Novel RNA construct and a method of preparing circular RNA using the same

Abstract

Provided is an RNA construct for producing a circular RNA of the present disclosure. A circular RNA with high stability and translation persistence is produced, thereby being applied to the production of various vaccines, therapeutic agents, etc.

Description

NOVEL RNA CONSTRUCT AND A METHOD OF PREPARING CIRCULAR RNA USING THE SAME

[0001] The present disclosure relates to a novel circular RNA construct for producing a circular RNA and a method of producing a circular RNA using the same.

[0002]

[0003] RNA is susceptible to degradation in the body and has low persistence, and thus it was considered to have limitations in developing as a therapeutic agent until recently. However, starting with the development of mRNA vaccines against COVID-19, research on vaccines and therapeutic agents using mRNA is actively being conducted. Since RNA can encode all types of proteins, it has the advantages of solving the problem of protein targeting, enabling rapid candidate identification, and lowering concerns about impurities during the production process.

[0004]

[0005] However, despite the rapidity and effectiveness of mRNA, mRNA has a limitation of having a short half-life due to degradation by nucleases, making it difficult to expect its sustained effect. To overcome this limitation, techniques such as capping, poly A tail modification, etc. have been studied to stabilize RNA.

[0006]

[0007] As another method, a method of developing circular RNA to increase persistence has recently emerged. Circular RNA has a structure in which both ends are linked to each other, making it resistant to nucleases, and thus it is expected to have the advantage of inducing protein expression in the body for a long time.

[0008]

[0009] Technologies for producing circular RNA are known to broadly comprise chemical methods of using cyanogen bromide or a similar condensing agent, enzymatic methods of using RNA or DNA ligases, or methods of performing RNA circularization using self-splicing intron ribozymes.

[0010] For example, there are known RNA circularization induced by comprising homology arms at both ends (WO 2019-236673 A1), and self-circularization RNA constructs, in which self-circularization reactions occur through self-targeting and splicing reactions (KR 10-2442946), etc.

[0011]

[0012] In particular, when ribozymes are used, it is expected that circular RNAs may be produced relatively easily at a reasonable cost. However, a technology capable of universally and easily producing circular RNAs has not yet been developed. Since the demand for RNA-based vaccines and therapeutics is expected to gradually expand, it is necessary to develop a method capable of efficiently producing circular RNAs.

[0013]

[0014] A technology capable of universally and easily producing circular RNAs has not yet been developed. There is still a need to develop a method capable of efficiently producing circular RNAs.

[0015]

[0016] An object of the present disclosure is to provide an RNA construct for producing a circular RNA, the RNA construct comprising a first module, an internal ribosome entry site (IRES), a protein coding region, and a second module.

[0017] Another object of the present disclosure is to provide a composition or kit for producing a circular RNA, the composition or kit comprising the first module and the second module.

[0018] Still another object of the present disclosure is to provide a composition or kit for producing a circular RNA, the composition or kit comprising the RNA construct.

[0019] Still another object of the present disclosure is to provide a method of producing a circular RNA, the method comprising the step of producing the circular RNA from the RNA construct throughin vitrotranscription.

[0020] Still another object of the present disclosure is to provide a circular RNA produced from the RNA construct.

[0021] Still another object of the present disclosure is to provide a vector comprising the RNA construct or circular RNA.

[0022] Still another object of the present disclosure is to provide a drug delivery system comprising the RNA construct or circular RNA.

[0023] An RNA construct comprising, at both ends, modules that are different from each other according to the present disclosure may produce a circular RNA by forming a dimer with each other and inducing self-splicing. The circular RNA produced by the RNA construct of the present disclosure is expected to have a long-term effect in the body because of its high stability, and on this basis, it may be applied to the production of various vaccines and therapeutic agents, etc.

[0024]

[0025] FIG.　1 illustrates a structure of an RNA construct derived fromTetrahymena thermophilaaccording to the present disclosure and the process of producing a circular RNA therefrom;

[0026] FIG.　2 is a diagram verifying splicing efficiency according to the length of the P6 domain of a module derived fromTetrahymena thermophilaof the present disclosure;

[0027] FIG.　3 is a diagram verifying whether or not a circular RNA is produced when changing a gene of interest (GOI) which is comprised in the RNA construct derived fromTetrahymena thermophilaof the present disclosure;

[0028] FIG.　4 is a diagram verifying the junction sequence of the circular RNA which is produced by the RNA construct derived fromTetrahymena thermophilaof the present disclosure;

[0029] FIG.　5 shows the results of purifying the circular RNA which is produced by the RNA construct derived fromTetrahymena thermophilaof the present disclosure (FIG.　5A: affinity chromatography, FIG.　5B: ion reversed-phase chromatography);

[0030] FIG.　6 shows translation efficiency and persistence of the circular RNA which is produced by the RNA construct derived fromTetrahymena thermophilaof the present disclosure;

[0031] FIG.　7 is a diagram showing a module derived fromAzoarcusaccording to the present disclosure;

[0032] FIG.　8 is a diagram showing a structure of an RNA construct derived fromAzoarcusaccording to the present disclosure;

[0033] FIG.　9 is a diagram showing the synthesis of a circular RNA by the RNA construct derived fromAzoarcusaccording to the present disclosure;

[0034] FIG.　10 is a diagram showing the circular RNA synthesis efficiency by changing the GC content and length of the P6 domain of the RNA construct derived fromAzoarcusaccording to the present disclosure;

[0035] FIG.　11 is a diagram verifying the junction sequence of the circular RNA which is produced by the RNA construct derived fromAzoarcusaccording to the present disclosure;

[0036] FIG.　12 is a diagram verifying circularization efficiency according to the GOI of the RNA construct derived fromAzoarcusaccording to the present disclosure; and

[0037] FIG.　13 is a diagram verifying the translation efficiency and persistence of the circular RNA which is produced by the RNA construct derived fromAzoarcusaccording to the present disclosure.

[0038]

[0039] One aspect of the present disclosure provides an RNA construct for producing a circular RNA, the RNA construct comprising a first module, an internal ribosome entry site (IRES), a protein coding region, and a second module.

[0040] In one specific embodiment, the first module and the second module are characterized by being a combination of domains derived from Group I introns that are different from each other.

[0041] In another specific embodiment, the Group I introns are characterized by being Group I introns derived fromTetrahymena thermophilaorAzoarcus.

[0042] In a specific embodiment according to any one of the previous specific embodiments, the Group I introns are characterized by comprising any one sequence of sequences of SEQ ID NOS: 1 to 3, and 22 to 24.

[0043] In a specific embodiment according to any one of the previous specific embodiments, the first module and the second module are characterized by having 55% or less base pairing with each other.

[0044] In a specific embodiment according to any one of the previous specific embodiments, the first module is characterized by comprising P1 to P6b domains of a first Group I intron and P6b to P9 domains of a second Group I intron, and the second module is characterized by comprising P1 to P6b domains of the second Group I intron and P6b to P9 domains of the first Group I intron.

[0045] In a specific embodiment according to any one of the previous specific embodiments, the first module is characterized by comprising P1 to P6a domains of the first Group I intron and P6a to P9 domains of the second Group I intron, and the second module is characterized by comprising P1 to P6a domains of the second Group I intron and P6a to P9 domains of the first Group I intron.

[0046] In a specific embodiment according to any one of the previous specific embodiments, the first module is characterized by comprising a nucleotide sequence of SEQ ID NO: 17.

[0047] In a specific embodiment according to any one of the previous specific embodiments, the second module is characterized by comprising a nucleotide sequence of SEQ ID NO: 18.

[0048] In a specific embodiment according to any one of the previous specific embodiments, the first module is characterized by comprising a nucleotide sequence of SEQ ID NO: 31.

[0049] In a specific embodiment according to any one of the previous specific embodiments, the second module is characterized by comprising a nucleotide sequence of SEQ ID NO: 32.

[0050] In a specific embodiment according to any one of the previous specific embodiments, the first module is characterized by comprising the nucleotide sequence of SEQ ID NO: 17, and the second module is characterized by comprising the nucleotide sequence of SEQ ID NO: 18.

