusion chromatography.

[0244]

[0218] FIG. IB shows gel images of overexpressed RNase R from exemplary mesophilic, piezophilic, psychrophilic and thermophilic organisms. FIG. IB is a gel showing that most homologs were expressed. Table 1 disclosed herein provides the RNaseR from thermophilic organisms tested in lanes 1-18, as compared to E. coli RNase R tested in lanes 19 and 20. Active RNase R was purified from mesophilic organisms, e.g., Bacillus subtilis (lane 1), Bacillus [multispecies] (lane 2), and E.coli (lane 19), and thermophilic organisms, e.g., Geobacillus stearothermophilus (lane 16). FIG. 1C shows gel images of Ni-NTA purification of RNase R from exemplary species, such as Bacillus subtilis (#1), Bacillus [multispecies] (#2), Synechocystis (#3), Shewanella piezotolerans (#7), Escherichia coli (#19), showing input supernatant of the sonicated cell extract, flowthrough and first elution of Ni-NTA.

[0245]

[0219] Overall, the data showed that recombinant RNase R derived from exemplary mesophilic and thermophilic organisms was successfully expressed in E. coli and purified by Nickel-NTA and size exclusion chromatography. Active RNase R was purified from mesophilic organisms, e.g., Bacillus subtilis, Bacillus [multispecies], and E. coli (lane 19), and an exemplary thermophilic organism, Geobacillus stearothermophilus.

[0246] Example 2. Activity of recombinant RNase R from mesophilic, psychrophilic and thermophilic organisms

[0247]

[0220] This example illustrates activity of recombinant RNase R derived from exemplary mesophilic, pyschrophilic and thermophilic organisms, expressed and purified from E. coli.

[0248]

[0221] RNase R purified as described in Example 1 using, for example, Ni-NTA purification and size exclusion chromatography was tested for RNase R activity on exemplary RNA samples comprising circular and linear RNA. Serial 10-fold dilutions of RNase R were carried out and tested at reaction temperatures of 20 °C, 37 °C, and 50 °C for RNase R activity.

[0249]

[0222] FIG. 2A shows gel images of linear and circular forms of RNA upon treatment with RNase R purified from Shewanella piezotolerans (SpRNR), Psychrobacter arcticus (PaRNR), Geobacillus stearothermophilus (GsRNR), and Escherichia coli (EcRNR). RNase R activity was denoted by digestion of linear RNA and disappearance or reduction of the linear RNA band, while circular RNA band was intact.

[0250]

[0223] FIG. 2B shows that upon digestion at 37 °C for 1 hour, RNase R from Bacillus subtilis and Bacillus [multispecies] was active, as seen from digestion of linear RNA band above the circular RNA band, while the circular RNA band remained intact; however, RNase R purified from Synechocystis and Shewanella was not active, as seen from the presence of linear RNA. The left panel shows a 4-12% SDS PAGE gel showing total protein.

[0251]

[0224] The results from FIG. 2A showed that G. stearothermophilus and E. coli RNase R were active. G. stearothermophilus RNR and E. coli RNR showed activity at both 37 °C and 50 °C.

[0252]

[0225] Overall, G. stearothermophilus RNase R recombinantly expressed and purified from E. coli showed activity at a temperature from 20°C to 50 °C, (and was also active at 65°C, data not shown). This data showed that G. stearothermophilus RNase R recombinantly expressed and purified from E. coli showed specificity for digesting linear RNA and not digesting circular RNA, which indicated efficiency for circular RNA manufacture and purification.

[0253] Example 3. Reduced endoribonuclease contamination using recombinant RNase R derived from thermophilic organisms

[0254]

[0226] This example describes methods for reducing endoribonuclease contamination using recombinant RNase R derived from thermophilic organisms.

[0255] Heat treatment

[0256]

[0227] Briefly, heat pre-treatment was carried out at temperatures between 65 °C to 100 °C on RNase R from G. stearothermophilus and E. coli prior to testing RNase R activity at 50 °C for 1 hour, 37 °C for 1 hour, or 37 °C for 21 hours. FIG. 3 shows gel images of RNase R activity in samples where the Ni-NTA purified RNase R was subjected to heat pretreatment in a last step of purification (See FIG. 1A) just prior to evaluating RNase R activity. FIG. 4 shows gel images of RNase R activity in samples that were subjected to heat pretreatment in an early step of purification (See FIG. 1A) after sonication of cells. The gel images in both FIG. 3 and FIG. 4 showed that RNase R activity in RNase R purified from heat-treated soluble fractions of G. stearothermophilus was intact and comparable to fractions that were not heat-treated. FIG. 3 and FIG. 4 also showed that at very high temperatures, greater than about 79 °C, GsRNase R was inactivated. Heat treatment at 72 °C or 79 °C reduced endoribonuclease activity. Further, overnight incubation at 50 °C resulted in loss of RNA stability.

