Self-circularizing RNA constructs

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] The present invention relates to an RNA construct with improved self-circularization efficiency.

[0002] This invention was completed with support from the Ministry of Science and ICT under the "Development of Basic Core Technology for Next-Generation Vaccines for Infectious Diseases" project of the Korea Research Foundation (Project Number: 2022M3E5F1017657). [Background technology]

[0003] Circular RNA (circRNA, cRNA) is a single-stranded transcript linked by a covalent bond. RNA-seq data and newly developed bioinformatics approaches have identified tens of thousands of circRNAs in various organisms. In eukaryotes, circRNAs are generated from mRNA via back-splicing and are known to function as microRNA sponges in vivo, regulating gene expression. It is unknown whether circRNAs cause immunogenicity, and their structural properties make them highly stable in vivo.

[0004] Recently, there has been active development of therapeutic drugs using messenger RNA (mRNA). However, mRNA has limitations, such as its easy degradation in vivo and its relatively short half-life. To overcome these limitations, research is underway to improve stability by adding a poly(A) tail to mRNA. Similarly, U.S. Patent No. 10,953,033 discloses circRNA for the purpose of in vivo gene expression based on the structural properties of circRNA. Summary of the Invention [Problem to be solved by the invention]

[0005] The technical problem to be solved by the present invention is to provide an RNA construct that undergoes targeting and splicing reactions by itself to be circularized.

[0006] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0007] In order to solve the above problems, the present invention provides a self-circularizing RNA construct having the following structure:

[0008] 5'-IGS (internal guide sequence)-ribozyme-gene of interest-target site-3'.

[0009] In one embodiment of the present invention, the IGS region forms a guanine (G):uracil (U) wobble base pair with the target site, the guanine that forms the wobble base pair is located at the 5' end of the IGS region, and the uracil that forms the wobble base pair is located at the 3' end of the target site region, and the IGS region consists of adenine (A) or uracil in addition to the base that forms the wobble base pair.

[0010] In another embodiment of the present invention, the IGS region may comprise or consist of a nucleotide sequence of 5'-GNNNNN-3', and the target site region may comprise or consist of a nucleotide sequence of 5'-N'N'N'N'N'U-3'. N in the IGS region and N' in the target site region may each independently be A or U, and the IGS region may contain A and U in a ratio of 2:3 or 3:2.

[0011] In a further embodiment of the present invention, the base sequence of the IGS region may be reverse complementary to the base sequence of the target site region, excluding the guanine.

[0012] In another embodiment of the present invention, the ribozyme may be a Group I intron ribozyme, and the ribozyme may comprise or consist of the nucleotide sequence of SEQ ID NO:6.

[0013] In a further embodiment of the present invention, the construct comprises nucleotides extending in the 5' direction of the IGS region to form a P1 helix and a P10 helix, wherein the P1 helix is ​​formed in a region where the IGS region and the target region bind complementary to each other with nucleotides extending in the 3' direction of the target region, and the P10 helix is ​​formed in a region where the nucleotides extending in the 5' direction of the IGS region bind complementary to a sequence reverse-complementary to the extended nucleotides located between the ribozyme and the GOI region. The length of the extended nucleotides forming the P1 helix may be 3 nt, and the length of the extended nucleotides forming the P10 helix may be 6 nt.

[0014] In a further embodiment of the present invention, the construct may form a P1 helix but not a P10 helix.

[0015] In a further embodiment of the present invention, the construct may include complementary binding regions and an ABS (antisense binding sequence) region at the 5' and 3' ends.

[0016] In a further embodiment of the present invention, the ABS region comprises a sequence that is reverse complementary to the AS region.

[0017] In a further embodiment of the present invention, the length of the AS region may vary depending on the GOI, but may be 10 to 500 nt, preferably more than 50 nt and less than 400 nt, and more preferably 150 to 350 nt.

[0018] In a further embodiment of the present invention, the GOI region may include an internal ribosome entry site (IRES) region at the 5' end, and may include a start codon and a stop codon.

[0019] In a further embodiment of the present invention, the construct may further include a spacer region consisting of a random base sequence, and the spacer region may be located between the IGS region and the gene of interest region and / or between the gene of interest region and the target site region.

[0020] In a further embodiment of the present invention, the spacer region may comprise or consist of poly(A), which is a polynucleotide in which adenine (A) is repeatedly linked, and the A may be repeatedly linked 10 to 50 times, preferably 30 times. [Effects of the Invention]

[0021] The self-circularizing RNA construct of the present invention can be expressed in a DNA vector and simultaneously circularized by self-targeting and splicing without additional GTP processing to form circRNA. The circRNA consists only of a target gene, which has the advantage of enabling rapid expression of a peptide or protein containing an IRES region, initiation codon, and termination codon. Furthermore, since the 5' and 3' ends of the circRNA are not exposed due to its circular structure, it has stability and a long half-life. Functional RNAs such as miRNA, anti-miRNA, shRNA, aptamer, mRNA vaccine, mRNA therapeutic, antibody, vaccine adjuvant, CAR-T mRNA, genome, or RNA editing-inducing RNA can be prepared using circRNA and exhibit high intracellular stability. [Brief explanation of the drawings]

[0022] [Figure 1a-1b] Figures 1a and 1b are schematic diagrams illustrating the process by which the self-circularizing RNA construct of the present invention, which contains only an IGS, a ribozyme, a gene of interest, and a target site region, is formed into a circRNA through a self-targeting and splicing (STS) reaction.

[0023] [Figure 2a] Figure 2a is a schematic diagram of the process by which the self-circularizing RNA structure of the present invention, which includes an AS, an IGS, a ribozyme, a gene of interest, a target site, and an ABS region, is formed into a circRNA by the STS reaction.

[0024] [Figure 2b] Figure 2b is a schematic diagram of a self-circularizing RNA construct of the present invention, which contains an IRES and a termination codon in the target gene region to enable translation of the transgene, and further contains nucleotides extended in the 5' direction to enable the formation of P1 helix and P10 helix, and the process by which the construct is converted into a circRNA by an STS reaction.