[0051] In a specific embodiment according to any one of the previous specific embodiments, the first module is characterized by comprising the nucleotide sequence of SEQ ID NO: 31, and the second module is characterized by comprising the nucleotide sequence of SEQ ID NO: 32.

[0052] In a specific embodiment according to any one of the previous specific embodiments, the RNA construct is characterized by further comprising a spacer or a translation enhancer.

[0053] In a specific embodiment according to any one of the previous specific embodiments, the RNA construct is characterized in that the first module and the second module form a heterodimer to function as a ribozyme.

[0054] In a specific embodiment according to any one of the previous specific embodiments, the length of the P6 domain of the RNA construct that forms the heterodimer is characterized by 10 nt to 80 nt.

[0055] In a specific embodiment according to any one of the previous specific embodiments, the GC content of the P6 domain of the RNA construct that forms the heterodimer is characterized by 40% to 85%.

[0056] In a specific embodiment according to any one of the previous specific embodiments, the RNA construct is characterized by not comprising homology arms at both ends.

[0057] In a specific embodiment according to any one of the previous specific embodiments, the internal ribosome entry site is characterized by being derived fromEncephalomyocarditis virus(ECMV),CoxsackievirusB3 (CVB3), orEnterovirus(EV-D94).

[0058] Another aspect of the present disclosure provides a composition for producing a circular RNA, the composition comprising the first module and the second module.

[0059] Still another aspect of the present disclosure provides a kit for producing a circular RNA, the kit comprising the first module and the second module.

[0060] Still another aspect of the present disclosure provides a composition for producing a circular RNA, the composition comprising the RNA construct.

[0061] Still another aspect of the present disclosure provides a method of producing a circular RNA, the method comprising the step of producing the circular RNA from the RNA construct throughin vitrotranscription.

[0062] In a specific embodiment, the method is characterized by further comprising the step of producing the circular RNA through self-splicing as the first module and the second module form a heterodimer.

[0063] In another specific embodiment, the method is characterized by further comprising the step of separating and / or purifying the produced circular RNA.

[0064] Still another aspect of the present disclosure provides a method of producing a circular RNA, the method comprising the step of expressing the RNA construct in an individual or a cell.

[0065] Still another aspect of the present disclosure provides a circular RNA produced by the RNA construct, the composition, or the method.

[0066] Still another aspect of the present disclosure provides a vector comprising the RNA construct or the circular RNA.

[0067] Still another aspect of the present disclosure provides a drug delivery system comprising the circular RNA or the RNA construct.

[0068]

[0069] The specific details for carrying out the present disclosure are as follows.

[0070] Meanwhile, each description and embodiment disclosed in this disclosure may also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in this disclosure fall within the scope of the present disclosure. Further, the scope of the present disclosure is not limited by the specific description described below.

[0071] Further, 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 disclosure described herein. Further, these equivalents should be interpreted to fall within the present disclosure.

[0072]

[0073] One aspect of the present disclosure provides an RNA construct for producing a circular RNA.

[0074] Specifically, the present disclosure provides an RNA construct capable of producing a circular RNA by inducing splicing using two modules derived from Group I intron, which are different from each other.

[0075] A specific embodiment of the present disclosure is, but not limited thereto, an RNA construct comprising a first module, an internal ribosome entry site (IRES), a protein coding region, and a second module. The RNA construct may further comprise a spacer comprising a splicing site, and / or a translation enhancer for enhancing the translation of a target protein, but is not limited thereto.

[0076]

[0077] As used herein, the term "circular RNA" refers to a single-stranded RNA in which the 3'-end and the 5'-end are connected to each other and have a circular structure. Circular RNA exhibits resistance to nucleases due to its circular structure, thereby having the advantage of high stability, as compared to existing linear RNA.

[0078] The circular RNA of the present disclosure may comprise an internal ribosome entry site (IRES), and a protein coding region, and may further comprise one or more spacer sequences and translation enhancers, etc. The circular RNA of the present disclosure may express a target protein in the body to obtain a desired effect from the protein (e.g., disease treatment, etc.).

[0079] The circular RNA of the present disclosure may be used in expressing and producing a protein to be used as a vaccine or therapeutic agent, and the use thereof may be determined according to the sequence comprised in the protein coding region. However, with respect to the object of the present disclosure, which is to provide a method of producing a circular RNA having high stability and long-lasting protein translation ability, and to provide a circular RNA produced thereby, the circular RNA is not limited to a specific use as long as it is a circular RNA having a closed loop structure.

[0080]

[0081] The circular RNA of the present disclosure is produced through splicing and circularization reactions by the RNA construct of the present disclosure, and specifically, the circular RNA may be produced and separated from the first module and the second module of the RNA construct. The circularization reaction may occur byin vitrotranscription (IVT) or in a cell.

[0082] In one embodiment of the present disclosure, it was confirmed that the circular RNA produced by the RNA construct of the present disclosure may exhibit protein translation ability for a longer time than existing linear RNA and circular RNA, which is advantageous for producing a target protein.

[0083]

[0084] The circular RNA of the present disclosure may comprise unmodified nucleotides, or may be in a modified form, wherein the modification refers to a modification in at least one of the position, pattern, ratio, or group with regard to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides. The modification may refer to, for example, chemical modifications comprising pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, or 2'-O-methyl uridine, etc. but is not limited thereto.

[0085]

[0086] The circular RNA of the present disclosure may comprise about 100 nt to about 30,000 nt, about 100 nt to about 25,000 nt, about 100 nt to about 20,000 nt, about 100 nt to about 15,000 nt, about 100 nt to about 10,000 nt, about 100 nt to about 5,000 nt, about 200 nt to about 4,000 nt, or about 300 nt to about 3,500 nt, but is not limited thereto, and a person skilled in the art may determine an appropriate length by considering the length of the target protein, structural stability, transcription and translation efficiency, etc.

[0087]

[0088] As used herein, the term "RNA construct" may comprise a module capable of inducing splicing, a protein coding sequence for expressing a target protein, and a nucleotide sequence that aids efficient circularization reaction and protein translation.

[0089] Specifically, the RNA construct of the present disclosure may comprise, but is not limited to, the first module, the internal ribosome entry site (IRES), the protein coding region, and the second module in sequence from the 5'-end.

[0090]

[0091] The RNA construct of the present disclosure may be produced through synthesis or throughin vitrotranscription (IVT) from a DNA template, but is not limited thereto.

[0092] The RNA construct of the present disclosure may be used in producing a circular RNA through induction of splicing and self-circularization. Furthermore, the RNA construct of the present disclosure may be used in producing functional RNAs, such as miRNA, anti-miRNA, shRNA, aptamer, CAR-T mRNA, in a circular form.

[0093]

[0094] The RNA construct of the present disclosure comprises two modules derived from Group I intron at both ends, which are different from each other, and the two modules are characterized in that they are not active alone, but when comprised in the RNA construct to form a heterodimer with each other, they become active as ribozymes of Group I intron.

[0095] Specifically, the RNA construct of the present disclosure performs splicing through two trans-esterification reactions, similar to Group I intron, one of which occurs in the modules at both ends, and the other in the upstream region of the internal ribosome entry site and the downstream region of the protein coding region within the RNA construct, and formation of the RNA construct undergoes the following process.

[0096] First, as the first module and the second module form a heterodimer, the domains of each module are formed and activated, and cleavage occurs in the exon region, which is the upstream region of the internal ribosome entry site connected to the first module by external guanosine triphosphate (GTP). Next, as P10 helix is formed, a structural change of the module occurs, and cleavage occurs in the downstream region of the protein coding region connected to the second module, and then the 5'- and 3'-ends of the two cleaved base sequences may be connected to form a circular RNA.

[0097]

[0098] In particular, the RNA construct of the present disclosure is characterized in that homology arms do not need to be comprised at both ends because splicing is induced by formation of a dimer by the above modules.