[0257] Size exclusion chromatography

[0258]

[0228] Size Exclusion Chromatography (data shown in FIG. 5B) was carried out as a purification step following a Ni-NTA purification (data shown in FIG. 5A), instead of a Strep-Tactin® purification.

[0259]

[0229] RNase R activity was tested at 50 °C for 1 hour and 37 °C for 21 hours. FIG. 5B showed that size exclusion chromatography further reduced endoribonuclease contamination. For example, A9 and A10 fractions collected after size exclusion chromatography (Superdex 200 10 / 300 GL column) showed digestion of linear RNA, while circular RNA remained intact. Fractions A8 to A10 correspond to about 80-100 kDa. As shown in FIG. 5B, the higher molecular weight fraction A8 corresponding to about 100 kDa showed endoribonuclease contamination; however, the lower molecular weight fractions A9 and A10 corresponding to about 80-90 kDa showed no endoribonuclease contamination. Fractions A9 and A10 were pooled to obtain an enriched sample of endoribonuclease-free RNase R.

[0260]

[0230] Overall, the results showed that both heat treatment and size exclusion chromatography using GsRNase R preparations reduced endoribonuclease enzyme contamination and endoribonuclease activity as compared to using E.coli RNase R.

[0261] Example 4. G. stearothermophilus RNase R activity was retained at high temperatures

[0262]

[0231] Briefly, G. stearothermophilus RNase R was tested for activity at various exemplary temperatures ranging from 50 °C to 74 °C, as shown in FIG. 6A [(specifically, at 50 °C (column 1), 55.4 °C (column 2), 59.4 °C (column 3), 64.9 °C (column 4), 69.2 °C (column 5), or 74 °C (column 6)] for a reaction time of 1 hour.

[0263]

[0232] FIG. 6A shows gel images that G. stearothermophilus RNase R retained activity up to about 65 °C (column 4), as shown by the digestion of the upper linear RNA bands as well as lower intron bands and selective retention of circular RNA bands.

[0264]

[0233] Further, FIG. 6B showed G. stearothermophilus RNase activity tested at 37 °C, 50 °C, 60 °C and 65 °C for 10 minutes, 30 minutes and 60 minutes, using IX and 10X concentrations of RNase R. FIG. 6B shows gel images depicting that RNase R digestion with a 10-fold higher concentration for 10 minutes was sufficient at temperatures of 50 °C and higher. As shown by the arrows in the gel images, when linear RNA and intron bands were not visible, this reflected that the linear RNA and introns had degraded. When the circular RNA band was visible, this reflected that the circular RNA had not degraded.

[0265]

[0234] Overall, the results showed that a thermophilic RNase R, for example, G. stearothermophilus RNase R, had activity up to 65 °C, which confered various advantages compared to RNase R from thermophilic organisms over, for example, E. coli RNase R in that reactions using a thermophilic RNase R were carried out at relatively higher temperatures, which provided for shorter reaction times, as well as resulting in reduced endoribonuclease contamination. Thus, a thermophilic RNase R, for example, G. stearothermophilus RNase R, increased the efficiency of an RNase R reaction in various applications, for example, for purifying circular RNA. Further, enzyme amounts did not need to be carefully titrated for RNase R from a thermophilic RNase R, e.g., G. stearothermophilus, in contrast to RNase R from E. coli, as there was little to no risk of digestion of circular RNA by using excess enzyme or letting the reaction proceed for a longer time period.

[0266]

[0235] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0267] EQUIVALENTS

[0268]

[0236] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMSWe claim:

1. A method for removing linear ribonucleic acid (RNA) molecules from a sample comprising contacting the sample comprising the linear RNA molecules with a recombinant Ribonuclease R (RNase R) from a thermophilic organism, thereby removing or reducing the linear RNA molecules in the sample.

2. The method of claim 1, wherein the thermophilic organism is a thermophilic bacterium.

3. The method of claim 2, wherein the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus.

4. The method of claim 1, wherein the sample is a RNA sample.

5. The method of claim 4, wherein the RNA sample comprises a circular RNA or a lariat RNA preparation sample.

6. The method of any one of the preceding claims, wherein at least over 85% of linear RNA molecules are removed from the sample.

