Self-circular RNA structures

Abstract

The self-circularizing RNA construct of the present invention can be expressed in a DNA vector and simultaneously circularized by self-targeting and splicing to form a circRNA. The circRNA is composed only of a target gene, and the target gene contains an IRES region, an initiation codon, and a termination codon, which allows for rapid expression of a peptide or protein.

Description

[Technical Field]

[0001] This invention relates to self-circularized RNA structures with improved circulation efficiency, and the like.

[0002] This invention was completed with funding from the Ministry of Science and ICT and support from the Korea Research Foundation's "Development of Core Technologies for Next-Generation Infectious Disease Vaccines" project (Project Number: 2022M3E5F1017657).

[0003] [Background technology]

[0004] Circular RNA (circRNA, cRNA) is a single-stranded transcript linked by covalent bonds. RNA-seq data and newly developed bioinformatics approaches have identified tens of thousands of different types of circRNA in various organisms. In eukaryotes, circRNA is generated from mRNA via back-splicing and is known to regulate gene expression by performing microRNA sponge functions in vivo. It is unknown whether circRNA causes immunogenicity, and due to its structural characteristics, it exists very stably in vivo.

[0005] On the other hand, the development of therapeutic drugs using messenger RNA (mRNA) has recently become active. However, mRNA has limitations, such as being easily degraded in vivo and having a relatively short half-life. To overcome these limitations, research is underway to improve stability by attaching poly(A) tails to mRNA. Similarly, U.S. Patent No. 10,953,033 discloses circRNA for the purpose of gene expression in vivo, based on the structural characteristics of circRNA.

[0006] [Overview of the project] [Problems that the invention aims to solve]

[0007] The technical problem that this invention aims to solve is to provide an RNA structure that is automatically targeted and spliced ​​to become circular.

[0008] However, the technical problems that this invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by an ordinary person skilled in the art from the following description.

[0009] [Means for solving the problem]

[0010] To solve the aforementioned problems, the present invention provides a self-circular RNA structure. The RNA structure of the present invention is

[0011] It has a structure of 5'-IGS (internal guide sequence)-ribozyme-target gene-target site-3' and can form a P1 helix including a bulge.

[0012] On the other hand, P1 helix refers to a helix structure formed by complementary bonding between a base sequence linked in the shear direction (5' direction) of the ribozyme and a base sequence in the 3' direction of the transcript, which is linked by the ribozyme during secondary structure formation of the group I intron ribozyme.

[0013] In one embodiment of the present invention, the IGS region includes or comprises the base sequence 5'-GNNNNN-3', the target site region includes or comprises the base sequence 5'-N'N'N'N'N'U-3', the IGS region may form a guanine (G):uracil (U) wobble base pair with the target site, and the P1 helix can be formed by complementary bonding between the IGS region and the target site.

[0014] As another embodiment of the present invention, the N of the IGS region and the N' of the target site region may each be independently A, G, C, or U, but preferably one or more nucleotides that can complementarily bind to the IGS region and the target site region, and more preferably, the N and N' excluding the fluctuating base pair may be inversely complementary nucleotides.

[0015] As a further embodiment of the present invention, the RNA structure includes a nucleotide sequence extended in the 5' direction of the IGS region and a nucleotide sequence extended in the 3' direction of the target site region, and can form a bulge in the P1 helix.

[0016] As another embodiment of the present invention, the base sequences extended in the 5' direction of the IGS region and the 3' direction of the target site region are designed not to complement each other, and the number of bases extended in the direction determined in each region may be independently 1 to 10 nt.

[0017] In another embodiment of the present invention, the length of the extended base sequence forming the bulge may be 1 to 10 nt, preferably 2 to 5 nt, and more preferably 3 to 5 nt.

[0018] In another embodiment of the present invention, the ribozyme may be a group I intron ribozyme, which may contain or consist of the nucleotide sequence of SEQ ID NO: 6.

[0019] As a further embodiment of the present invention, the construct comprises a nucleotide extended toward the 5' direction of the IGS region, and forms a P1 helix and a P10 helix, wherein the P1 helix is formed in a region where complementary binding between the IGS region and the target site occurs, together with a nucleotide extended toward the 3' direction of the target site, and the P10 helix can be formed in a region where complementary binding occurs between the nucleotide extended toward the 5' direction of the IGS region and a reverse complementary sequence of the extended nucleotide located between the ribozyme and the GOI region. The length of the extended nucleotide forming the P1 helix may be 3-nt, and the length of the extended nucleotide forming the P10 helix may be 6-nt.

[0020] As a further embodiment of the present invention, the construct forms a P1 helix, but does not necessarily form a P10 helix.

[0021] As a further embodiment of the present invention, the construct can comprise a region capable of complementary binding to each other at the 5' end and the 3' end, and an ABS (antisense binding sequence) region.

[0022] As a further embodiment of the present invention, the ABS region consists of a reverse complementary sequence to the AS region.

[0023] As a further embodiment of the present invention, the length of the AS region is variable depending on the GOI, and may be 10 to 500-nt, preferably more than 50-nt and less than 400-nt, more preferably 150 to 350 nt.

[0024] As a further embodiment of the present invention, the GOI region may comprise an IRES (internal ribosome entry site) region at the 5' end, and can comprise a start codon and a stop codon.

[0025] As a further embodiment of the present invention, the structure may further comprise a Spacer region consisting of a random nucleotide sequence, and the spacer region may be arranged between the IGS region and the target gene region, and / or between the target gene region and the target site region.

[0026] As a further embodiment of the present invention, the spacer region may comprise or consist of poly(A), wherein poly(A) is a polynucleotide formed by repeatedly linking adenine (A), and A may be repeatedly linked 10 to 50 times, preferably, may be repeatedly linked 30 times.

[0027] Effects of the Invention

[0028] The self-circularizing RNA structure of the present invention can be expressed by a DNA vector, and at the same time, can be circularized by a self-targeting & splicing reaction without additional GTP treatment to form circRNA. The circRNA is composed only of the target gene, and the target gene has the advantage of enabling rapid expression of a peptide or protein comprising an IRES region, an initiation codon, and a termination codon. In addition, circRNA has a stable structure with no exposed 5' and 3' ends, thus it has high stability and a long half-life. When functional RNAs such as miRNA, anti-miRNA, shRNA, aptamer, mRNA vaccine, mRNA therapeutic agent, antibody, vaccine adjuvant, and CAR-T mRNA are prepared as circRNA, they can exhibit high stability in cells.

[0029] Brief Description of the Drawings

[0030] [Figure 1a-1b]Figures 1a and 1b are schematic diagrams illustrating the process by which the self-circular RNA structure of the present invention, which includes only IGS, ribozyme, target gene, and target site region, is formed into circRNA through self-targeting and splicing (STS) reactions.