[0099]

[0100] As described, when a circular RNA is produced using the RNA construct comprising two modules capable of forming a dimer, there are advantages in that the circular RNA may be synthesized using modules different from each other, and the introduction of large-sized genes is also possible.

[0101]

[0102] A specific example of the RNA construct of the present disclosure may comprise an RNA construct comprising the first module, the internal ribosome entry site, the protein coding region, and the second module, and optionally, the RNA construct may further comprise a spacer, and / or a translation enhancer, etc., but is not limited thereto.

[0103]

[0104] As used herein, the term "Group I intron" is a term that may be used interchangeably with "Group I intron ribozyme", and refers to a self-splicing ribozyme through cleavage and ligation. The Group I intron is known to have the activity of trans-splicing an RNA precursor in two steps, and is known to consist of P1 to P9 domains. The domains form a substrate domain, a scaffold domain, and a catalytic domain, and it is known that splicing occurs while cleaving the phosphodiester bond at the P1 site in the substrate domain.

[0105]

[0106] The Group I intron of the present disclosure may be derived from various organisms, for example, all Group I introns derived fromTetrahymena thermophila, T4 phage,Anabaena,Azoarcus,Pneumocystis carinii,Didymium, etc., but is not limited thereto. Specifically, it may be a Group I intron derived fromTetrahymena thermophilaorAzoarcus, but is not limited thereto.

[0107] The Group I introns derived fromTetrahymena thermophilaandAzoarcusare found in various types of tRNA, mRNA, and rRNA precursors, and are used in various ways in RNA-related research.

[0108] Specifically, the Group I intron derived fromTetrahymena thermophilamay comprise a nucleotide sequence of SEQ ID NO: 1. Alternatively, a Group I intron having a change of some sequence in the Group I intron of SEQ ID NO: 1 may be used. The change of some sequence may comprise, but is not limited to, substitution, insertion, deletion, or modification of nucleotides, or a combination thereof.

[0109] For example, Group I intron variants of SEQ ID NO: 2 and 3, which have change of some sequence from the Group I intron of wild-typeTetrahymena thermophila, may be exemplified, but the Group I intron is not limited to a specific sequence as long as it may function as a Group I intron.

[0110] For another example, the Group I intron derived fromAzoarcusmay comprise a nucleotide sequence of SEQ ID NO: 22. Alternatively, a Group I intron having change of some sequence in the Group I intron of SEQ ID NO: 22 may be used. The change of some sequence may comprise, but is not limited to, substitution, insertion, deletion, or modification of nucleotides, or a combination thereof.

[0111] As a specific example, Group I intron variants of SEQ ID NO: 23 and 24, which have change of some sequence from the Group I intron of wild-typeAzoarcus, may be exemplified, but the Group I intron is not limited to a specific sequence as long as it may function as a Group I intron.

[0112] In particular, the module derived fromAzoarcushas a length of 424 nt, which is smaller in size than the module derived fromTetrahymena thermophilahaving a length of 890 nt, and therefore, it is expected to be highly useful in circular RNA production.

[0113]

[0114] In one embodiment of the present disclosure, domains of two different Group I introns are combined to produce two modules which do not have ribozyme activity alone but may function as ribozymes by forming a dimer, thereby constructing an RNA construct comprising the same at both ends.

[0115]

[0116] As used herein, the terms "first module" and "second module" refer to the components comprised at both ends of the RNA construct of the present disclosure, which have a structure comprising a substrate domain, a scaffold domain, and a catalytic domain, similar to the Group I intron, but which do not undergo splicing with only one module.

[0117] As an example of the first module and the second module according to the present disclosure, the first module and the second module may be a combination of domains derived from different Group I introns, or a hybrid thereof, but are not limited thereto.

[0118] Specifically, the first module and the second module may comprise a hybrid of any one or more domains of P1 to P10 domains that are comprised in different Group I introns, but are not limited thereto.

[0119] In this regard, the Group I intron may be a natural type or Group I intron in which some sequence of the natural type is modified by insertion, substitution, deletion, modification, or a combination thereof, but is not limited thereto. The modification of some sequence may be a modification of a sequence within a scaffold domain of the Group I intron, but is not limited thereto. The modification of the sequence is advantageous in terms of increasing circularization efficiency.

[0120]

[0121] For example, one or more domains may be selected from the first Group I intron, and one or more domains may be selected from the second Group I intron (which does not have a sequence 100% identical to that of the first Group I intron), and then they may be combined. A domain may be selected from third, fourth, or more Group I introns that are different from the first Group I intron and the second Group I intron.

[0122] In this regard, the selected domains may be consecutive domains or non-consecutive domains, but are not limited thereto. The selected domains may be combined to form the first module and the second module, each comprising a substrate domain, a scaffold domain, and a catalytic domain, but are not limited thereto.

[0123] With respect to the objects of the present disclosure, the first module and the second module may be referred to as hybrid modules.

[0124]

[0125] The first module and the second module of the present disclosure may have the same or different lengths, specifically, each module may have a length of 100 nt to 1000 nt, 150 nt to 600 nt, 200 nt to 500 nt, but is not limited to a specific length as long as the first module and the second module form a heterodimer to exhibit activity as a Group I intron ribozyme.

[0126]

[0127] The first module and the second module, which comprise a combination of domains derived from different Group I introns, may have low base pairing, homology or identity of, specifically, about 60% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51% or less, 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 40% or less, 30% or less and 1% or more, but are not limited thereto.

[0128]

[0129] As used herein, the term "about" comprises all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and comprises all numerical values of the ranges equivalent to or similar to the numerical value following the term "about", but is not limited thereto.

[0130]

[0131] As described above, the RNA construct of the present disclosure is characterized in that the first module and the second module having such low base pairing, homology, or identity form a heterodimer to function as a ribozyme.

[0132]

[0133] As a specific example of the first module and the second module according to the present disclosure, the first module and the second module may be formed by crossing and connecting the upstream region and the downstream region, based on the respective P6 domains in two different Group I introns, but are not limited thereto.

[0134]

[0135] As another specific example, the first module may comprise P1 to P6b domains of the first Group I intron and P6b to P9 domains of the second Group I intron, and the second module may comprise P1 to P6b domains of the second Group I intron and P6b to P9 domains of the first Group I intron, but are not limited thereto.

[0136]

[0137] As still another specific example, the first module may comprise P1 to P6a domains of the first Group I intron and P6a to P9 domains of the second Group I intron, and the second module may comprise P1 to P6a domains of the second Group I intron and P6a to P9 domains of the first Group I intron, but are not limited thereto.

[0138]

[0139] As still another specific example, the first module may comprise a nucleotide sequence of SEQ ID NO: 17, and the second module may comprise a nucleotide sequence of SEQ ID NO: 18, but are not limited thereto.

[0140]

[0141] As still another specific example, the first module may comprise a nucleotide sequence of SEQ ID NO: 31, and the second module may comprise a nucleotide sequence of SEQ ID NO: 32, but are not limited thereto.

[0142]

[0143] With respect to the objects of the present disclosure, the splicing site where cleavage occurs adjacent to each of the first module and the second module may be comprised, but is not limited thereto. The splicing site may be comprised in the RNA construct by existing within a spacer comprising any or defined additional sequence that may provide structural stability for the module or RNA construct and may assist transcription and / or translation.

[0144]

[0145] In the present disclosure, even though a nucleotide sequence (base sequence) is defined as a specific SEQ ID NO, as long as it has a function and activity identical or corresponding to that of the nucleotide sequence of the corresponding SEQ ID NO, it does not exclude the addition of meaningless sequences upstream and downstream the corresponding sequence, or a naturally occurring mutation, or a silent mutation thereof, and it is obvious that those having such a sequence addition or mutation fall within the scope of the present invention.

[0146]

[0147] Specifically, the nucleotide sequence defined in the present disclosure may have or comprise a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the nucleotide sequence of the corresponding SEQ ID NO, or may consist of or essentially consist of a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the nucleotide sequence of the corresponding SEQ ID NO, but is not limited thereto.