[0025] [Figure 2c] FIG. 2c shows one embodiment of a self-circularizing RNA construct of the present invention.

[0026] [Figure 2d] FIG. 2d shows the base sequence of a DNA template for preparing an expression vector for the self-circularizing RNA construct of the present invention.

[0027] [Figure 3] Figure 3 shows the results of polyacrylamide gel electrophoresis to confirm the generation of circRNAs immediately after in vitro transcription of the self-circularizing RNA construct expression vector of the present invention without additional GTP treatment. Samples collected immediately after transcription (direct STS) and samples after the primary and secondary STS reaction steps, in which additional GTP is added to induce the primary and secondary circularization reactions, were used. A portion of each sample was treated with RNase R to remove linear RNA (linear RNA) and concentrate the circRNAs within the sample, followed by electrophoresis.

[0028] [Figure 4] Figure 4 shows the verification that Candidate 1, which is presumed to be a circRNA based on the results shown in Figure 3, is a circRNA. Figure 4a shows the results of RT-PCR performed on RNA extracted from the Candidate 1 band, and Figure 4b shows the results of analysis of the base sequence of the RNA extracted from the Candidate 1 band.

[0029] [Figure 5] Figure 5 shows the results of verifying that the RNA in the candidate 1 band is a monomer based on the results shown in Figure 4. It shows the results of electrophoresis after RNA was extracted from the candidate 1 and 2 bands, which were presumed to be circRNAs in the results shown in Figure 3, and nicked by treating with Mg2+.

[0030] [Figure 6]Figure 6 shows the results of confirming that the self-circularizing RNA construct expression vector of the present invention generates circRNA in cells and expresses a transgene contained in the circRNA. Specifically, Figure 6a shows the structure of the self-circularizing RNA construct 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 in the circRNA.

[0031] [Figure 7-8] Figures 7 and 8 show the circRNA purification conditions and results by HPLC.

[0032] [Figures 7a-8b] Specifically, Figure 7a shows the HPLC analysis conditions using an Ultra HPLC system, Figure 7b shows the results of column purification of a sample collected immediately after in vitro transcription, and Figure 7c shows the results of column purification of the sample after treatment with RNase R. Also, Figure 8a shows the results of confirming peaks in fractions collected through column purification, and Figure 8b shows the results of electrophoresis of fractions 7, 10, 11, 12, and 13, which have prominent peaks in Figure 8a.

[0033] [Figure 9-10] Figures 9 and 10 confirm the effect of the AS region on the STS reaction and circularization in the self-circularizing RNA construct of the present invention. Figure 9 shows the structure of self-circularizing RNA containing AS regions of different lengths, and Figure 10 shows the results of electrophoresis of a sample obtained after in vitro transcription of a vector expressing the RNA.

[0034] [Figure 11-12]Figures 11 and 12 confirm the effect of the spacer region in the self-circularization and RNA constructs of the present invention on the STS reaction and circularization. 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 results of electrophoresis of samples obtained after in vitro transcription of a vector expressing the RNA.

[0035] [Figure 13] Figure 13 shows the base sequences and each region near the region cleaved by the ribozyme in a self-circularizing RNA containing only the P1 region; a self-circularizing RNA containing only the P1 and P10 regions; and a self-circularizing RNA containing the P1, P10, and AS regions.

[0036] [Figure 14] FIG. 14 shows the base sequence of a DNA template for preparing a self-circularizing RNA expression vector containing only the P1 region, without the P10 and AS regions.

[0037] [Figure 15] FIG. 15 shows the results of electrophoresis of samples obtained after in vitro transcription of each of the self-circularized RNA expression vectors shown in FIG.

[0038] [Figures 16a-16b] Figures 16a and 16b show the results of electrophoresis confirming that a self-circularizing RNA structure was formed into circRNA after in vitro transcription in a self-circularizing RNA expression vector containing only the P1 region, but not the P10 or AS region.

[0039] [Figure 17-19]Figures 17 to 19 show the effects of the base sequence of the P1 region in the self-circularization and RNA constructs of the present invention on the STS reaction and circularization. Specifically, Figure 17 shows P1 regions with different base sequences, Figure 18 shows the results of electrophoresis of samples obtained after in vitro transcription of a self-circularization RNA expression vector having the P1 region of Figure 17, and Figure 19 shows the results of electrophoresis of samples obtained after in vitro transcription of a self-circularization RNA expression vector having an AU-rich P1 region and a self-circularization RNA expression vector having a 2-site P1 region, as well as the results of RT-PCR electrophoresis confirming the circular RNA.

[0040] [Figure 20] Figure 20 briefly illustrates the essential components of a self-circularizing RNA construct. Figure 20a shows that circRNAs can be prepared using only an RNA construct containing an IGS, ribozyme, and GOI region and only a uracil base at the 3' end. Figure 20b shows that circRNAs can be prepared using only an RNA construct containing only an IGS, ribozyme, and GOI region if the GOI contains a uracil base at its 3' end. In this case, the resulting circRNA is composed solely of the GOI. In other words, Figure 20b shows that if any part of the GOI contains a uracil after a unique five-base sequence, that part is used as the 3' end, and the remaining 3' end of the GOI is sent immediately after the ribozyme, resulting in a circular RNA composed solely of the GOI without any additional uracil.

[0041] [Figure 21] FIG. 21 shows various combinations of AU-rich target sites designed.

[0042] [Figure 22a-22b] Figures 22a and 22b show denaturation PAGE results confirming circular RNA as a result of electrophoresis of a sample obtained after in vitro transcription of a self-circularizing RNA expression vector having the AU-rich target site and the corresponding IGS region shown in Figure 21.

[0043] [Figure 23a] Figure 23a is a simple schematic diagram illustrating that circRNAs consisting only of a GOI can be prepared by selecting a target site within a gene of interest and reconstructing the GOI based on that site.

[0044] [Figure 23b] Figure 23b is a schematic diagram of a reconstructed GOI structure in which a target site is selected within a gene of interest and the GOI is reconstructed based on that site.