[0031] [Figure 2a] Figure 2a is a schematic diagram illustrating the process by which the self-circular RNA structure of the present invention, which includes AS, IGS, ribozyme, target gene, target site, and ABS region, is formed into circRNA by the STS reaction.

[0032] [Figure 2b] Figure 2b shows the self-circular RNA structure of the present invention, which includes an IRES and a termination codon in the target gene region to enable translation of a transgene, and further includes nucleotides extended in the 5' direction to form P1 and P10 helices, and a schematic diagram of the process by which the structure is formed into circRNA by an STS reaction.

[0033] [Figure 2c] Figure 2c shows one embodiment of the self-circular RNA structure of the present invention.

[0034] [Figure 2d] Figure 2d shows the nucleotide sequence of the DNA template for preparing the self-circular RNA structure expression vector according to the present invention.

[0035] [Figure 3] Figure 3 shows the results of electrophoresis on a polyacrylamide gel to confirm that circRNA is generated immediately after in vitro transcription of the self-circulating RNA structure expression vector of the present invention, without additional GTP treatment. Samples secured immediately after transcription (direct STS) and samples after the primary and secondary STS reaction steps, in which primary and secondary circulation reactions are induced by additional GTP treatment, were used. A portion of each sample was enriched for circRNA by removing linear RNA (linear RNA) with RNase R and then electrophoresed.

[0036] [Figure 4] Figure 4 shows the verification that Candidate 1, which was presumed to be circRNA based on the results shown in Figure 3, is indeed circRNA. Figure 4a shows the results of RT-PCR performed on RNA extracted from Candidate 1 band, and Figure 4b shows the results of the nucleotide sequence analysis of the RNA extracted from Candidate 1 band.

[0037] [Figure 5] Figure 5 shows the results from Figure 4, verifying that the RNA in candidate band 1 is a monomer. In Figure 3, RNA was extracted from candidate bands 1 and 2, which were presumed to be circRNA, treated with Mg2+ to induce nicks, and then subjected to electrophoresis.

[0038] [Figure 6] Figure 6 shows the results of confirming that the self-circular RNA structure expression vector of the present invention generates circRNA in cells and that the transgene contained in the circRNA is expressed. Specifically, Figure 6a shows the structure of the self-circular RNA structure expression vector, Figure 6b shows the results of confirming transgene expression via luciferase activity assay, and Figures 6c and 6d show the results of RP-PCR and nucleotide sequence analysis verifying that the transgene was expressed via circRNA.

[0039] [Figure 7-8] Figures 7 and 8 show the circRNA purification conditions and results by HPLC.

[0040] [Figure 7a]Specifically, Figure 7a shows the HPLC analysis conditions using the Ultra HPLC system, Figure 7b shows the results of column purification of the sample immediately after in vitro transfer, and Figure 7c shows the results of column purification after treating the sample with RNase R. Furthermore, Figure 8a shows the results of identifying peaks in the fractions obtained through column purification, and Figure 8b shows the electrophoresis results of fractions 7, 10, 11, 12, and 13, which showed prominent peaks in Figure 8a.

[0041] [Figure 9-10] Figures 9 and 10 show the effects of the AS region on the STS reaction and circulation in the self-circular RNA structure of the present invention. Figure 9 shows the structure of the self-circular RNA containing AS regions of different lengths, and Figure 10 shows the electrophoresis results of the sample obtained after in vitro transcription of the vector expressing the RNA.

[0042] [Figure 11-12] Figures 11 and 12 show the effects of the spacer region in the self-circular and RNA structures of the present invention on the STS reaction and circulation. Figure 11 shows the structure of self-circularized RNA containing a control spacer and poly(A) spacers of various lengths, and Figure 12 shows the electrophoresis results of samples obtained after in vitro transcription of the vector expressing the RNA.

[0043] [Figure 13] Figure 13 shows the nucleotide sequences near the regions cleaved by ribozymes in self-circular RNA containing only the P1 region; self-circular RNA containing only the P1 and P10 regions; and self-circular RNA containing the P1, P10, and AS regions, along with each region.

[0044] [Figure 14]Figure 14 shows the nucleotide sequence of a DNA template for preparing a self-circular RNA expression vector, which lacks the P10 and AS regions and contains only the P1 region.

[0045] [Figure 15] Figure 15 shows the electrophoresis results of samples obtained after in vitro transcription of each self-circular RNA expression vector shown in Figure 13.

[0046] [Figures 16a-16b] Figures 16a and 16b show the results of electrophoresis confirming that a self-circular RNA structure is formed on circRNA after in vitro transcription in a self-circular RNA expression vector containing only the P1 region and lacking the P10 and AS regions.

[0047] [Figure 17-19] Figures 17 to 19 show the effects of the P1 region sequence in the self-circular and RNA structures of the present invention on the STS reaction and circulation. Specifically, Figure 17 shows P1 regions designed with different nucleotide sequences, Figure 18 shows the electrophoresis results of samples obtained after in vitro transcription of the self-circular RNA expression vector having the P1 region of Figure 17, and Figure 19 shows the electrophoresis results of samples obtained after in vitro transcription of the self-circular RNA expression vector having an AU-rich P1 region and the self-circular RNA expression vector having a 2-site P1 region, as well as the RT-PCR electrophoresis results to confirm the circular RNA.

[0048] [Figure 20]Figure 20 shows a simplified representation of the essential components of a self-circular RNA structure. Figure 20a shows that circRNA can be prepared using only an RNA structure containing IGS, ribozyme, and GOI region with only uracil bases at the 3' end. Figure 20b shows that if uracil bases are present at the 3' end of the GOI, circRNA can be prepared using only an RNA structure containing IGS, ribozyme, and GOI region, in which case the resulting circRNA consists solely of the GOI. In other words, Figure 20b is a schematic diagram showing that if uracil is present after five unique base sequences at any point in the GOI, that portion becomes the 3' end, and the rest of the GOI on the 3' side is moved immediately after the ribozyme, resulting in circular RNA composed solely of the GOI without additional uracil.

[0049] [Figure 21a] Figure 21a shows various P1 helix variants designed by adding bases in the 5' direction of the 5'-GNNNNN-3' IGS region, Figure 21b is a schematic diagram of the structure and preparation of an expression vector of a self-circularized RNA structure containing the various P1 helix variants, and Figure 21c is a primer for amplification of the vector. In Figure 21a, the red arrow indicates the expected position of the STS junction.

[0050] [Figure 22a] Figure 22a shows the electrophoresis results of the STS reaction products under IVT conditions for self-circular RNA structure expression vectors containing various P1 helix variants; Figure 22b shows the results of electrophoresis after RNase R treatment of the STS reaction products under IVT conditions for the P1 bulge-AS structure expression vector, which is the structure with the highest circular RNA generation efficiency in Figure 22a; Figure 22c shows the results of RT-PCR performed on RNA samples from the circRNA bands based on the electrophoresis results after RNase R treatment; and Figure 22d shows the results of the nucleotide sequence analysis of the RNA extracted from the bands.