[0148]

[0149] As used herein, the term 'homology' or 'identity' means the degree of similarity between two given nucleotide sequences and may be expressed as a percentage. The terms 'homology and identity' may often be used interchangeably.

[0150] Methods of determining sequence similarity or identity between two or more nucleotide sequences or amino acid sequences are known in the art.

[0151] The sequence similarity or identity may be determined using known computer algorithms such as the "FASTA" program, for example, using default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the sequence similarity or identity may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (comprising GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, the homology or identity may be determined using BLAST or ClustalW of the National Center for Biotechnology Information.

[0152]

[0153] The first module and the second module comprised in the RNA construct of the present disclosure may form a dimer centered on the P6 domain. The P6 domain plays an important role in the dimer formation of the two modules and the splicing process of the RNA construct.

[0154] The dimer formation of the two modules may partially or completely form a dimer with the P2, P3, P7, or P8 domain of each module while one module and the other module form a dimer structure centered on the P6 domain, and the tertiary structure formed through this, e.g., P5b-P6a or P5-P9b or P4-P5a or P13, etc., partially forms a dimer between respective modules, and forms the structure of the intron I ribozyme.

[0155]

[0156] The scaffold domain within the above module comprises the P6 domain, P6a and P6b domains, and the first module and the second module of the present disclosure may be formed by hybridizing the upstream and downstream regions, based on P6a or P6b of two different Group I introns, but are not limited thereto. The circular RNA production efficiency may be controlled according to the length and GC content of the P6 domain.

[0157]

[0158] In one embodiment of the present disclosure, by extending the length of the P6 (P6a and / or P6b) domain of the RNA construct, it was confirmed that the length of the P6 domain affects the splicing efficiency.

[0159] For example, the length may be extended by adding 1 nt to 50 nt, 5 nt to 40 nt, 10 nt to 30 nt, or 10 nt to 20 nt to the P6 (P6a and / or P6b) domain of natural Group I intron, but is not limited thereto.

[0160] Specifically, the total length of the P6 domain comprised in the first module and the second module in the present disclosure may be 10 nt to 80 nt, 30 nt to 80 nt, 40 nt to 75 nt, 50 nt to 75 nt, 50 nt to 70nt or 55 nt to 70 nt, but is not limited thereto. When the length of P6 is shorter than the above range, splicing may not properly occur due to structural interference between adjacent domains, and when the length is longer than the above range, there is a problem that the size of the RNA construct becomes large.

[0161] Specifically, the sequence of the P6 domain may comprise or (essentially) consist of any one nucleotide sequence of SEQ ID NOS: 11 to 16, but is not limited thereto.

[0162]

[0163] In one embodiment of the present disclosure, it was confirmed that the circularization efficiency is controlled depending on the GC content in the P6 (P6a and / or P6b) domain of the RNA construct. Specifically, the GC content of the P6 domain of the RNA construct of the present disclosure may be, but is not limited to, 85% or less, specifically 40% to 85%, 45% to 80%, or 45% to 75%.

[0164]

[0165] Further, P6 domains of the first module and the second module may be the same as or different from each other. Specifically, P6 domains of the first module and the second module may have a sequence having 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 100% or less, or less than 100% base pairing, homology or identity, but are not limited thereto.

[0166]

[0167] As used herein, the term "internal ribosome entry site (IRES)" refers to an RNA element involved in translation initiation, which may be derived from a virus or mammal, but is not limited thereto, and may comprise, for example, a sequence of an internal ribosome entry site derived from an aptamer for Encephalomyocarditis virus, Human coxsackievirus B3, Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Drosophila C Virus, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BNS, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G, or a modified sequence thereof, but is not limited thereto. Specifically, the internal ribosome entry site of the present disclosure may be derived from ECMV B3, or Enterovirus (EV-D94), but is not limited thereto. The sequence of the internal ribosome entry site comprised in the RNA construct of the present disclosure may comprise those described in the database available athttp: / iresite.org / . The sequence of the internal ribosome entry site may comprise or (essentially) consist of a sequence of SEQ ID NO: 6 or 27, but is not limited thereto.

[0168]

[0169] As used herein, the term "protein coding region" refers to a sequence encoding a protein to be expressed from the circular RNA of the present disclosure. The protein to be expressed is called a target protein, and may be a vaccine, a protein drug for disease treatment, an antibody, a protein for diagnosis, etc.

[0170] This target protein may be two or more proteins for a multimeric protein, and therefore, an IRES sequence may be added. In addition, a self-cleavage amino acid sequence may be comprised within the target protein (e.g., 2A; T2A, P2A, E2A, F2A, etc.).

[0171] When the target protein is two or more, the RNA construct of the present disclosure may comprise two or more protein coding regions corresponding to respective target proteins, and the respective protein coding regions may be connected and comprised in the RNA construct, or may be comprised in the RNA construct with a sequence added between the respective protein coding regions. In addition, the two or more target proteins may be homologous or heterologous, and may be independently selected. A person skilled in the art may appropriately select the number of target proteins and protein coding regions encoding the same which are comprised in the RNA construct of the present disclosure, and the method of introduction.

[0172] The target protein is a protein that may exhibit physiological activity in the body, and examples thereof may comprise, but are not limited to, F luciferase, protein drugs (e.g., enzymes, hormones, functional peptides, recombinant proteins, interferons, cytokines, receptor proteins, albumin, growth factors, cell surface antigens, virus-derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, antibody fragments, etc.), chimeric antigen receptors, cell surface receptors, cell membrane proteins, etc.

[0173]

[0174] Depending on the target protein, the sequence of the protein coding region may be appropriately prepared by a method known in the art, and the sequence of the protein coding region may be a natural sequence or a sequence in which one or more bases are modified. Specifically, the sequence of the protein coding region of the present disclosure may comprise various modifications in consideration of codon degeneracy or codons preferred by cells or organisms in which the target protein is expressed. The protein coding region of the present disclosure may have a length of 500 nt to 5000 nt, 600 nt to 4000 nt, 700 nt to 4000 nt, or 1000 nt to 10000 nt, but is not limited thereto.

[0175] In addition, the protein coding region may comprise a start codon and a stop codon.

[0176]

[0177] In one embodiment of the present disclosure, it was confirmed that the RNA construct comprising the first module and the second module is able to produce a circular RNA regardless of the specific type of target protein, and thus the RNA construct of the present disclosure may be universally used.

[0178]

[0179] In addition, in one embodiment of the present disclosure, it was confirmed that even though various sizes of protein coding regions were introduced into the RNA construct according to the present disclosure, they may be all produced as circular RNAs.

[0180]

[0181] The RNA construct of the present disclosure may comprise additional elements to improve the structural stability and splicing efficiency of the RNA construct or one or more additional elements to improve the sequence stability and translation efficiency of the protein coding region of the circular RNA.

[0182] For example, the RNA construct of the present disclosure may further comprise, but is not limited to, a spacer or a translation enhancer (e.g., aptamer, poly A, poly AC) or a miRNA capture sequence for cell-specific expression.

[0183]

[0184] As used herein, the term "spacer" may be a sequence which is added to provide structural stability for the RNA construct and to minimize interference between adjacent components, and further, may be a sequence comprising a site where splicing occurs. The RNA construct of the present disclosure may comprise one or more independent spacer sequences.

[0185] In the present disclosure, the length of the spacer may be 10 nt to 500 nt, 20 nt to 300 nt, 30 nt to 200 nt, or 50 nt to 100 nt, may be a non-translated sequence, and may comprise any sequence or a defined sequence, but is not limited thereto. Specifically, the spacer sequence may comprise or (essentially) consist of a nucleotide sequence of SEQ ID NO: 8, 9, 29, or 30, but is not limited thereto.

[0186]

[0187] As used herein, the term "translation enhancer" refers to a factor capable of regulating expression of a target protein. Specifically, it refers to a factor capable of increasing expression of the target protein. The translation enhancer is a regulatory element that increases expression of a target sequence. The translation enhancer may be endogenous, exogenous, or heterologous, but is not limited thereto. The sequence of the translation enhancer may be appropriately determined and prepared by a person skilled in the art using a known method. Specifically, the translation enhancer sequence may comprise or (essentially) consist of a nucleotide sequence of SEQ ID NO: 10, but is not limited thereto.