[0045] [Figure 23c] Figure 23c shows the region of the target site in the base sequence of the target gene (CVB3 IRES-sGFP).

[0046] [Figure 23d] Figure 23d shows the DNA template for expression of a self-circularizing RNA construct containing a reconstituted gene of interest (CVB3 IRES-sGFP), with each region indicated.

[0047] [Figure 23e] Figure 23e shows the PAGE results after IVT using a self-circularizing RNA construct expression vector containing a reconstituted gene of interest (CVB3 IRES-sGFP).

[0048] [Figure 24] Figure 24 shows the results of HPLC analysis after IVT using a self-circularizing RNA construct expression vector containing a reconstituted gene of interest (CVB3 IRES-sGFP).

[0049] [Figure 24a-24b]Specifically, Figures 24a and 24b show the HPLC results after IVT of a self-circularizing RNA construct expression vector using a reconstructed gene of interest (CVB3 IRES-sGFP) rearranged based on the AU11 target site. Figures 24c and 24d show the HPLC results after IVT of a self-circularizing RNA construct expression vector using a reconstructed gene of interest (CVB3 IRES-sGFP) rearranged based on the AU19 target site. Figure 24e shows the HPLC conditions.

[0050] [Figure 25] FIG. 25 shows the results of 4% denatured PAGE of the HPLC peak of the IVT results using a self-circularizing RNA construct expression vector containing a reconstructed gene of interest (CVB3 IRES-sGFP).

[0051] [Figure 26a] Figure 26a shows the region of the target site in the base sequence of the target gene (CVB3 IRES-R.Luciferase mutant).

[0052] [Figure 26b] Figure 26b shows the DNA template for expressing a self-circularizing RNA construct containing a reconstituted gene of interest (CVB3 IRES-R. Luciferase mutant), with each region indicated.

[0053] [Figure 26c] Figure 26c shows the PAGE results after IVT using a self-circularizing RNA construct expression vector containing a reconstituted gene of interest (CVB3 IRES-R. Luciferase mutant).

[0054] [Figure 26d]Figure 26d shows the results of 4% denatured PAGE of the HPLC peak of the IVT results using a self-circularizing RNA construct expression vector containing a reconstructed target gene (CVB3 IRES-R. Luciferase mutant).

[0055] [Figure 27a] Figure 27a shows the region of the target site in the base sequence of the target gene (CVB3 IRES-F. luciferase).

[0056] [Figure 27b] Figure 27b shows the DNA template for expressing a self-circularizing RNA construct containing a reconstructed gene of interest (CVB3 IRES-F. luciferase), with each region indicated.

[0057] [Figure 27c] Figure 27c shows the PAGE results after IVT using a self-circularizing RNA construct expression vector containing the reconstituted gene of interest (CVB3 IRES-F. luciferase). DETAILED DESCRIPTION OF THE INVENTION

[0058] To prepare circRNA, the inventors devised a circularization system by self-targeting and splicing reaction using a trans-splicing ribozyme (T / S ribozyme) to circularize RNA carrying a target gene through a self-targeting and splicing reaction (Figure 1a and Figure 1b).

[0059] Group I intron ribozymes cleave target RNAs through two successive transesterification reactions, and then ligate the cleaved 3' ends of the target RNAs to induce trans-splicing.

[0060] Here, the system of the present invention constructs an internal guide sequence (IGS) in the 5' direction of a gene of interest (GOI) and a target site in the 3' direction, allowing the IGS to bind complementarily to the target site, forming a guanine (G):uracil (U) wobble base pair to induce cleavage and joining by a ribozyme located between the GOI and IGS, thereby preparing circRNA (Figures 2a and 2b).

[0061] Meanwhile, the present inventors focused on previous research to improve the trans-splicing efficiency of group I intron ribozymes (Mol Ther. 2005 Nov;12(5):824-34.), and designed a self-circularizing RNA construct so that it contained an AS (antisense sequence) region and an ABS (antisense binding sequence) at the 5' and 3' ends, which could bind to each other in a complementary manner, as shown in Figure 2c, and so that P1 and P10 helices were formed before and after the secondary structure of the ribozyme, and prepared a DNA template (DNA template) capable of expressing the RNA construct under the control of a T7 promoter (Example 1).

[0062] Next, we prepared a vector containing a DNA template, transcribed it in vitro (IVT), and confirmed the formation of circRNA. The vector expressed a self-circularizing RNA construct, which immediately formed a monomeric circRNA through a self-targeting and splicing (STS) reaction after transcription, even without the addition of GTP (Example 2).

[0063] Furthermore, the inventors confirmed that the self-circularizing RNA construct expression vector can express the RNA construct of the present invention in 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) in cells, and a plasmid vector was prepared using Gaussian luciferase as a transgene in the target gene. The plasmid vector was then transformed into cells to confirm the generation of circRNA and luciferase activity. As a result, it was confirmed at the molecular level that the prepared plasmid vector expressed the self-circularizing RNA construct in cells, the construct was circularized into circRNA by ribozyme, and the target gene was expressed via circRNA (Example 3).

[0064] Next, the inventors attempted to optimize the RNA structure so that circRNAs could be efficiently formed by direct STS reactions on the IVT.

[0065] First, we performed specific experiments to examine the direct STS reaction rate depending on the length of the AS region in IVT. Specifically, we prepared self-circularizing RNA expression vectors with AS and ABS regions of 50, 100, 150, 200, 250, or 300 nt in length. After IVT, the vectors were analyzed for direct STS efficiency. The results confirmed that the self-circularization efficiency significantly decreased with lengths of 200 nt or more. Meanwhile, while the self-circularization efficiency was similar for 50, 100, and 150 nt, the in vitro transcription reaction itself decreased when the AS and ABS regions were 50 or 100 nt long. From the perspective of cricRNA preparation efficiency, we confirmed that a 150-nt length for the AS and ABS regions was preferable (Example 5).