[0051] [Figure 23] Figure 23 illustrates the structure of one embodiment of the P1 bulge. Best mode for carrying out the invention

[0052] The inventors have devised a circularization system (Figures 1a and 1b) in which RNA carrying a target gene undergoes self-targeting and splicing reactions using a trans-splicing ribozyme (T / S ribozyme) to prepare circRNA (Figures 1a and 1b).

[0053] Group I intron ribozymes can induce trans-splicing by cleaving target RNA through two consecutive trans-esterification reactions, and then linking transcripts already present at the cleaved 3' ends to each other.

[0054] In this system, an internal guide sequence (IGS) is configured in the 5' direction of the gene of interest (GOI), and a target site is configured in the 3' direction, so that the IGS binds complementarily to the target site. By forming a guanine (G):uracil (U) fluctuation base pair, cleavage and conjugation by a ribozyme located between the GOI and the IGS can be induced to prepare circRNA (Figures 2a and 2b).

[0055] On the other hand, the inventors focused on previous research to improve the trans-splicing efficiency of group I intronic ribozymes (Mol Ther. 2005 Nov;12(5):824-34.) and designed a self-circular RNA structure such that, as diagrammed in Figure 2c, the 5' and 3' ends of the self-circular RNA structure have AS (antisense sequence) regions and ABS (antisense binding sequence) regions that can bind complementaryly to each other, and P1 and P10 helices are formed before / after the secondary structure of the ribozyme. A DNA template was then prepared that can express the RNA structure under a T7 promoter (Example 1).

[0056] Next, a vector with an inserted DNA template was prepared and transcribed in vitro (IVT), after which the formation of circRNA was confirmed. As a result, it was found that the vector expressed a self-circulating RNA structure, and that the RNA structure immediately formed monomeric circRNA after transcription through self-targeting and splicing (STS) reactions, even without the addition of GTP (Example 2).

[0057] Furthermore, the inventors decided to confirm that the self-circular RNA structure expression vector can express the RNA structure of the present invention even within cells, and that the expressed RNA can express the target gene in the form of circRNA. Specifically, an IRES and a termination codon were included in the target gene to enable translation of the gene (transgene) within cells, and a plasmid vector was prepared using gaussian luciferase as the transgene within the target gene. The plasmid vector was then transformed into cells, and the generation of circRNA and luciferase activity were confirmed. As a result, it was confirmed at the molecular level that the prepared plasmid vector expressed a self-circular RNA structure within cells, that the structure was circularized into circRNA by a ribozyme, and that the target gene was expressed in the form of circRNA (Example 3).

[0058] Next, the inventors attempted to optimize the RNA structure so that circRNA could be efficiently formed immediately on IVT by a direct STS reaction.

[0059] First, the direct STS reaction rate based on the length of the AS region on the IVT was confirmed through specific tests. Specifically, self-circularized RNA expression vectors were prepared so that the lengths of the AS and ABS regions were 50, 100, 150, 200, 250, or 300 nt. After incubating the vectors in the IVT, the direct STS efficiency was checked. As a result, it was confirmed that the self-circularization efficiency decreased significantly with lengths of 200 nt or more. On the other hand, the self-circularization efficiency was similar for lengths of 50, 100, and 150 nt, but it was confirmed that the transcription reaction itself in the test tube decreased when AS and ABS regions of 50 or 100 nt length were included. From the viewpoint of cricRNA preparation efficiency, it was confirmed that a length of 150 nt for the AS and ABS regions is preferable (Example 5).

[0060] On the other hand, similar to how the efficiency of circRNA preparation was confirmed based on the length of the AS and ABS regions, the efficiency of circRNA preparation was confirmed on IVT based on the length and base sequence of the spacer region. The results showed that although the length and base sequence did not have a significant effect on the efficiency of circRNA preparation, the linkage of 30 adenine (A) units was sufficient to prevent structural collisions that could occur due to the narrow spacing between the ribozyme and the IRES in the case of ribozyme and EMCV IRES (Example 6).

[0061] In this invention, a group I intronic ribozyme capable of continuous transesterification was used. Group I intronic ribozymes induce trans-splicing by linking transcripts already present at the cleaved 3' ends after target site cleavage. In self-circularized RNA structures, the P1 and P10 helix regions, which are known to enhance trans-splicing efficiency by linking the 5' region of a non-transcript GOI to the cleaved 3' end, and the AS and ABS regions at both ends of the self-circularized RNA structure were investigated to see if they also positively influence the efficiency of circulation.

[0062] Specifically, the inventors prepared self-circular RNA expression vectors containing only the P1 helix region, only the P1 and P10 helix regions, or all of the P1 and P10 helices and the AS region, and after in vitro testing (IVT) of the vectors, they confirmed the direct STS efficiency. Surprisingly, the results showed that the P10 helix region in the self-circular RNA structure reduced the efficiency of circRNA preparation in the presence of the P1 and P10 helix regions. On the other hand, even when only the P1 helix region was included without the AS region, excellent circRNA preparation efficiency was observed (Example 7).

[0063] Furthermore, the inventors designed a self-circular RNA structure such that only the IGS region forms a P1 helix, so that only the target gene can be retained in the final product, circRNA, and confirmed that the STS reaction occurs with various IGS sequences. Surprisingly, the results showed that even when only the IGS region forms a P1 helix, the STS reaction of the self-circular RNA structure expressed in DNA was induced, and circRNA was formed even though the IGS region and the target site region were not complementary to each other. However, when the IGS region and the target site region are not complementary to each other, nonspecific reactions may occur at undesirable sites, potentially generating undesirable products. It was found that the efficiency of circRNA preparation increased as the ratio of complementary binding base sequences between the 5'-GNNNNN-3' IGS region and the 5'-N'N'N'N'U-3' target site region increased, and that higher circRNA preparation efficiency was observed with specific sequences (Example 8).

[0064] Furthermore, the inventors investigated whether the efficiency of circular RNA preparation could be improved by preparing various P1 helix variants by adding bases in the 5' direction of the 5'-GNNNNN-3' IGS. Specifically, they constructed structures in which the added bases in the 5' direction of the IGS form an extension of the P1 helix (P1 extension) and a bulge (P1 bulge), and compared the efficiency of circular RNA preparation of the P1 extension structure, the P1 bulge-AS150 structure, and the P1 extension & bulge-AS150 structure with that of the P1 structure and the P1-P10 structure. As a result, it was confirmed that, unlike the extension of the P1 helix, the formation of a bulge improved the efficiency of self-circularization. Moreover, the P1 bulge-AS150 structure showed a higher circular RNA generation rate than the P1-P10_AS150 structure. From the above, it was found that when a bulge (where RNA appears to be swelling due to the formation of a secondary structure) is present within the P1 helix, the STS reaction induced by ribozymes is more efficiently induced (Example 9).