[0188]

[0189] The spacer and the translation enhancer may or may not be comprised in the RNA construct depending on the module and the target protein, and the spacer and the translation enhancer may be appropriately introduced into the RNA construct according to a method known in the art.

[0190]

[0191] In addition, the nucleotide sequence comprised in the RNA construct of the present disclosure may be optimized. For example, it may comprise modifications such as formation of an appropriate folding structure, increase of circular RNA stability, regulation of transcription and translation, regulation of post-translational processes, and insertion or deletion of protein transport sequences, etc., but is not limited thereto.

[0192]

[0193] A specific embodiment of the RNA construct according to the present disclosure may comprise the first module and the second module at both ends, and one end of the first module may be connected to one end of the internal ribosome entry site and the protein coding region, and one end of the second module may be connected to the other end of the internal ribosome entry site and the protein coding region, but is not limited thereto.

[0194] Further, with respect to the objects of the present disclosure, a splicing site, where splicing occurs, may be comprised between the first module and the internal ribosome entry site and between the protein coding region and the second module. The site where splicing occurs may be present within a spacer region existing in the RNA construct, and its length may be 1 nt to 50 nt, 1 nt to 30 nt, or 1 nt to 20 nt, but is not limited thereto. A person skilled in the art may appropriately select the sequence where splicing may occur by the first module and the second module of the present disclosure.

[0195] In addition, a spacer and a translation enhancer may be further comprised, and as long as a circular RNA may be produced from the RNA construct, the connection site and connection order of the respective components are not limited to a specific position and order.

[0196]

[0197] The respective components comprised in the RNA construct of the present disclosure may be operably linked. The term "operably linked" as used herein, means that respective sequences are functionally linked so that the respective components may perform their original function in the transcription and translation processes.

[0198]

[0199] The above description may be equally applied to all aspects of the present disclosure described in this specification.

[0200]

[0201] Another aspect of the present disclosure provides a composition for producing a circular RNA, the composition comprising the first module and the second module.

[0202] Still another aspect of the present disclosure provides a kit for producing a circular RNA, the kit comprising the first module and the second module.

[0203] Still another aspect of the present disclosure provides a composition for producing a circular RNA, the composition comprising the RNA construct comprising the first module and the second module.

[0204] Still another aspect of the present disclosure provides a kit for producing a circular RNA, the kit comprising the RNA construct comprising the first module and the second module.

[0205] The "first module", the "second module", the "RNA construct" and the "circular RNA" are as described above.

[0206]

[0207] The composition or kit for producing a circular RNA of the present disclosure, where the first module and the second module form a dimer to function as a ribozyme, thereby inducing splicing, may be used in producing the circular RNA.

[0208] Specifically, the RNA construct of the present disclosure may be transcribedin vitroto comprise the first module, the internal ribosome entry site, the protein coding region, the second module, etc., and a circular RNA comprising the internal ribosome entry site and the protein region may be produced by splicingin vitro, and the first module and the second module may be separated and / or removed from the circular RNA.

[0209] Alternatively, the RNA construct of the present disclosure may be introduced into a cell in order to produce a circular RNA, and a person skilled in the art may appropriately create a reaction environment so that the circular RNA may be produced from the RNA construct by splicing and circularization.

[0210]

[0211] A person skilled in the art may produce a circular RNAin vitroby inserting an appropriate internal ribosome entry site sequence, protein coding region sequence, and other necessary sequences between the first module and the second module of the present disclosure.

[0212] The first module and the second module may be inserted into one vector or cassette to be used for producing a circular RNA, but are not limited thereto.

[0213] The vector is a vector for cloning or a vector for expression, and those commonly used in the art to express foreign proteins in plants, animals, or microorganisms may be used. The vector may be constructed through various methods known in the art.

[0214]

[0215] The composition or kit of the present disclosure may comprise a stabilizer (e.g., buffer, aqueous or non-aqueous solution, suspension, emulsion, etc.) or a protective agent to stabilize the first module, the second module, and / or the RNA construct, and may further comprise, but is not limited to, a component to assistin vitrotranscription, splicing, and circularization.

[0216]

[0217] Still another aspect of the present disclosure provides a method of producing a circular RNA from the RNA construct throughin vitrotranscription. The "RNA construct" is as described above.

[0218] In vitrotranscription (IVT) is a biochemical method of synthesizing RNA in a test tube, and the method is well known to those skilled in the art. One method is to produce a large amount of DNA as a template, and then to synthesize RNA therefrom using RNA polymerase and to purify the RNA.

[0219]

[0220] In the method of producing a circular RNA of the present disclosure, the RNA construct of the present disclosure is prepared through anin vitrotranscription reaction, and the circular RNA may be produced from the prepared RNA construct through self-splicing. However, the method is not limited to a specific method, as long as it is able to produce the circular RNA through self-splicing of the RNA construct.

[0221]

[0222] The method of producing a circular RNA may further comprise the step of producing the circular RNA through self-splicing while forming a heterodimer by the first module and the second module, but is not limited thereto.

[0223]

[0224] Specifically, the method may further comprise, but is not limited to, the step of adding water, a buffer, and / or GTP and performing a heat treatment, after producing RNA byin vitrotranscription. More specifically, the method of producing a circular RNA may further comprise, but is not limited to, the step of performing a heat treatment at 50°C to 65°C for 5 minutes to 30 minutes in a buffer comprising Mg2+and GTP and then cooling.

[0225] In addition, the method of producing a circular RNA of the present disclosure may further comprise the step of removing DNA which is used in transcription. The step of removing DNA may be a purification process using DNAse I or oligo dT, but is not limited thereto.

[0226]

[0227] In addition, the method may further comprise, but is not limited to, the step of isolating and / or purifying the produced circular RNA.

[0228] The step of isolating and / or purifying may be performed by, but is not limited to, any one or more methods of anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, reversed phase column chromatography, ultrafiltration / dialysis filtration, salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fraction precipitation using acetone, ethanol, etc.), dialysis, and gel filtration.

[0229]

[0230] Still another aspect of the present disclosure provides a vector for producing a circular RNA, the vector comprising the RNA construct. The "RNA construct" is as described above.

[0231] As used herein, the term "vector" refers to a carrier for a nucleic acid, which may be used to introduce the nucleic acid into a cell. The vector may be used to introduce the RNA construct into a cell and to produce the circular RNA within the cell.

[0232] Examples of the vectors of the present disclosure may comprise, but are not limited to, plasmids, nanoparticles, liposomes, viruses, phages, BACs, YACs, baculoviruses, retroviruses, adenoviruses, lentiviruses, plasmids, nanoparticles, polymeric nanoparticles, lipid nanoparticles, etc., more specifically, lentiviruses, retroviruses, herpesviruses, adenoviruses, and Adeno-Associated Virus (AAV), plasmid DNA (pDNA), but are not limited thereto. Other examples may comprise, but are not limited to, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc., which may be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series, etc., which may be used as plasmid vectors, and any known expression vector may be used.

[0233]

[0234] With respect to the objects of the present disclosure, the vector may be in a form in which the elements (first module, internal ribosome entry site, protein coding region, and second module) constituting the RNA construct of the present disclosure are operably linked to each other. The operably linkage to the vector may be achieved using a genetic recombination technique known in the art, and the site-specific DNA cleavage and linkage may be achieved using restriction enzymes and ligases known in the art.

[0235]

[0236] The vector may further comprise a sequence for producing a circular RNA in a cell, but the sequences within the vector are operably linked to each other.

[0237]

[0238] Still another aspect of the present disclosure provides a method of producing a circular RNA, the method comprising the step of introducing the RNA construct into an individual or a cell.

[0239] The step of introducing the RNA construct of the present disclosure into the individual or cell may be, but is not limited to, transforming using a vector comprising the RNA construct. The "RNA construct" and the "vector" are as described above.