[0066] On the other hand, similar to the confirmation of the efficiency of circRNA preparation depending on the length of the AS and ABS regions, we confirmed the efficiency of circRNA preparation depending on the length and base sequence of the spacer region on IVT. As a result, we found that while the length and base sequence do not have a significant effect on the efficiency of circRNA preparation, in the case of a ribozyme and EMCV IRES, the linkage of 30 adenines (A) is sufficient to prevent structural conflicts that may occur due to the narrow spacing between the ribozyme and IRES (Example 6).

[0067] In the present invention, a group I intron ribozyme capable of sequential transesterification was used. Group I intron ribozymes induce trans-splicing by ligating separately present transcripts at the cleaved 3' end after cleavage of the target site. However, in the case of a self-circularizing RNA construct, the 5' end of the GOI, which is not a separately present transcript, is ligated to the cleaved 3' end. This is known to increase trans-splicing efficiency, so we investigated whether the P1 and P10 helix regions and the AS and ABS regions at both ends of the self-circularizing RNA construct also have a positive effect on circularization efficiency.

[0068] Specifically, the inventors prepared self-circularizing RNA expression vectors containing only the P1 helix region, only the P1 and P10 helices, or both the P1 and P10 helices and the AS region, and then performed IVT on the vectors to confirm the direct STS efficiency. Surprisingly, the P10 helix region in the self-circularizing RNA constructs was found to reduce the efficiency of circRNA preparation in the presence of the P1 and P10 helices. On the other hand, excellent circRNA preparation efficiency was also observed when only the P1 helix region was included without the AS region (Example 7).

[0069] Furthermore, the inventors designed self-circularizing RNA constructs so that only the IGS region forms a P1 helix, leaving only the target gene in the final circRNA product. They confirmed that STS reactions occur with various IGS sequences. Surprisingly, even when only the IGS region forms a P1 helix, STS reactions of self-circularizing RNA constructs expressed in DNA were induced, and circRNAs were formed even when the IGS region and target site region were not complementary to each other. However, when the IGS region and target site region are not complementary to each other, nonspecific reactions can occur at undesired sites, resulting in the generation of undesired products. It was found that the efficiency of circRNA preparation increased with increasing proportion of complementary base sequences between the 5'-GNNNNN-3' IGS region and the 5'-N'N'N'N'N'U-3' target site region (Example 8-1). Furthermore, based on the results showing high circularization efficiency in AU-rich samples, the inventors sought to determine whether specific IGS sequences could increase the efficiency of circRNA preparation. Here, we designed various combinations of AU-rich IGSs, excluding bases that form wobble base pairs, and prepared DNA templates to express self-circularizing RNA constructs with complementary target sites to confirm the extent of circRNA formation. As a result, circRNA generation was confirmed for all combinations of AU-rich IGSs. In particular, we confirmed that AU-rich IGS sequences consisting of two A bases and three U bases exhibited relatively high self-circularization efficiency (Example 8-2). Based on the above, we confirmed that circRNAs can be obtained through STS reactions using only the RNA constructs diagrammed in Figure 1a. Furthermore, the self-circularizing RNA constructs diagrammed in Figure 20a can form circRNAs consisting solely of the target gene containing a U base at the 3' end of the GOI as a circRNA precursor, as shown in the schematic diagram in Figure 20b.

[0070] On the other hand, if the 3' end of the GOI ends with a U base, the IGS region can be designed to be reverse-complementary to the GOI, allowing the final circRNA to be composed solely of the GOI region (Figure 20b). However, if the 3' end of the GOI does not end with a U base, it is difficult to prepare a circRNA consisting solely of the GOI. The inventors confirmed that by selecting a target site within the GOI and reconstructing the GOI based on this target site, it is possible to obtain a circRNA consisting solely of the GOI even when the 3' end of the GOI does not end with a U base (Example 9).

[0071] When the target site is selected within a gene of interest, the portion of the GOI located in the 3' direction relative to the uracil base of the target site is designated the "3' region GOI," and the GOI excluding the 3' region GOI is designated the "5' region GOI." Here, the reconstructed gene of interest is designed by linking the 3' region GOI to the 5' direction of the 5' region GOI. That is, the reconstructed gene of interest has a 5'-[3' region GOI]-[5' region GOI]-3' structure, where the [3' region GOI] and [5' region GOI] are directly linked.

[0072] To prepare circRNAs for protein expression, the inventors designed a gene of interest containing an IRES and a transgene encoding the protein. Then, they selected a target site within the gene of interest and then reconstructed the gene of interest so that the circRNA would be formed solely from the IRES and transgene. They then prepared vectors expressing the self-circularizing RNA constructs and confirmed circular RNA production. Specifically, they selected a target site within the gene of interest containing CVB3 as an IRES and sGFP (superfold GFP), RLuc M185V / Q253A, or FLuc as a transgene. They then designed a reconstructed GOI based on the target site. They then prepared a circular RNA precursor expression vector containing the reconstructed GOI. They then performed IVT and confirmed circular RNA production and yield via PAGE and HPLC (Examples 9-1 to 9-3). The results showed that circular RNA precursors containing the reconstructed gene of interest produced circular RNAs regardless of the type of transgene, and that the selection of the target site affected the circular RNA production yield.

[0073] On the other hand, P1 helix refers to the helical structure formed by the complementary binding between the base sequence linked in the shearing (5') direction of the ribozyme and the base sequence in the 3' direction of the transcript to which joining is induced by the ribozyme when the secondary structure of the group I intron ribozyme is formed, and P10 helix refers to the helical structure formed by the complementary binding between the shearing region of the ribozyme and the base sequence in the 5' direction of the transcript to be cleaved by the ribozyme.

[0074] In the present invention, the P1 helix can be formed by the complementary binding between the IGS at the 5' end and the target site at the 3' end in the self-circularizing RNA structure of the present invention, and the P10 helix refers to a helical structure formed by the complementary binding between the extended base sequence at the 5' end and the base sequence linked to the rear end (3' direction) of the ribozyme.

[0075] In this specification, the base sequence that forms the P1 helix is ​​referred to as the P1 region or P1 helix region, and similarly, the base sequence that forms the P10 helix is ​​named the P10 region or P10 helix region.