[0065] On the other hand, a P1 helix refers to a helix structure formed by complementary binding between the base sequence linked in the shear direction (5' direction) of the ribozyme and the base sequence in the 3' direction of the transcript, which is induced to join by the ribozyme during secondary structure formation of a group I intron ribozyme. A P10 helix refers to a helix structure formed by complementary binding between the shear region of the ribozyme and the base sequence in the 5' direction of the transcript, which is cleaved by the ribozyme.

[0066] In the present invention, a P1 helix can be formed by the complementary binding of the IGS at the 5' end and the target site at the 3' end of the self-circular RNA structure of the present invention, and a P10 helix refers to a helix structure formed by the complementary binding of an extended base sequence at the 5' end and a base sequence linked to the trailing end (3' direction) of the ribozyme.

[0067] In this specification, the nucleotide sequence that forms a P1 helix is ​​referred to as the P1 region or P1 helix region, and similarly, the nucleotide sequence that forms a P10 helix is ​​named the P10 region or P10 helix region.

[0068] In the present invention, the nucleotide sequence of the IGS region is 5'-GNNNNN-3', the nucleotide sequence of the target site region is 5'-N'N'N'N'N'U-3', and the P1 helix can be formed by the complementary binding of the IGS region and the target site region. Herein, in order to eliminate redundant representations, the P1 helix region technique may be used in combination with the nucleotide sequence of the IGS region.

[0069] In the present invention, the P1 helix is ​​formed to include an extended nucleotide sequence in the 5' direction of the IGS region, where the nucleotide sequence in the 3' direction of the target site opposite the extended nucleotide sequence may include an inversely complementary sequence to form an extension of the P1 helix, or a bulge may be formed on the P1 helix without including an inversely complementary sequence; however, a bulge may be formed on the P1 helix if preferred.

[0070] In this specification, if a nucleotide sequence is present that is extended in the 5' direction of the IGS region, the extended nucleotide sequence region will be designated as P1 for distinction from the IGS region. The same applies to the P10 helix. In this specification, P1 helix variants modified by a nucleotide sequence extended in the 5' direction of the IGS region will be described as P1 extension and P1 bulge, respectively. An example of a P1 bulge can be diagrammed as shown in Figure 23.

[0071] In the present invention, the number of bases extended in the 5' direction of the IGS region forming the P1 bulge and in the 3' direction of the target site may be independently 1 to 10 nt. That is, the length of the extended bases in each region may be the same or different within the aforementioned numerical range.

[0072] Furthermore, in the present invention, the nucleotide sequences extended in the 5' direction of the IGS region forming the P1 bulge and in the 3' direction of the target site are designed so that not all or part of them complementarily bind to each other, thereby enabling the formation of the bulge.

[0073] On the other hand, in the present invention, the target site region is intended to indicate a region containing bases that form a fluctuating base pair with the IGS region, and may be inversely complementary to the IGS region. Furthermore, the target site region may overlap with the target gene (GOI) region depending on the base sequence design of the IGS region. In this case as well, in order to identify the region that binds complementaryly to the IGS region, the region within the target gene that binds complementaryly to the IGS region is divided and named as the target site. In other words, the target site in the present invention may overlap with part or all of the target gene region, or it may exist separately from the target gene.

[0074] In this invention, the AS (antisense sequence) region is located at the 5' end of the self-circularized RNA structure and is intended to form hydrogen bonds with the base sequence of the ABS (antisense binding sequence) region located at the 3' end of the RNA structure. In this invention, the AS region and the ABS region must coexist, and the absence of the AS region is understood to include the absence of the ABS region. Similarly, an RNA structure containing the AS region is understood to also contain the ABS region. In this invention, the length of the AS may affect the self-circularization efficiency depending on the target gene, and the length of the AS is indicated by the letters "AS" followed by a number. For example, AS150 means an AS region with a length of 150 nt.

[0075] On the other hand, the inventors confirmed the effect of the presence or absence of three types of structures—P1 region, P10 region, and AS region—on the circulation efficiency of self-circular RNA structures by STS reaction. As a result, they confirmed that the amount of circRNA produced is highest when all three regions (P1, P10, and AS) are present, followed by when only the P1 region is present, and then when only the P1 and P10 regions are present. They also confirmed that self-circular RNA structures containing only the P1 region are circulated with sufficient efficiency and produce circRNA. Therefore, the present invention provides a self-circular RNA structure containing only the P1 region.

[0076] Therefore, in this specification, a self-circular RNA structure containing only the P1 region (P1 structure) means that the P10 region and AS region are absent, and is not used to mean that other components are excluded. Similarly, a self-circular RNA structure containing only the P1 region and AS region (P1-AS structure) means that the P10 region is absent, and is not used to mean that other components other than the P10 region are excluded.

[0077] [Modes for carrying out the invention]

[0078] The embodiments will be described in detail below with reference to the attached drawings. However, since various modifications can be made to the embodiments, the scope of the patent application will not be limited or restricted by these embodiments. All modifications, equivalents, or substitutes to the embodiments should be understood to be included within the scope of the patent.

[0079]

[0080] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as “includes” or “having” specify the presence of features, figures, processes, operations, components, parts, or combinations thereof described in the specification, and should not be understood as preemptively excluding the possibility of the presence or addition of one or more other features, figures, processes, operations, components, parts, or combinations thereof.

[0081]

[0082] Unless otherwise defined, all terms used in this invention, including technical or scientific terms, have the same meaning as they would be generally understood by a person of ordinary skill in the art to which the examples belong. Any commonly used terms should be interpreted as having the meaning consistent with their meaning in the context of the relevant art, and not as ideal or overly formal unless explicitly defined in this application.

[0083]

[0084] Furthermore, when explaining with reference to the drawings, the same reference numerals will be used for identical components, regardless of the numerals used in the drawings, and redundant explanations for these components will be omitted. In the description of the embodiments, if it is determined that a specific explanation of related prior art would unnecessarily obscure the gist of the embodiments, such detailed explanations will be omitted.

[0085]

[0086] [Examples]

[0087] Example 1. Design of self-circularized RNA and preparation of RNA expression vectors

[0088] The self-circular RNA was designed as shown in Figure 2c, and the DNA template for its expression further included a T7 promoter sequence for in vitro transcription (Figure 2d). The DNA template was amplified by PCR using T7 Circular Forward primer: 5'-GGGATTCGAACATCGATTAATACGACTCACTATAGGGGCATCGATTGAATTGTCGA-3' (Tm=77.5°C) and T7 Circular Reverse primer: 5'-AGATCTCTCGAGCAGCGCTGCTCGAGGCAAGCTT-3' (Tm=79.4°C). The amplified DNA template product was inserted into a pTOP TAV2 cloning vector (Enzynomics) using PstI restriction enzyme to prepare a self-circular RNA expression vector.