[0240] The method of producing a circular RNA may utilize a method of introducing the vector of the present disclosure into the individual or host cell, and the method of transforming using the vector may comprise any method of introducing a nucleic acid into an individual or a cell, and may be performed by selecting a suitable standard technique known in the art. Specifically, electroporation, calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran, a cationic liposome method, and a heat shock method may be comprised, but are not limited thereto.

[0241]

[0242] In the present disclosure, the individual refers to an animal, typically, a mammal, and may comprise any suitable mammal. Examples of the mammal may comprise humans, non-human primates (apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), livestock (dogs and cats), farm animals (horses, cows, goats, sheep, pigs) and laboratory animals (mice, rats, bats, rabbits, guinea pigs), etc., for example, humans. For a more specific example, the individual may refer to an individual having a disease which may be treated with the target protein.

[0243]

[0244] In the present disclosure, the cell may be a cell capable of stably and continuously cloning and expressing the vector of the present disclosure, and may be any known host cell. For example, the cell may be a eukaryotic cell, more specifically, a mammalian cell or a human cell. Specifically, the cell may comprise, but is not limited to, HeLa cells, A549 cells, human embryonic kidney (HEK) 293 cells or derivatives thereof (HEK293T or HEK293F), Vero cells, Chinese hamster ovary (CHO) cells, and NIH 3T3 cells.

[0245] The circular RNA may be produced by culturing cells transformed with the vector of the present disclosure under appropriately controlled environmental conditions, but is not limited thereto. The culturing procedure of the present disclosure may be performed according to appropriate media and culturing conditions known in the art. Such culturing procedure may be easily adjusted and used by those skilled in the art depending on the selected cell.

[0246]

[0247] Meanwhile, the method of producing a circular RNA of the present disclosure may further comprise the step of producing the circular RNA through self-splicing while forming a heterodimer by the first module and the second module, but is not limited thereto.

[0248]

[0249] In addition, the method may further comprise the step of isolating and / or purifying the produced circular RNA from the cell, but is not limited thereto.

[0250] The step of isolating and / or purifying may be performed by, but is not limited to, any one or more methods of anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, reverse phase column chromatography, ultrafiltration / dialysis filtration, salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fraction precipitation using acetone, ethanol, etc.), dialysis, and gel filtration, but is not limited thereto.

[0251]

[0252] Still another aspect of the present disclosure provides a circular RNA produced by using the vector, composition, or kit. The "circular RNA" is as described above.

[0253] The circular RNA of the present disclosure is characterized in that it has high stability, as compared to not only the known linear RNAs but also the existing known circular RNAs, allowing long-term protein translation.

[0254] Specifically, the circular RNA may comprise a protein coding region for expressing a target protein and an internal ribosome entry site, and optionally, may further comprise a miRNA capture sequence for regulating translation, a translation enhancer for enhancing translation, such as poly A, poly AC, an aptamer, etc..

[0255]

[0256] Still another aspect of the present disclosure provides a vector comprising the circular RNA. The "circular RNA" and "vector" are as described above.

[0257]

[0258] The vector of the present disclosure may be an expression vector for expressing the circular RNA in a host cell, but is not limited thereto.

[0259]

[0260] The vector of the present disclosure may comprise a nucleic acid construct comprising a nucleotide sequence of a protein coding region operably linked to a suitable expression control region (or expression control sequence) so as to express a target protein in a suitable host.

[0261] The vector may be capable of replicating or functioning independently of the host genome, after being transformed into the suitable host cell, and may be integrated into the genome itself.

[0262] The vector used in the present disclosure is not particularly limited, and any vector known in the art may be used. Examples of vectors commonly used may comprise natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. may be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series and pET series, etc. may be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. may be used.

[0263] In addition, the vector may further comprise a selection marker. The selection marker is for selecting the cells transformed with vectors, i.e., for confirming the insertion of a target nucleic acid molecule, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, and thus transformed cells may be selected.

[0264]

[0265] As used herein, the term "transformation" means that a vector comprising a nucleotide sequence encoding a target protein is introduced into an individual or a cell so that the target protein may be expressed. The nucleotide sequence may be introduced in any form as long as it may be introduced into an individual or a cell and expressed. For example, the nucleotide sequence may be introduced in the form of an expression cassette, which is a gene construct comprising all elements required for self-expression. The expression cassette may be in the form of an expression vector capable of self-replicating.

[0266]

[0267] Still another aspect of the present disclosure provides a drug delivery system comprising the circular RNA. The "circular RNA" is as described above.

[0268]

[0269] The drug delivery system of the present disclosure is to safely and stably deliver the circular RNA capable of expressing the target protein to an individual in need thereof, and as long as the circular RNA may be effectively introduced into the individual to express the target protein, it may be comprised in the present disclosure without limitation on its type. For example, the drug delivery system of the present disclosure may be a viral vector or a non-viral vector, but is not limited thereto.

[0270] Examples of viral vectors comprise adenoviruses and retroviruses, etc., and examples of non-viral vectors comprise lipidoids, liposomes, lipoplexes, lipid nanoparticles, compound polymers, peptides / proteins, cells, nanoparticle mimics, or nanotubes, etc.

[0271] Additional components for increasing the stability of circular RNA and for increasing protein translation may be comprised, but are not limited thereto.

[0272]

[0273] The drug delivery system may be administered to an individual in need thereof to express the target proteinin vivofrom the circular RNA.

[0274] Specifically, the drug delivery system of the present disclosure may be administered by transdermal, oral, or parenteral routes, for example, intradermal, subcutaneous, intravenous, intramuscular, intranodal and / or intraperitoneal, or nasal (e.g., tracheal inhalation), intranasal, vaginal, rectal, oral, or transdermal route, and may be administered by, but is not limited to, a "needle-free" delivery system.

[0275]

[0276] The drug delivery system of the present disclosure is to administer or deliver a pharmaceutically effective amount of circular RNA to an individual, and methods of determining the most effective administration means and dosage are known to those skilled in the art, and may be appropriately determined by those skilled in the art according to the composition used in the therapy, the purpose of the therapy, the target cell to be treated, and the subject to be treated. The "pharmaceutically effective amount" means an amount sufficient to produce a beneficial or desired result with no or minimal side effects, and the effective amount may be administered in one or more administrations, applications, or dosages. Such delivery depends on a number of variables comprising the time period for which the individual dosage unit is used, the bioavailability of the therapeutic agent, the route of administration, etc. However, dose levels of the circular RNA of the present disclosure for any particular subject depend upon a variety of factors comprising the activity thereof, bioavailability, the route of administration, the individual's age and body weight, general health, sex, and diet, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and mode of administration.

[0277]

[0278] Still another aspect of the present disclosure provides use of a nucleotide sequence comprising the first module and the second module in producing a circular RNA. The "first module", the "second module", and the "circular RNA" are as described above.

[0279]

[0280] Still another aspect of the present disclosure provides use of a nucleotide sequence comprising the RNA construct in producing a circular RNA. The "RNA construct" and the "circular RNA" are as described above.

[0281]

[0282] The redundant contents are omitted in consideration of complexity of the present specification, and it is obvious that the description described in one aspect also applies to other aspects. The terms not otherwise defined in the present specification have the meanings commonly used in the technical field to which the present disclosure pertains.

[0283]

[0284] Hereinafter, the present invention will be described in more detail with reference to the following exemplary embodiments. However, the following exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not intended to be limited thereby.

[0285]

[0286] Example 1: Production ofTetrahymena thermophila-derived RNA construct

[0287]

[0288] To produce an RNA construct capable of producing a circular RNA, a module of Group I intron derived fromTetrahymena thermophilaand an RNA construct comprising the same were produced through anin vitrotranscription reaction (IVT reaction) as follows.