[0076] In the present invention, the base sequence of the IGS region is 5'-GNNNNN-3', and the base sequence of the target site region is 5'-N'N'N'N'N'U-3'. The P1 helix can be formed by the complementary binding of the IGS region and the target site region. Here, to avoid redundant expression, the technology of the P1 helix region may be used in combination with the base sequence of the IGS region.

[0077] In the present invention, the P1 helix is ​​formed to include a base sequence extending in the 5' direction of the IGS region, and a base sequence that is reverse complementary to the naturally extended base sequence is extended in the 3' direction of the target site to form the P1 helix. Herein, if an extended base sequence is present, the extended base sequence region is referred to as P1 to distinguish it from the IGS region. The same applies to the P10 helix.

[0078] On the other hand, in the present invention, the target site region indicates a base sequence that binds complementarily to the IGS region, and can overlap with a gene of interest (GOI) region depending on the design of the base sequence of the IGS region. Even in this case, in order to identify the region that binds complementarily to the IGS region, the region in the gene of interest that binds complementarily to the IGS region is classified and named as the target site. In other words, the target site in the present invention may overlap part or all of the gene of interest region, or may exist separately from the gene of interest.

[0079] In the present invention, the AS (antisense sequence) region is located at the 5' end of the self-circularizing RNA construct 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 construct. In the present invention, the AS region essentially coexists with the ABS region, and the absence of an AS region is understood to mean the absence of an ABS region. Similarly, an RNA construct containing an AS region is understood to also contain an ABS region.

[0080] Meanwhile, the present inventors confirmed the effect of the presence or absence of the three components of the P1 region, P10 region, and AS region on the circularization efficiency of self-circularizing RNA constructs by STS reaction. As a result, they confirmed that the amount of circRNA produced was greatest in the following order: when all of the P1 and P10 regions and the AS region were contained, when only the P1 region was contained, and when only the P1 and P10 regions were contained. They also confirmed that self-circularizing RNA constructs containing only the P1 region were circularized with sufficient efficiency to produce circRNA. The present invention provides a self-circularizing RNA construct containing only the P1 region.

[0081] Therefore, in this specification, a self-circularizing RNA construct containing only the P1 region means that the P10 region and AS region are absent, and is not intended to exclude other constructs. Similarly, a self-circularizing RNA construct containing only the P1 region and AS region means that the P10 region is absent, and is not intended to exclude other constructs other than the P10 region.

[0082]

[0083] Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various modifications can be made to the embodiments, the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, or alternatives to the embodiments are included in the scope of the patent.

[0084]

[0085] The terms used in the examples are used merely for the purpose of description and should not be construed as being limiting. The singular term includes the plural term unless the context clearly dictates otherwise. In this specification, the terms "comprise" or "have" and the like specify the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0086]

[0087] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments belong. Commonly used terms should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and should not be interpreted as having an ideal or overly formal meaning unless expressly defined in this application.

[0088]

[0089] In addition, when describing the embodiments with reference to the drawings, the same components will be given the same reference numerals regardless of the reference numerals in the drawings, and redundant descriptions thereof will be omitted. In describing the embodiments, if a detailed description of related prior art is determined to unnecessarily obscure the gist of the embodiments, the detailed description thereof will be omitted.

[0090]

[0091] [Example]

[0092] Example 1. Self-circularizing RNA design and RNA expression vector preparation

[0093] The self-circularizing RNA was designed as shown in Figure 2c, and the DNA template for its expression further contained a T7 promoter sequence for in vitro transcription (Figure 2d). The DNA template was amplified by PCR using a T7 circular forward primer: 5'-GGGATTCGAACATCGATTAATACGACTCACTATAGGGGCATCGATTGAATTGTCGA-3' (Tm = 77.5°C) and a T7 circular reverse primer: 5'-AGATCTCTCGAGCAGCGCTGCTCGAGGCAAGCTT-3' (Tm = 79.4°C). The DNA template amplification product was inserted into the pTOP TAV2 cloning vector (Enzynomics) using PstI restriction enzyme to prepare the self-circularizing RNA expression vector.

[0094]

[0095] Example 2. Confirmation of in vitro transcription and self-circularization

[0096] 2-1. In vitro transcription (IVT)

[0097] The self-circularizing 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 20μL scale (1μg T7 DNA template, 1X Reaction buffer, 10mM each of ATP, UTP, CTP, GTP, and 2μL of T7 RNA polymerase mix) was incubated at 37°C for 3 hours. After incubation, 29μL of nuclease-free water was added, followed by 1μL of RNase-free DNase I (10U / μL) and incubation at 37°C for 30 minutes. This directly induced circularization (direct STS) after transcription.

[0098] Next, to confirm the steps of the self-cyclization reaction, an additional cyclization reaction was induced. Specifically, the initial self-targeting and splicing (1 st To induce the second 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 concentration: 2 mM) were added to make a volume of 100 μL, and the self-cyclization reaction was carried out at 37°C for 1 hour. After heating at 55°C for 15 minutes, column purification was carried out using the Monarch RNA cleanup kit (NEB). 20 μL of 5X STS buffer, 100 mM GTP (final concentration: 2 mM), and 28 μL of nuclease-free water were added to the column-purified 50 μL sample to make a final volume of 100 μL, and this was reacted at 37°C for 3 hours to obtain 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) instrument.

[0099] To remove linear RNA from the reaction products, a portion of the column-purified samples from the first and second STS reactions was 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: 0.2 M Tris-HCl pH 8.0, 1 M KCl, 1 mM MgCl2) and 20 units of RNase R (adjusted to 100 μL with water). The mixture was incubated at 37°C for 30 minutes, followed by the addition of an additional 10 units of RNase R and further incubation for 30 minutes. The RNA was then purified using the Monarch RNA cleanup kit (NEB), and the RNA concentration was measured using a Nanodrop™ system.

[0100] 250 ng of RNA obtained from each STS step and samples obtained by treating the RNA obtained from each step with RNase R were mixed 1:1 with 10 M urea-BPB (1X TBE) dye and heated at 75°C for 5 minutes, followed by 4% Polyacrylamide-7 M urea denature PAGE (electrophoresis was carried out for 2 hours at 50°C and 50 W). The gel was stained with SYBR Gold Nucleic Acid Stain (Thermo Fisher Scientific) and analyzed using an ImageQuant 800 (Cytiva).