[0089]

[0090] Example 2. Confirmation of in vitro transfer and auto-cyclization.

[0091] 2-1. In vitro transcription (IVT)

[0092] The auto-circular RNA expression vector prepared in Example 1 above was transcribed in vitro using NEB's HiScribe T7 High Yield RNA Synthesis Kit according to the manufacturer's protocol. Specifically, a 20 μL scale (1 μg T7 DNA template, 1X Reaction buffer, 10 mM each ATP, UTP, CTP, GTP, T7 RNA polymerase mix 2 μL) was reacted at 37°C for 3 hours, then 29 μL of nuclease-free water was added, followed by 1 μL of RNase-free DNase I (10 U / μL), and the mixture was reacted at 37°C for 30 minutes to induce a direct STS reaction immediately after transcription.

[0093] Next, to confirm the completion of the autocyclization reaction, an additional cyclization reaction was induced. Specifically, the initial autotargeting and splicing (1 st To induce the STS reaction, 28 μL of nuclease-free water, 20 μL of 5X STS buffer (50 mM Hepes (pH 7.0), 150 mM NaCl, 5 mM MgCl2), and 2 μL of 100 mM GTP (final 2 mM) were added to create a 100 μL volume, and the auto-cyclization reaction was carried out at 37°C for 1 hour. After heating at 55°C for 15 minutes, column purification was performed using the Monarch RNA cleanup kit (NEB). To the column-purified 50 μL sample, 20 μL of 5X STS buffer, 100 mM GTP (final 2 mM), and 28 μL of nuclease-free water were added again to create a final 100 μL volume, and the mixture was reacted at 37°C for 3 hours to produce the second STS(2 nd The STS reaction was induced. After the reaction, the mixture was heated at 55°C for 8 minutes, and column purification was performed using the Monarch RNA cleanup kit (NEB). The concentration was measured using a Nanodrop (Thermo Fisher Scientific product).

[0094] On the other hand, to remove linear RNA from the reaction product, a portion of the column-purified samples after the first and second STS reactions were treated with RNase R. Specifically, up to 100 μg of column-purified IVT RNA was treated with 10 μL of 10X RNase R reaction buffer (10X: 0.2M Tris-HCl pH 8.0, 1M KCl, 1 mM MgCl2), 20 units of RNase R (adjusted to 100 μL with water), and reacted at 37°C for 30 minutes. Then, another 10 units of RNase R were added and the reaction was continued for another 30 minutes. After that, column purification was performed using the Monarch RNA cleanup kit (NEB), and the concentration was measured with Nanodrop.

[0095] 250 ng each of the RNA obtained from each STS step and the RNA obtained from each step treated with RNase R were mixed in a 1:1 ratio with 10M Urea-BPB (1X TBE) dye, heated at 75°C for 5 minutes, and then subjected to 4% Polyacrylamide-7M Urea denature PAGE (electrophoresis at 50°C for 2 hours under 50W conditions). The gel was stained with SYBR Gold Nucleic Acid Stain (Thermo Fisher Scientific) and analyzed using ImageQuant 800 (Cytiva).

[0096] As a result, as can be seen in Figure 3, when treated with RNase R, an RNA band appeared that was not properly cleaved by RNase R and became abundant (Candidate 1), and in addition, bands that were clearly observable despite RNase R treatment were identified (Candidate 2 and a band presumed to be Nicked circular RNA). Furthermore, an additional 1 st and 2 nd It has been confirmed that even without performing an STS reaction, sufficient amounts of substances presumed to be circRNA can be produced through a direct STS reaction, i.e., a transcription reaction in a test tube alone.

[0097]

[0098] 2-2. Confirmation of self-circularization

[0099] Next, to verify that candidate 1, among the RNA bands not cleaved by RNase R as a result of PAGE, was circRNA, RT-PCR sequencing analysis was performed. Specifically, after cleaving and crushing the band at the position of candidate 1 in PAGE, the circular RNA sample was purified by elution in water at 37°C for 3 to a maximum of 16 hours and ethanol precipitation. A linear RNA that lacked ribozyme and antisense sites and could not be circularized was used as a control group, and RT-PCR was performed using primers that allowed PCR amplification only when circRNA was formed.

[0100] Reverse transcription (RT) was performed using OneScript Plus RTase (Abm). 125 ng of each RNA (control RNA and presumed circular RNA, treated with RNase R or untreated) was heated at 70°C for 5 minutes, then placed on ice. 4 μL of 5X RT buffer, 1 μL of 10 mM dNTP mix, 1 μL of 2 μM reverse primer (Circular STS R), and 200U of OneScript Plus RTase were added in that order. The final volume was 20 μL and reacted at 50°C for 15 minutes. The enzyme was then deactivated by heating at 95°C for 5 minutes before being stored on ice.

[0101] Next, PCR was performed using AccuPower Taq PCR premix (BIONEER). 2 μL of RT sample and 1 μL each of 20 μM Circular STS F (5'-CCCTGAGTGGCTGAGCTCAGG-3') and Circular STS R (5'-CAGCAAGCATACTAAATTGCCAG-3') were added, and the volume was adjusted to 20 μL with water. PCR amplification was performed at 95°C for 1 minute, followed by 35 cycles of [95°C for 30 seconds, 65°C for 30 seconds, 72°C for 30 seconds], and then at 72°C for 5 minutes. 5 μL of the PCR product was placed in 1 μL of 6X DNA loading die and subjected to electrophoresis at 150V for 35 minutes. The images were then analyzed using a gel imaging system (Davinch-Gel product from YOUNG IN SCIENTIFIC). The expected length of the STS PCR product was 479 bp, and when analyzed on a 1.5% agarose gel (containing Intron Bio's RedSafe Nucleic Acid Staining Solution at 1X concentration) using a GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific), a specific PCR product of the predicted size was observed only in RNA samples presumed to be circular RNA, regardless of RNase R treatment (Figure 4a).

[0102] Furthermore, in the bands of the expected size obtained, PCR products were isolated and purified using a gel extraction kit (COSMO Genetech product) according to the manufacturer's protocol, cloned using a TOPcloner TA-Blunt kit (Enzynomics product), transformed into DH5alpha Escherichia coli (Chemically competent E. coli, Enzynomics product), obtained E. coli colonies on an LB-Agar (containing Kanamycin) plate, and plasmid DNA was extracted and purified using a DNA purification kit (COSMO Genetech product) according to the manufacturer's protocol. The results of the sequencing analysis (COSMO Genetech's Sanger sequencing service, using M13R(-40) or M13F(-20) universal primer provided by the supplier) confirmed that the 3' of Gaussia Luciferase and the 5' of IRES were accurately linked at the STS junction site (Figure 4b).

[0103] From the results above, it can be seen that candidate 1 is a circular RNA.