[0289]

[0290] Example 1-1: Production of module ofTetrahymena thermophila-derived Group I intron

[0291]

[0292] First, two mutants of SEQ ID NOS: 2 and 3 were prepared from Group I intron (WT, SEQ ID NO: 1) derived fromTetrahymena thermophila. Two hybrid Group I introns (SEQ ID NOS: 4 and 5) were prepared by combining the domains of the two prepared mutants.

[0293]

[0294] In detail, a first module comprising P1 to P6b domains of SEQ ID NO: 2 and P6b to P9 domains of SEQ ID NO: 3, and a second module comprising P1 to P6b domains of SEQ ID NO: 3 and P6b to P9 domains of SEQ ID NO: 2 were produced, and the modules alone do not have activity as a ribozyme of Group I intron.

[0295]

[0296] Example 1-2: Production ofTetrahymena thermophila-derived RNA construct

[0297]

[0298] In order to produce an RNA construct capable of inducing circular RNA production, an RNA construct was produced, in which the first module and the second module produced above were located at both ends, and an internal ribosome entry site (IRES) and a protein coding region were comprised therebetween.

[0299]

[0300] In detail, produced was an RNA construct comprising the first module (SEQ ID NO: 4), a first spacer sequence (SEQ ID NO: 8), IRES (SEQ ID NO: 6), a protein coding region encoding eGFP (SEQ ID NO: 7), a translation enhancer (SEQ ID NO: 10), a second spacer sequence (SEQ ID NO: 9), and the second module (SEQ ID NO: 5) from the 5'-end (FIG. 1). The first spacer sequence and the second spacer sequence are sequences in which P1-P10 pairing may occur within the module.

[0301]

[0302] The RNA construct comprising the above composition was produced by expressing the same from a vector throughin vitrotranscription (IVT), and the produced RNA construct was heat-treated at 55℃ to 70℃ for 5 minutes to 30 minutes under condition to which a buffer comprising magnesium (50 mM Tris-HCl (pH 7.4), 1 mM DTT, 10 mM MgCl2) and guanosine triphosphate (GTP) were added, followed by a cooling process to induce self-splicing and circularization, thereby producing the circular RNA.

[0303]

[0304] Example 2: Verification of circularization byTetrahymena thermophila-derived RNA construct

[0305]

[0306] To verify the production of circular RNA by the RNA construct produced in Example 1, an agarose gel electrophoresis experiment was performed. Additionally, the length of P6 domain was determined to increase the efficiency of the splicing process involved in the production of circular RNA.

[0307]

[0308] Example 2-1:Adjustment of length of scaffold domain to increase splicing efficiency and Production of circular RNA

[0309]

[0310] In order for the RNA construct produced in Example 1-2 to be able to produce a circular RNA with high efficiency, splicing efficiency was intended to be increased by producing a module capable of minimizing the structural hindrance of Group I intron. To this end, the length of P6 domain of the module was adjusted as follows, and the splicing efficiency was confirmed.

[0311]

[0312]

[0313]

[0314] Example 2-2: Adjustment of length of scaffold domain to increase splicing efficiency and Production of circular RNA

[0315]

[0316] After thein vitrotranscription reaction, the circularized RNA was subjected to agarose gel electrophoresis to confirm that the circular RNA was produced by the modules produced in the present disclosure.

[0317]

[0318] Furthermore, the circularization efficiency according to the migration degree of the electrophoretic band was examined to confirm that the circularization efficiency varied depending on the length of the P6 domain. In particular, it was confirmed that the circularization efficiency was the highest when a module comprising 20 additional nucleotides in the P6 domain was used (FIG. 2). As a result, the modules of SEQ ID NOS: 17 and 18 were finally determined as the optimized modules, and the versatility and translation efficiency of the RNA construct comprising the modules were additionally confirmed.

[0319]

[0320] Example 3: Production of circular RNA byTetrahymena thermophila-derived RNA construct after CDS modification

[0321]

[0322] The circularization efficiency was examined by introducing three different genes of interest (GOI) into the RNA construct produced in Example 2, respectively.

[0323]

[0324] In detail, RNA constructs comprising sequences corresponding to three GOIs (eGFP, FXR, SSADH) of SEQ ID NOS: 19 to 21 were cloned and synthesized by IVT, and changes in bands through agarose gel electrophoresis were examined.

[0325]

[0326] As a result, it was confirmed that circular RNAs were produced regardless of the type of gene of interest (FIG. 3).

[0327]

[0328] Example 4: Identification of junction sequence of circular RNA produced byTetrahymena thermophila-derived RNA construct

[0329]

[0330] The structure of circular RNA was identified through a junction sequence of the synthesized circular RNA.

[0331]

[0332] In detail, the circular RNA produced in Example 1-2 was synthesized again into cDNA and sequenced to confirm the sequence information. As a result, the junction sequence in which P1 and P10 were paired and spliced was identified, confirming that splicing was induced by the modules of the present disclosure, and circular RNA was produced with the 5'-end and the 3'-end connected to each other (FIG. 4).

[0333]

[0334] Example 5: Purification of circular RNA produced fromTetrahymena thermophila-derived RNA construct

[0335]

[0336] The circular RNA produced in the above Examples was purified from by-products which was produced during the splicing process and precursors before production of the circular RNA. In detail, the modules were removed through affinity chromatography specific to the circular RNA sequence (FIG. 5A), and the remaining precursor and nicked circular RNA were removed through Ion pair reversed-phase chromatography (IP-RP) (FIG. 5B). The purified circular RNA was examined by agarose gel electrophoresis to identify a single band.

[0337]

[0338] From this, it was confirmed that circular RNA may be stably produced with a small amount of impurities from the RNA construct of the present disclosure.

[0339]

[0340] Example 6: Evaluation of cell translation persistence of circular RNA produced fromTetrahymena thermophila-derived RNA construct

[0341]

[0342] The translation persistence of circular RNA produced and purified through the above Examples was evaluated in comparison with the existing linear RNA and oRNA' circular RNA (Wesselhoeft, R.A., Kowalski, P.S. & Anderson, D.G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun 9, 2629 (2018)) in A549 cells. To evaluate translation persistence, protein expression levels and total protein expression rates (AUC) over time were measured using eGFP.

[0343]

[0344] As a result, in A549 cells, the circular RNA according to the present disclosure showed significantly superior translation persistence and total protein expression rate (AUC), as compared to linear RNA and existing known circular RNA, indicating high translation efficiency (FIG. 6).

[0345]

[0346] Meanwhile, in order to confirm whether an RNA construct produced from Group I intron derived from a different origin, other than the Group I intron derived fromTetrahymena thermophilaof Example 1, is able to also produce a circular RNA, an RNA construct was produced from Group I intron derived fromAzoarcusas follows, and its circular RNA production and translation persistence, etc. were evaluated.

[0347]

[0348] Example 7: Production of Azoarcus-derived RNA construct

[0349]

[0350] In the similar manner as Example 1, Group I first module and second module derived fromAzoarcus, and an RNA construct comprising the same were produced.

[0351]

[0352] Example 7-1: Production of modules ofAzoarcus-derived Group I intron

[0353]

[0354] Two mutants of SEQ ID NOS: 23 and 24 were produced from Group I intron (WT, SEQ ID NO: 22) derived fromAzoarcus. The domains of the two mutants were combined to produce two hybrid Group I introns (SEQ ID NOS: 25 and 26).

[0355]

[0356] In detail, a first module (SEQ ID NO: 25) comprising P1 to P6a domains of SEQ ID NO: 23 and P6a to P9 domains of SEQ ID NO: 24, and a second module (SEQ ID NO: 26) comprising P1 to P6a domains of SEQ ID NO: 24 and P6a to P9 domains of SEQ ID NO: 23 were produced, and the above modules alone do not have activity as a ribozyme of Group I intron (FIG. 7).

[0357]

[0358] Example 7-2: Production ofAzoarcus-derived RNA construct

[0359]

[0360] In order to produce an RNA construct capable of inducing circular RNA production, an RNA construct was produced in the same manner as in Example 1-2, in which theAzoarcus-derived first module and second module produced were located at both ends, and an internal ribosome entry site (IRES), and a protein coding region were comprised therebetween.