[0101] As a result, as can be seen in Figure 3, when treated with RNase R, an RNA band that was not properly cleaved by RNase R and became abundant appeared (Candidate 1), and other bands that were clearly observable despite RNase R treatment were confirmed (Candidate 2 and a band presumed to be nicked circular RNA). st and 2 nd It was confirmed that even without performing an STS reaction, substances presumed to be circRNAs could be sufficiently produced by a direct STS reaction, i.e., an in vitro transcription reaction alone.

[0102]

[0103] 2-2. Confirmation of self-cyclization

[0104] Next, RT-PCR base sequence analysis was performed to verify that candidate 1, one of the RNA bands not cleaved by RNase R in the PAGE analysis, was circRNA. Specifically, the band at the position of candidate 1 in PAGE was cleaved and crushed, and then eluted in water at 37°C for 3 to 16 hours. The circular RNA sample was purified by ethanol precipitation, and RT-PCR was performed using a primer that can only be amplified when circRNA is produced. The control group was linear RNA that does not contain ribozyme or antisense sites and cannot be circularized.

[0105] Reverse transcription (RT) was performed using OneScript Plus RTase (Abm). 125 ng of each RNA (control RNA and putative circular RNA treated with or without RNase R) 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 200 U of OneScript Plus RTase were added, and the mixture was incubated at 50°C for 15 minutes in a final volume of 20 μL. The enzyme was inactivated by heating at 95°C for 5 minutes and then stored on ice.

[0106] 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 under the following conditions: 95°C for 1 minute, 35 cycles of [95°C for 30 seconds, 65°C for 30 seconds, 72°C for 30 seconds], and 72°C for 5 minutes. 5 μL of PCR product was added to 1 μL of 6X DNA loading dye and electrophoresed at 150 V for 35 minutes. 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. When analyzed on a 1.5% agarose gel (containing 1X Intron Bio RedSafe Nucleic Acid Staining Solution) using a GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific), a specific PCR product of the expected size was observed only in the case of RNA samples presumed to be circular RNA, regardless of RNase R treatment (Figure 4a).

[0107] Furthermore, for the band of the expected size obtained, the PCR product was isolated and purified using a gel extraction kit (COSMO Genetech) according to the manufacturer's protocol, cloned using a TOPcloner TA-Blunt kit (Enzynomics), and transformed into DH5alpha E. coli (chemically competent E. coli, Enzynomics). E. coli colonies were grown on LB-Agar (containing kanamycin) plates. Plasmid DNA was extracted and purified using a DNA purification kit (COSMO Genetech) according to the manufacturer's protocol, and sequenced (using COSMO Genetech's Sanger sequencing service, using the M13R(-40) or M13F(-20) universal primers provided by the manufacturer). This confirmed that the 3' end of Gaussia luciferase and the 5' end of the IRES were correctly linked at the STS junction site (Figure 4b).

[0108] The above results indicate that candidate 1 is a circular RNA.

[0109]

[0110] 2-3. Re-examination of self-cyclization

[0111] Although RT-PCR confirmed that candidate 1 was a circRNA, theoretically, the possibility that candidate 1 was in the form of a dimer could not be ruled out. Therefore, we performed a nicking test to reconfirm that candidate 1 was a circRNA.

[0112] Purified circular RNA candidates 1 and 2 (100 ng) were mixed with MgCl2 at final concentrations of 0, 2.5, and 5 mM, respectively. The samples in a final volume of 10 μL of water were heated at 65°C for 30 min, then stored on ice for a while, and then mixed with 10 μL of 10 M urea-BPB (1X TEB) loading dye.

[0113] After heating at 75°C for 5 minutes, 4% Polyacrylamide-7 M Urea denature PAGE (electrophoresis was performed for 2 hours at 50°C and 50 W) was performed. The gel was stained with SYBR Gold Nucleic Acid Stain (Thermo Fisher Scientific) and analyzed using ImageQuant 800 (Cytiva). The results are shown in Figure 5.

[0114] For candidate 1, Mg 2+ In the absence of 2.5 mM Mg, a band of the size corresponding to nicked circular RNA (1092 nt) was observed. 2+ In the case of the condition, the band at the position of candidate 1, which is thought to be the position of circular RNA, was confirmed to be reduced, and a band of the size of nicked circular RNA was still confirmed to be present. 2+ Under these conditions, it was confirmed that even the nicked circular RNA band disappeared completely upon hydrolysis. This confirmed that candidate 1, i.e., circular RNA passes through the expected 1092-nt size monomer when nicking occurs (2.5 mM Mg 2+ ), and eventually all of it is decomposed (5mM Mg 2+ ), we were able to reaffirm that candidate 1 is a circular RNA and a monomer.

[0115] On the other hand, candidate 2 has a size similar to that of the intact RNA (1874 nt) (around 2000 nt of the marker), and was isolated from the intact RNA in 2.5 mM Mg 2+Under mild nicking conditions, a 1092-nt band, which is a different size from candidate 1 and is a nicked circular RNA, disappeared without being generated, indicating that it was not a circular RNA.

[0116]

[0117] Example 3. Confirmation of intracellular transcription and self-circularization

[0118] In Example 2, we confirmed that the self-circularizing RNA expression vector prepared in Example 1 was transcribed in vitro to form circRNA. We then further confirmed whether the vector functioned similarly within cells and whether the target gene contained in the circRNA was smoothly expressed within cells.

[0119] To express the target gene in cells, the target gene was designed with a 5'-EMCV IRES-transgene-stop codon-3' structure, and Gaussia luciferase (G. luci) was used as the transgene to easily confirm the expressed target gene.

[0120] The self-circular and RNA constructs containing the target gene were designed, and the DNA template capable of expressing them was inserted into a plasmid for expression under the pCMV promoter (Figure 6a). The plasmid vector was transfected into 293A cells, and circRNA production was confirmed by RT-PCR and nucleotide sequence analysis. Transgene expression was confirmed by luciferase activity analysis.