[0104]

[0105] 2-3. Re-examining self-circularization

[0106] RT-PCR confirmed that candidate 1 is a circRNA, but theoretically, the possibility that candidate 1 is in the form of a dimer cannot be ruled out. Therefore, we decided to perform a nicking test to re-verify that candidate 1 is a circRNA.

[0107] Purified circular RNA candidate 1 and candidate 2 (100 ng each) were mixed with MgCl₂ to final concentrations of 0, 2.5 and 5 mM respectively. Samples dissolved in a final volume of 10 µL water were heated at 65°C for 30 minutes, stored on ice for a while, then mixed with 10 µL of 10 M Urea-BPB (1X TEB) loading dye.

[0108] After heating at 75°C for 5 minutes, 4% polyacrylamide-7 M urea denature PAGE (maintained at 50°C, electrophoresis performed at 50 W for 2 hours) was carried out. The gel was stained with SYBR Gold Nucleic Acid Stain (Thermo Fisher Scientific) and analyzed with ImageQuant 800 (product of Cytiva). The results are shown in Figure 5.

[0109] For candidate 1, Mg 2+ In the absence of Mg²⁺, a faint band corresponding in size to nicked circular RNA (1092-nt) was observed. Under the 2.5 mM Mg 2+ condition, it was confirmed that the intensity of the band at the position of candidate 1, which is considered the position of circular RNA, decreased, and a band corresponding to the size of nicked circular RNA was still present. Under the 5 mM Mg 2+ condition, it was confirmed that even the nicked circular RNA band completely disappeared via hydrolysis. Accordingly, when nicking occurs to candidate 1, i.e., the circular RNA, it was observed that the product passes through the 1092-nt size (at 2.5 mM Mg 2+ ), which is predicted to be the monomer size, and finally completely degrades (at 5 mM Mg 2+ ). Thus, it can be reconfirmed that candidate 1 is circular RNA and is a monomer.

[0110] On the other hand, candidate 2 has a size similar to 1874-nt, which is the size of intact RNA (around 2000-nt of the marker), and at 2.5 mM Mg 2+Under mild nicking conditions, the 1092-nt band, which represents a different size of nicked circular RNA than candidate 1, was not generated and disappeared, indicating that it was not a circular RNA.

[0111]

[0112] Example 3. Confirmation of intracellular transcription and autocyclic formation.

[0113] In Example 2, we confirmed that the self-circular RNA expression vector prepared in Example 1 was transcribed in vitro and formed circRNA. We then decided to confirm whether the vector functions similarly in cells, and further, whether the target gene contained in the circRNA is smoothly expressed in cells.

[0114] To express the target gene within the cell, the target gene was designed with the structure 5'-EMCV IRES-transgene-stop codon-3', and Gaussial luciferase (G. luci) was used as the transgene for easy confirmation of the expressed target gene.

[0115] Autocyclic and RNA structures containing the target gene described above were designed, and a DNA template capable of expressing these structures was inserted into a plasmid under the influence of a pCMV promoter (Figure 6a). The plasmid vector was transfected into 293A cells, and the generation of circRNA was confirmed by RT-PCR and sequencing analysis. Transgene expression was confirmed by luciferase activity analysis.

[0116] Specifically, 2 x 10 in a 6-well plate 5293A cells were seeded in a single well and transformed with a plasmid vector using lipofectamine 2000 transfection reagent 24 hours later. The culture medium was changed 6 hours after transformation. Then, 100 μL of culture medium was collected at 12, 24, and 48 hours after transformation and G. luci activity was measured. G. luci activity was detected in the culture medium and increased over time, confirming that the transgene G. luci gene was expressed in the intracellularly introduced vector (Figure 6b).

[0117] We confirmed at the molecular level whether transgene expression was due to circRNA formation using RT-PCR and nucleotide sequence analysis. 48 hours after transformation, total RNA was extracted from 293A cells into which the plasmid vector had been introduced using trizol reagent, and then RT-PCR was performed to confirm the generation of circular RNA.

[0118] Reverse transcription (RT) was performed using OneScript Plus RTase (Abm product). 1 μg of total RNA was heated at 70°C for 5 minutes, then placed on ice. 4 μL of 5X RT buffer, 1 μL of 10 mM dNTP mix, 1 μL of 2 μM reverse primer (Circular STS R), and 200U of OneScript Plus RTase were added in sequence. Finally, a total volume of 20 μL was reacted at 50°C for 15 minutes, then heated at 95°C for 5 minutes to deactivate the enzyme, and the mixture was stored on ice.

[0119] Next, PCR was performed using AccuPower Taq PCR premix (BIONEER product). 2 μL of RT sample was mixed with 1 μL each of 20 μM Circular STS primer F (5'-caaggacttggagcccatggagcag-3') and primer R (5'-tgtgccgcctttgcaggtgtatc-3'), and the volume was adjusted to 20 μL with water. PCR amplification was performed at 95°C for 1 minute, followed by 35 cycles of [95°C for 30 seconds, 65°C for 30 seconds, 72°C for 30 seconds], and then at 72°C for 5 minutes. The expected length of the STS PCR product was 479 bp. Analysis on a 1.5% agarose gel (containing Intron Bio's RedSafe Nucleic Acid Staining Solution at 1X concentration) using a GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific product) confirmed a specific PCR product of the size predicted only when circular RNA is present. Here, 5 μL of PCR product was placed in 1 μL of 6X DNA loading die and subjected to electrophoresis at 150V for 35 minutes, after which the images were analyzed using a gel imaging system (Davinch-Gel product from YOUNG IN SCIENTIFIC) (Figure 6c).

[0120] Furthermore, the bands of the expected size obtained were isolated and purified as PCR products using a gel extraction kit (COSMO Genetech product) according to the manufacturer's protocol, and cloned using a TOPcloner TA-Blunt kit (Enzynomics product). These were transformed into DH5alpha Escherichia coli (Chemically competent E. coli, Enzynomics product), and E. coli colonies were obtained on an LB-Agar (containing Kanamycin) plate. Plasmid DNA was extracted and purified using a DNA purification kit (COSMO Genetech product) and manual, and the results of the sequencing analysis (COSMO Genetech's Sanger sequencing service, using M13R(-40) or M13F(-20) universal primer provided by the vendor) confirmed that the nucleotide sequence in which the 3' of Gaussia Luciferase and the 5' of IRES were accurately linked at the STS junction site was confirmed (Figure 6d).

[0121] From the above, we were able to confirm at the molecular level that the prepared plasmid vector expresses a self-circulating RNA structure in cells, that the structure is circularized into circRNA by a ribozyme, and that the target gene is expressed in the circRNA.

[0122]

[0123] Example 4. CircRNA purification

[0124] 4-1. Confirmation of the possibility of circRNA purification by HPLC.