7. The method of any one of the preceding claims, wherein at least over 90% of linear RNA molecules are removed from the sample.

8. The method of any one of the preceding claims, wherein at least over 95% of linear RNA molecules are removed from the sample.

9. The method of any one of the preceding claims, wherein at least over 99% of linear RNA molecules are removed from the sample.

10. The method of any one of the preceding claims, wherein the method is carried out at a temperature between 50°C to 65°C.

11. The method of any one of the preceding claims, wherein the sample is treated with the RNase R for less than two hours.

12. The method of claim 11, wherein the sample is treated for about one hour.

13. The method of claim 12, wherein the sample is treated for about 10 minutes, about 20 minutes, or about 30 minutes.

14. The method of any one of the preceding claims, wherein the sample is treated with RNase R in the presence of about 0.1 mM to about 1 mM MgCh.

15. The method of any one of the preceding claims, wherein the sample is treated with RNase R in the presence of 0.2 mM to 20 mM Tris buffer.

16. The method of any one of the preceding claims, wherein the sample is treated with RNase R in the presence of 100 mM to 1000 mM KC1, NaCl and / or LiCl.

17. The method of any one of the preceding claims, wherein the sample is treated with RNase R at a pH of between 6.5 to 8.

18. The method of any one of the preceding claims, wherein the sample is treated with RNase R in the presence of 20 mM Tris-HCl at pH 8.0, 100 mM KC1, and 0.1 mM MgCl2.

19. A method for producing circular RNA comprising circularizing linear RNA molecules and removing the uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products using an RNase R isolated or purified from a thermophilic organism.

20. The method of claim 19, wherein the thermophilic organism is a thermophilic bacterium.

21. The method of claim 20, wherein the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus.

22. The method of any one of claims 1-21, wherein the uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products are present in a circular RNA preparation.

23. The method of any one of the preceding claims, wherein the production of circular RNA and the removal of uncircularized linear RNA, linear RNA byproducts, or linear RNA side products is performed at a temperature of between 50°C to 65°C.

24. A method of producing circular RNA, the method comprising:(i) transcribing a nucleic acid molecule comprising a sequence of interest to produce a linear RNA molecule,(ii) circularizing the linear RNA molecule, thereby producing a circular RNA molecule, and(iii) removing uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products using an RNase R from a thermophilic organism.

25. The method of claim 24, wherein the thermophilic organism is a thermophilic bacterium.

26. The method of claim 25, wherein the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus.

27. The method of any one of the preceding claims, wherein the RNase R is at a concentration of 0.1 pg / pl to 10 pg / pl.

28. The method of any one of the preceding claims, wherein the step of removing uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products occurs in a buffer comprising a pH of between 7.0 and 8.0.

29. The method of claim 28, wherein the buffer comprises a pH of about 7.5.

30. The method of claims 28 or 29, wherein the buffer comprises 20 mM Tris-HCl at pH 8.0, 100 mM KC1, and 0.1 mM MgCk31. The method of any one of the preceding claims, wherein the step of removing uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products is carried out in the absence of adenosine triphosphate (ATP).

32. The method of any one of the preceding claims, wherein the step of removing uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products is carried out in less than two hours.

33. The method of any one of the preceding claims, wherein the step of removing uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products is carried out in about one hour.

34. A purified circular RNA prepared according to any one of the preceding claims.

35. A composition comprising the purified circular RNA of claim 34.

36. A method of purifying RNase R from a thermophilic organism in E. coli, wherein the method comprises heat treatment at between 72°C to 79°C.

37. The method of claim 36, wherein the method further comprises using size exclusion chromatography .

38. The method of claims 36 or 37, wherein the RNase R from a thermophilic organism has reduced endoribonuclease as compared to RNase R from E. coli.

39. The method of claim 38, wherein endoribonuclease activity is 0.1%, 1%, or 10% as compared to RNase R from E.coli.

40. The method of any one of claims 36-39, wherein the thermophilic organism is a thermophilic bacterium.

41. The method of claim 40, wherein the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans, and / or Thermus thermophilus.

42. A kit comprising a recombinant RNase R from a thermophilic bacterium, wherein the thermophilic bacterium is Geobacillus stearothermophilus, Fervidobacterium pennivorans and / or Thermus thermophilus, and at least one buffer.

43. The kit of claim 42, wherein the buffer comprises 20 mM Tris-HCl at pH 8.0, 100 mM KC1, and 0.1 mM MgCl2.

44. The kit of claim 42 or 43, wherein the kit comprises RNase R from Geobacillus stearothermophilus.

Images