[0121] Specifically, 2x10 cells were placed in a 6-well plate. 5293A cells were seeded at 100 μL / well and transfected with the plasmid vector 24 hours later using Lipofectamine 2000 transfection reagent. The culture medium was replaced 6 hours after transfection. 100 μL of culture medium was then collected at 12, 24, and 48 hours after transfection, and G. luci activity was measured. G. luci activity was detected in the culture medium and confirmed to increase over time, confirming that the transgene G. luci gene was expressed in the cells (Figure 6b).

[0122] To confirm whether transgene expression was due to the formation of circRNA, RT-PCR and base sequence analysis were performed. 48 hours after transfection, total RNA was extracted from 293A cells transfected with the plasmid vector using Trizol reagent, and RT-PCR was performed to confirm the formation of circular RNA.

[0123] Reverse transcription (RT) was performed using OneScript Plus RTase (Abm). 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 200 U of OneScript Plus RTase were added in that order, and the reaction was carried out at 50°C for 15 minutes in a final volume of 20 μL. The enzyme was inactivated by heating at 95°C for 5 minutes, and then stored on ice.

[0124] Next, PCR was performed using AccuPower Taq PCR premix (BIONEER). 2 μL of the RT sample was mixed with 1 μL each of 20 μM Circular STS primer F (5'-caaggacttggagcccatggagcag-3') and primer R (5'-tgtgccgcctttgcaggtgtatc-3'). The mixture was diluted to 20 μL with water and subjected to PCR amplification conditions of 95°C for 1 minute, 35 cycles of [95°C for 30 seconds, 65°C for 30 seconds, 72°C for 30 seconds], and 72°C for 5 minutes. The expected STS PCR product was 479 bp in length. Analysis of the PCR product on a 1.5% agarose gel (containing 1X Intron Bio RedSafe Nucleic Acid Staining Solution) using a GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific) confirmed the presence of a specific PCR product of the expected size only in the presence of circular RNA. Here, 5 μL of PCR product was added to 1 μL of 6X DNA loading dye and subjected to electrophoresis at 150 V for 35 minutes, and the image was analyzed using a gel imaging system (Davinch-Gel product, YOUNG IN SCIENTIFIC) (Figure 6c).

[0125] The PCR product was isolated and purified using a gel extraction kit (COSMO Genetech) according to the manufacturer's protocol. The PCR product was then cloned using the TOPcloner TA-Blunt kit (Enzynomics). The product was transformed into DH5alpha E. coli (chemically competent E. coli, Enzynomics). Colonies were grown on LB-Agar plates containing kanamycin. Plasmid DNA was extracted and purified using a DNA purification kit (COSMO Genetech) and the manufacturer's instructions. Sequence analysis was performed using COSMO Genetech's Sanger sequencing service, using the M13R(-40) or M13F(-20) universal primers provided by the manufacturer. This confirmed the precise junction sequence between the 3' end of Gaussia luciferase and the 5' end of the IRES at the STS junction (Figure 6d).

[0126] From the above, we were able to confirm at the molecular level that the prepared plasmid vector expressed a self-circularizing RNA construct in cells, the construct was circularized into circRNA by ribozymes, and the target gene was expressed by the circRNA.

[0127]

[0128] Example 4. circRNA purification

[0129] 4-1. Confirmation of the feasibility of circRNA purification by HPLC

[0130] To minimize the risk of immunogenicity in preparing 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 shown in Figure 7A. Analysis was performed using the "Analytical" gradient condition, and sample fractionation was performed using the "Fraction collection" condition.

[0131] After IVT, we analyzed RNA that had only been column-purified using the Monarch RNA cleanup kit (NEB) (Figure 7B) and RNA that had been treated with RNase R (Figure 7C). An increased peak was observed in the RNase R-treated sample, indicating that circRNAs could be isolated by HPLC without RNase R treatment.

[0132]

[0133] 4-2. circRNA purification by HPLC

[0134] The fractions were collected via HPLC using the gradient indicated in the fraction collection conditions, and the individual fractions were collected as shown in Figure 8a. Fractions 7 and 10-13, which had clear peaks, were run on 4% denature PAGE, with 200 ng of each fraction, and electrophoresis was performed in the same manner as above.

[0135] As a result, as can be seen in Figure 8b, clean circular RNA was isolated and purified in fraction 12.

[0136]

[0137] Example 5. Optimization of the AS (ANTISENSE SEQUENCE) region

[0138] We attempted to determine the effect of the length of the AS (ANTISENSE SEQUENCE) region and its reverse-complementary ABS (ANTISENSE BINDING SEQUENCE) region on the immediate circularization reaction during in vitro transcription. DNA templates were prepared with AS and ABS regions of 50, 100, 150, 200, 250, or 300 nt in length (Figure 9). Expression vectors for each RNA construct were prepared as in Example 1. In vitro transcription was performed at 37°C for 3 hours as in Example 2. The extent of the STS reaction was immediately assessed by PAGE on a 4% polyacrylamide-7 M urea gel (20 × 20 cm, 1 mm) and the relative band intensity was compared.

[0139] The base sequences of each AS region are shown in Table 1 below.

[0140] [Table 1]

[0141] As shown in Figure 10 and Table 2 below, the self-circularization efficiency of AS50, AS100, and AS150 was relatively superior to that of lengths longer than AS200. However, AS50 and AS100 produced significantly less total RNA after in vitro transcription. Therefore, AS150 was found to be the optimal length for the expression of self-circularizing RNA constructs and the preparation of circular RNA.

[0142] [Table 2]

[0143] Example 6. Optimization of the spacer region

[0144] Next, to confirm the effect of the length or type of spacer region on the immediate circularization 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 construct were prepared as in Example 1. In vitro transcription was performed at 37°C for 3 hours as in Example 2. The STS reaction was immediately evaluated by PAGE on a 4% Polyacrylamide-7 M Urea gel (20 x 20 cm, 1 mm) and the relative band intensity was compared. The base sequences of each spacer region are shown in Table 3 below. In this study, the A10, A30, and A30 spacers had a restriction site added to the 3' end for insertion of an IRES immediately after the spacer region. In this study, the spacers had an AatII site (GACGTC) added to the 3' end.