[0125] To minimize immunogenicity during the preparation of circRNA for human injection, analysis and purification were performed using HPLC. Specifically, an agilent 1290 Infinity II Bio UHPLC system was used, and the analytical conditions are as shown in Figure 7A. The [Analytical] gradient condition was used for analysis, and the [Fraction collection] condition was used for sample preparation.

[0126] After performing IVT, we analyzed RNA purified only by column purification using the Monarch RNA cleanup kit (NEB) (Figure 7B) and RNA treated with RNase R (Figure 7C). We found an increased peak in the RNase R-treated sample, indicating that circRNA can be separated by HPLC without RNase R treatment.

[0127]

[0128] 4-2. CircRNA purification by HPLC

[0129] Fraction collection was performed via HPLC using the gradient specified in the fraction collection conditions, and each fraction was obtained as shown in Figure 8a. Fractions 7 and 10-13, which had clearly defined peaks, were subjected to 4% denature PAGE (200 ng each), and electrophoresis was performed in the same manner as described above.

[0130] As a result, clean circular RNA was isolated and purified in fraction 12, as can be seen in Figure 8b.

[0131]

[0132] Example 5. Optimization of the AS (ANTISENSE SEQUENCE) region

[0133] We attempted to investigate the influence of the lengths of the AS (ANTISENSE SEQUENCE) region and its inversely complementary ABS (ANTISENSE BINDING SEQUENCE) region on the immediate cyclization reaction during in vitro transcription. Here, DNA templates with different AS and ABS region lengths of 50, 100, 150, 200, 250, or 300 nt were prepared (Figure 9). Vectors capable of expressing each RNA structure were prepared in the same manner as in Example 1, and in vitro transcription was performed at 37°C for 3 hours in the same manner as in Example 2. Immediately afterward, the degree of STS reaction was examined by PAGE on 4% Polyacrylamide-7 M Urea gel (20 × 20 cm, 1 mm) and compared using relative band intensity.

[0134] The nucleotide sequences of each AS region are shown in Table 1 below.

[0135] [Table 1]

[0136] As a result, as can be seen in Figure 10 and Table 2 below, the self-circulation efficiency was relatively superior for AS50, AS100, and AS150 compared to lengths of AS200 or longer. On the other hand, AS50 and AS100 produced significantly less total RNA after the transcription reaction in vitro. Therefore, it was found that AS150 is the optimal length for the expression of self-circulated RNA structures and for the preparation of circular RNA.

[0137] [Table 2]

[0138] Example 6. Optimization of the spacer area

[0139] Next, to confirm the effect of spacer region length or type on the immediate cyclization reaction during in vitro transcription, DNA templates containing spacer regions of different lengths and base sequences were prepared (Figure 11). Vectors capable of expressing each RNA structure were prepared in the same manner as in Example 1, and in vitro transcription was performed at 37°C for 3 hours, as in Example 2. The degree of STS reaction was immediately assessed by PAGE on 4% Polyacrylamide-7 M Urea gel (20 × 20 cm, 1 mm) and compared using relative band intensity. The base sequences of each spacer region were as shown in Table 3 below. In this study, A10, A30, and A30 spacers were used with a restriction site added to the 3' end immediately after the spacer region for IRES insertion. In this study, an AatIIsite (GACGTC) was added to the 3' end of the spacer.

[0140] [Table 3]

[0141] As a result, as can be seen in Figure 12 and Table 4 below, despite differences in spacer length and sequence, there was no significant difference in the immediate circulation efficiency during the in vitro transcription process. However, it was found that A30 acts as the optimal spacer for the expression of self-circularized RNA structures and the preparation of circular RNA.

[0142] [Table 4]

[0143] Example 7. Optimization of auto-circular RNA structure

[0144] 7-1. AS region, P1 helix, and P10 helix region

[0145] The self-circularized RNA structure designed in Example 1 includes an AS region, a P1 helix, and a P10 helix region. To confirm the effect of each configuration on the immediate circulation reaction during the transcription process in vitro, DNA templates containing only the P1 helix region, only the P1 and P10 helix regions, or all of the P1 and P10 helices and the AS region were prepared, as illustrated in Figure 13. Vectors capable of expressing each RNA structure were prepared in the same manner as in Example 1, and then in vitro transcription was performed at 37°C for 3 hours, as in Example 2. The degree of the STS reaction was immediately checked by PAGE on 4% Polyacrylamide-7 M Urea gel (20 × 20 cm, 1 mm) and compared using relative band intensity.

[0146] The nucleotide sequence of the T7 DNA template containing only the P1 helix region is shown in Figure 14.

[0147] As a result, as can be seen in Figure 15 and Table 5 below, there was no significant difference in the total RNA generated by each vector in vitro. However, it was found that the amount of circular RNA generated was highest when the vector contained all of the P1 and P10 helices and the AS region, followed by when it contained only the P1 helix region, and then when it contained only the P1 and P10 regions.

[0148] [Table 5]

[0149]

[0150] 7-2. Validation of Circularization of Self-Circularized RNA Structures that Do Not Contains P10 and AS Regions

[0151] The results of Example 7-1 show that sufficient circRNA can be prepared using a DNA template (P1 structure) that does not contain the P10 and AS regions, without any additional circularization steps during the in vitro transcription process. To verify this, the post-STS reaction product of Example 7-1 was treated with RNase R to remove linear RNA, PAGE was performed as in the previous test, and RT-PCR and nucleotide sequence analysis were performed as in Example 2-2.

[0152] As a result, as can be seen in Figures 16a and 16b, even when self-circulation was performed using the P1 structure, an abundant RNA band was observed that was not properly cleaved by RNase R upon treatment with RNase R. The STS reaction product of the band was then subjected to RT-PCR and nucleotide sequence analysis, confirming that it was circRNA.

[0153]

[0154] Example 8. Optimization of the P1 helix region

[0155] The results of Example 7 confirmed that self-circularized structures without P10, AS, and ABS can also sufficiently produce circRNA. Here, we hypothesized that circular RNA would be generated if only the internal guide sequence (IGS) located at the 5' end of the P1 helix and the base sequence of the target site located at the 3' end were joined complementaryly (U and G are fluctuation base pairs), and we decided to verify this.

[0156] The IGS region is 5'-GNNNNN-3', and the target site sequence is 5'-N'N'N'N'N'U-3'. Therefore, by adding only one U nucleotide to the GOI, the resulting circRNA consists only of one U base and the GOI region (Figures 1a and 1b). Furthermore, if the 3' end of the GOI ends with a U base, the IGS region sequence can be designed to be inversely complementary to the GOI, so that the resulting circRNA consists only of the GOI region (Figures 20a and 20b).

[0157] As shown in Figure 17, various arrangements of IGS and target sites were designed, and after preparing the vector using the method of Example 1, in vitro transfer was performed as in Example 2-1.