[0361]

[0362] In detail, produced was an RNA construct comprising the first module (SEQ ID NO: 25), a first spacer sequence (SEQ ID NO: 29), IRES (SEQ ID NO: 27), a protein coding region encoding eGFP (SEQ ID NO: 28), a second spacer sequence (SEQ ID NO: 30), and the second module (SEQ ID NO: 26) from the 5'-end (FIG. 8).

[0363]

[0364] The produced RNA construct was prepared by expressing the same from a vector throughin vitrotranscription (IVT), and the produced RNA construct was heat-treated at 55℃ to 70℃ for 5 minutes to 30 minutes under conditions to which a buffer comprising magnesium (50 mM Tris-HCl (pH 7.4), 1 mM DTT, 10 mM MgCl2) and guanosine triphosphate (GTP) were added, followed by a cooling process to induce self-splicing and circularization, thereby producing the circular RNA.

[0365]

[0366] Example 8: Verification of circularization byAzoarcus-derived RNA construct

[0367]

[0368] To verify circular RNA production by the RNA construct produced in Example 7, an agarose gel electrophoresis experiment was performed.

[0369]

[0370] In detail, after inducing IVT and circularization reaction by the producedAzoarcus-derived RNA construct, it was confirmed through agarose gel analysis that a precursor band changed into a circular RNA band in the circularization reaction (FIG. 9a) and the circular RNA band was concentrated when treated with RNaseR (FIG. 9b), indicating that the synthesis of circular RNA using theAzoarcus-derived RNA construct was possible.

[0371]

[0372] Example 9: Adjustment of length and change of GC content of scaffold domain inAzoarcus-derived RNA construct to improve circularization efficiency

[0373]

[0374] In order for the RNA construct produced in Example 7 to be able to produce a circular RNA with high efficiency, it was attempted to improve circularization efficiency. To this end, the circularization efficiency was evaluated by adjusting the length and GC content of the scaffold domain (P4 to P6) of the module (FIG. 10).

[0375]

[0376] As a result, it was confirmed that circularization may be induced when the GC content of the P6 domain was 45% to 85% and the length was 16 nt to 36 nt. It was confirmed that, as compared to the P6 domain of the natural Group I intron, the lower the GC content and the longer the length, the lower the precursor band and the higher the circular RNA band and the spliced intron band.

[0377]

[0378] Based on these results, the modules of SEQ ID NO: 31 and 32 were finally determined as the optimized modules, and the versatility and translation efficiency of an RNA construct comprising the same were additionally verified.

[0379]

[0380] Example 10: Verification of junction sequence of circular RNA produced byAzoarcus-derived RNA construct

[0381]

[0382] The structure of circular RNA was verified through the junction sequence of the synthesizedAzoarcus-derived circular RNA.

[0383]

[0384] In detail, the circular RNA produced in Example 7-2 was synthesized again into cDNA and sequenced to confirm the sequence information. As a result, the junction sequence in which P1 and P9 were paired and spliced was identified, confirming that splicing was induced by the modules of the present disclosure, and circular RNA was produced with the 5'-end and the 3'-end connected to each other (FIG. 11).

[0385]

[0386] Example 11: Verification of versatility ofAzoarcus-derived RNA construct

[0387]

[0388] In order to verify the versatility of theAzoarcus-derived RNA construct of the present disclosure, five different GOIs in Table 2 below were introduced, and then IVT and circularization reactions were performed to evaluate the circularization efficiency for each GOI. As a result, it was confirmed that a precursor band changed into a circular RNA band after the circularization reaction in the RNA construct comprising five types of GOI (FIG. 12).

[0389] This suggests that theAzoarcus-derived RNA construct according to the present disclosure may be used universally regardless of the type of GOI and is not limited by its size.

[0390]

[0391]

[0392]

[0393] Example 12: Evaluation of cell translation persistence of circular RNA produced fromAzoarcus-derived RNA construct

[0394]

[0395] In order to evaluate the translation persistence of the circular RNA produced from theAzoarcus-derived RNA construct of the present disclosure, the translation persistence was evaluated over time using eGFP in three types of cells: HeLa, A549, and Hep3B. As a control, the oRNA's circular RNA of Example 6 was used, which is known to have a longer translation efficiency persistence than existing linear mRNA.

[0396]

[0397] The circular RNA produced from the RNA construct according to the present disclosure (AZ-LoopRNA 5: comprising modules of SEQ ID NOS: 31 and 32 and IRES sequence of SEQ ID NO: 39) and the oRNA's circular RNA as the control were evaluated for up to 5 days, and the translation efficiency was compared using area under the curve (AUC). As a result, it was confirmed that the RNA construct according to the present disclosure showed translation efficiency equivalent to or higher than the control in three types of cells (FIG. 13).

[0398]

[0399] From the above Examples, the present inventors confirmed that the RNA construct comprising modules produced as a hybrid of different Group I introns according to the present disclosure may induce the production of circular RNA by circularization through self-splicing of the modules at both ends, and the circular RNA produced therefrom exhibits high stability and high translation efficiency, as compared to the existing known linear RNAs or other circular RNAs. In particular, it was confirmed that the circular RNA may be produced by using two modules produced as a hybrid regardless of the specific origin of Group I intron.

[0400]

[0401] Based on the above description, it will be understood by those skilled in the art that the present disclosure may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the disclosure is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.

Claims

1.An RNA construct for producing a circular RNA, the RNA construct comprising a first module, an internal ribosome entry site (IRES), a protein coding region, and a second module,wherein the first module and the second module are a combination of domains derived from Group I introns which are different from each other.2.The RNA construct of claim 1, wherein the Group I intron is a Group I intron derived fromTetrahymena thermophilaorAzoarcus.3.The RNA construct of claim 2, wherein the Group I introns comprise any one sequence of sequences of SEQ ID NOS: 1 to 3, and 22 to 24.4.The RNA construct of claim 1, wherein the first module and the second module have 55% or less base pairing with each other.5.The RNA construct of claim 1, comprising:i) the first module comprising P1 to P6b domains of a first Group I intron and P6b to P9 domains of a second Group I intron, and the second module comprising P1 to P6b domains of the second Group I intron and P6b to P9 domains of the first Group I intron; orii) the first module comprising P1 to P6a domains of the first Group I intron and P6a to P9 domains of the second Group I intron, and the second module comprising P1 to P6a domains of the second Group I intron and P6a to P9 domains of the first Group I intron.6.The RNA construct of claim 5, wherein the first module comprises a nucleotide sequence of SEQ ID NO: 17, and the second module comprises a nucleotide sequence of SEQ ID NO: 18.7.The RNA construct of claim 5, wherein the first module comprises a nucleotide sequence of SEQ ID NO: 31, and the second module comprises a nucleotide sequence of SEQ ID NO: 32.8.The RNA construct of claim 1, further comprising a spacer or a translation enhancer.9.The RNA construct of claim 1, wherein the first module and the second module form a heterodimer to function as a ribozyme.10.The RNA construct of claim 9, wherein the length of the P6 domain of the RNA construct forming the heterodimer is 10 nt to 80 nt.11.The RNA construct of claim 9, wherein the GC content of the P6 domain of the RNA construct forming the heterodimer is 40% to 85%.12.The RNA construct of claim 1, wherein the RNA construct does not comprise homology arms at both ends.13.The RNA construct of claim 1, wherein the internal ribosome entry site is derived fromEncephalomyocarditisvirus (ECMV),CoxsackievirusB3 (CVB3), orEnterovirus(EV-D94).14.A composition for producing a circular RNA, the composition comprising the RNA construct of any one of claims 1 to 13.15.A method of producing a circular RNA, the method comprising the step of producing the circular RNA from the RNA construct of any one of claims 1 to 13 throughin vitrotranscription.16.The method of claim 15, further comprising the step of producing the circular RNA through self-splicing as the first module and the second module form a heterodimer.17.A circular RNA produced by the method of claim 15.18.A drug delivery system comprising the circular RNA of claim 17.

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