[0158] As a result, as can be seen in Figures 18-19 and Table 6 below, when both ends have complementary sequences, an increase in circRNA generated by the STS reaction can be observed immediately after in vitro transcription, and it was found that vectors containing a no-complement IGS region generate very low levels of circRNA. From the above, it can be seen that if the IGS and target site are complementary to each other, both can efficiently induce the auto-circulation reaction.

[0159] [Table 6]

[0160] Example 9. Deformation of the P1 helix region

[0161] In Example 7, it was confirmed that the P10 region reduces the efficiency of circular RNA preparation in the absence of the AS region, and in Example 8, it was confirmed that the auto-circularization reaction can be efficiently induced when the IGS forming the P1 helix and the target site are designed to be inversely complementary sequences.

[0162] Next, we modified the P1 helix region generated by adding bases in the 5' direction of the 5'-GNNNNN-3' IGS region and in the 3' direction of the 3'-N'N'N'N'N'U-3' target site region to see if it affected the efficiency of circular RNA preparation. Specifically, we decided to check the effect on circular RNA preparation efficiency when the length of the P1 helix was extended by designing the bases extended in the 5' direction of the IGS region and the 3' direction of the target site region to be inversely complementary to each other (hereinafter referred to as "P1 extension"), or when the extended bases were designed not to complement each other to form a P1 helix containing a bulge (hereinafter referred to as "P1 bulge").

[0163] Here, as shown in Figure 21a, we designed P1 extension, P1 bulge + AS structure, and P1 extension & bulge + AS structure by adding bases in the 5' direction of the IGS region. To prepare the expression vectors for each of the above structures, we first synthesized the respective gene sections using gBlock and then inserted them into the In-fusion HD cloning kit (TAKARA) using restriction enzymes (PstI, NheI, Pml, and SpeI) (Figure 21b). The prepared expression vectors for each of the above structures were amplified by PCR using the primers shown in Figure 21c.

[0164] Next, the in vitro transcription and direct STS reaction of Example 2-1 were induced using the prepared vectors for expressing each of the structures, and column purification of Example 2-1 was performed without RNase R treatment. The concentration was measured using Nanodrop equipment. 250 ng of each sample was mixed 1:1 with 10 M Urea-BPB (1X TBE) dye, heated at 75°C for 5 minutes, and then subjected to 4% Polyacrylamide-7 M Urea denature PAGE (electrophoresis at 50W for 2 hours while maintaining a temperature of 50°C). The gel was stained with SYBR Gold Nucleic Acid Stain (Thermo Fisher Scientific) and analyzed using ImageQuant 800 (Cytiva).

[0165] As a result, as can be seen in Figure 22a, higher circular RNA generation was observed with the P1 bulge-AS150 structure compared to the P1 structure, and furthermore, the P1 bulge-AS150 structure was superior in circular RNA preparation efficiency to the P1-P10-AS150 structure. On the other hand, the extension of the P1 helix did not appear to affect the self-circularization efficiency of the structure. Neither the P1 extension nor the P1 extension-bulge-AS150 structure showed a meaningful improvement in self-circularization efficiency.

[0166] On the other hand, in order to reconfirm the high round RNA generation result of the P1 bulge-AS150 structure as described above, a portion of the sample purified by column to remove linear RNA with the reaction product after in vitro transcription and direct STS reaction induction was treated with RNase R and PAGE was performed (Figure 22b). Then, the band at the circRNA position was selected from the PAGE results and RT-PCR as in Example 2-2 was performed (Figure 22c), and the base sequence was analyzed (Figure 22d). From the above results, it was confirmed that the P1 bulge-AS150 structure forms round RNA after the direct STS reaction under IVT conditions.

[0167]

[0168] Although embodiments of the present invention have been described in detail above with reference to the drawings, a person with ordinary skill in the art can apply various technical modifications and variations based on the above. For example, the described techniques may be performed in a different order than described, and / or the components of the described systems, structures, devices, circuits, etc. may be combined or assembled in a different manner than described, and substituted or replaced by other components or equivalents, and still the appropriate results may be achieved.

[0169] Therefore, other embodiments, other examples, and those equivalent to the claims described below also fall within the scope of the claims.

[0170] [Industrial applicability]

[0171] The present invention can be used for the preparation of circRNA for protein expression in vitro, intracellularly, and in vivo, and can be used to prepare functional RNAs such as miRNA, anti-miRNA, shRNA, aptamer, mRNA vaccines, mRNA therapeutics, antibodies, vaccine adjuvants, and CAR-T mRNA using circRNA.

Claims

1. A self-circular RNA structure, The aforementioned structure has the structure 5'-IGS (internal guide sequence)-ribozyme-target gene-target site-3', The IGS region includes 5'-GNNNNNN-3', the target site includes 5'-N'N'N'N'U-3', and the guanine (G) in the IGS region forms a fluctuating base pair with the uracil (U) in the target site. The 5'-GNNNNN-3' base sequence of the IGS region, excluding the guanine, consists of a sequence that is inversely complementary to the base sequence of the target site region, forming a secondary structure (P1 helix). The IGS region includes a base sequence that is extended in the 5' direction of 5'-GNNNNN-3', The target region includes a base sequence that is extended in the 3' direction of 5'-N'N'N'N'U-3', The IGS and the extended base sequence region in the target site region form a bulge in the P1 helix, The ribozyme is a group I intronic ribozyme. Self-circularizing RNA structure.

2. The self-circularized RNA structure according to claim 1, wherein the target site region overlaps with the target gene region.

3. The self-circularized RNA structure according to claim 1, characterized in that the lengths of the base sequences extended in the 5' direction of the IGS region forming a bulge in the P1 helix and in the 3' direction of the target site are each independently 1 to 10 nt.

4. The ribozyme comprises the nucleotide sequence of Sequence ID No. 6, as described in claim 1, for the self-circular RNA structure.

5. The self-circular RNA structure according to claim 1, wherein the structure does not form a P10 helix.

6. The aforementioned structure has an AS (antisense sequence) region in the 5' direction of the IGS region, An ABS (antisense binding sequence) region that can bind complementaryly to the AS region in the 3' direction of the target site, The self-circularized RNA structure according to claim 1, further comprising:

7. The self-circular RNA structure according to claim 6, wherein the length of the AS region is 50 to 400 nt.

8. The self-circular RNA structure according to claim 1, wherein the target gene region includes an IRES (internal ribosome entry site) region at its 5' end.

9. The self-circular RNA structure according to claim 1, wherein the structure is such that the ribozyme region and the target gene region are linked to a spacer region consisting of a random base sequence.

10. The self-circular RNA structure according to claim 1, wherein the structure is linked to a spacer region consisting of a random base sequence, wherein the target gene region and the target site region are connected.

11. A vector for expressing the self-circular RNA structure described in claim 1.

12. The vector according to claim 11, wherein the vector comprises a promoter operably linked to a gene that encodes a self-circular RNA structure.

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