[0145] [Table 3]

[0146] 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 circularization efficiency during the in vitro transcription process, and it was found that A30 acts as the optimal spacer for the expression of self-circularizing RNA constructs and the preparation of circular RNA.

[0147] [Table 4]

[0148] Example 7. Optimization of self-circularizing RNA constructs

[0149] 7-1. AS region, P1 helix, and P10 helix region

[0150] The self-circularizing RNA construct designed in Example 1 contains an AS region, a P1 helix, and a P10 helix region. To confirm the effect of each construct on the immediate circularization reaction during in vitro transcription, DNA templates containing only the P1 helix region, only the P1 and P10 helices, or both the P1 and P10 helices and the AS region were prepared as shown in Figure 13. Vectors capable of expressing each RNA construct were prepared as in Example 1, and in vitro transcription was performed at 37°C for 3 hours as in Example 2. The extent of the STS reaction was immediately assessed by PAGE on a 4% polyacrylamide-7 M urea gel (20 x 20 cm, 1 mm) and the relative band intensity was compared.

[0151] The base sequence of the T7 DNA template containing only the P1 helix region is shown in FIG.

[0152] As a result, as can be seen in Figure 15 and Table 5 below, there was no significant difference in the total RNA produced by in vitro transcription of each vector, but the amount of circular RNA produced was found to be greater in the case where the vector contained both the P1 and P10 helices and the AS region, the vector containing only the P1 helix region, and the vector containing only the P1 and P10 regions.

[0153] [Table 5]

[0154]

[0155] 7-2. Circularization verification of self-circularizing RNA constructs that do not contain P10 or AS regions

[0156] The results of Example 7-1 indicate that sufficient circRNA can be prepared using a DNA template (P1 construct) that does not contain the P10 and AS regions during the in vitro transcription process without an additional circularization step. To verify this, the STS reaction product of Example 7-1 was treated with RNase R to remove linear RNA, and PAGE was performed as in the previous test, followed by RT-PCR and base sequence analysis as in Example 2-2.

[0157] As can be seen in Figures 16a and 16b, even when self-cyclization was performed using the P1 structure, an RNA band was observed that was not properly cleaved by RNase R and became abundant after treatment with RNase R. The STS reaction product of this band was subjected to RT-PCR and base sequence analysis, confirming that it was circRNA.

[0158]

[0159] Example 8. Optimization of the P1 helix region

[0160] Example 8.1. P1 Helix Region and Self-Circularization

[0161] The results of Example 7 confirmed that a self-circularization construct lacking P10, AS, and ABS can also produce circRNA. Here, we hypothesized that circular RNA would be generated if only the internal guide sequence (IGS) at the 5' end of the P1 helix and the target site sequence at the 3' end were complementarily bound (U and G are wobble base pairs), and we verified this.

[0162] The IGS region has a sequence of GNNNNN and a target site sequence of N'N'N'N'N'U. Therefore, adding a single U nucleotide to the GOI results in a circRNA consisting of only one U nucleotide and the GOI region (Figures 1a and 1b). Furthermore, if the 3' end of the GOI ends with a U nucleotide, the IGS region can be designed to be reverse-complementary to the GOI, allowing the circRNA to be composed solely of the GOI region (Figures 20a and 20b).

[0163] As shown in FIG. 17, the sequences of IGS and target site were variously designed, and the vectors were prepared by the method of Example 1, followed by in vitro transcription (IVT) as in Example 2-1.

[0164] As shown in Figures 18-19 and Table 6 below, when both ends have complementary sequences, an increase in circRNA generated by the STS reaction was immediately observed after in vitro transcription. In particular, when the IGS and target site were AU-rich sequences, the efficiency of circRNA generation was confirmed to be higher. On the other hand, vectors containing no-complement IGS regions were found to generate very low levels of circRNA. From the above, it can be seen that the IGS and target site, if they are complementary to each other, can both efficiently induce self-circularization.

[0165] [Table 6]

[0166] 8-2. P1 helix region optimization

[0167] When the IGS and target site sequences are complementary, an immediate STS reaction occurs after in vitro transcription. It was found that the STS reaction is particularly active when the IGS and target site sequences are AU-rich. Next, to determine whether specific sequences among the A and U bases constituting the P1 helix affect self-cyclization efficiency, a total of 32 combinations of AU-rich IGSs were designed. Target sites were prepared containing sequences complementary to the IGSs, and the test was performed in the same manner as in Example 8-1 (Figure 21). Using the same conditions, the GOI was selected so that none of the 32 AU-rich sequences were present. The AU-rich sequences complementary to the IGSs were counted in the self-cyclization and RNA constructs prepared so that the IGSs could complement other regions of the target site. One sequence complementary to IGSs 17 and 18 was identified within the ribozyme sequence; no other sequences complementary to other IGSs were identified within the RNA construct. On the other hand, since ribozymes cannot target and splice their own internal sequences, the circular RNA produced by an expression vector for a self-circularizing RNA structure containing IGSs 17 and 18 is generated by targeting and splicing by the ribozyme through complementary binding between the IGS and the target site.

[0168] A 20 μL scale (1 μg T7 DNA template, 1X Reaction buffer, 10 mM each of ATP, UTP, CTP, GTP, and 2 μL of T7 RNA polymerase mix) was incubated at 37°C for 3 hours, followed by the addition of 29 μL of nuclease-free water and 1 μL of RNase-free DNase I (10 U / μL) for 30 minutes at 37°C. Next, column purification was performed using the Monarch RNA cleanup kit (NEB), and the concentration was measured using a Thermo Fisher Scientific Nanodrop instrument. 250 ng of each sample was mixed with 10 M urea-BPB (1X TBE) dye at a minimum sample:dye ratio of 1:1 or a higher ratio, heated at 75°C for 5 minutes, then held at 50°C. The resulting circular RNA was confirmed by electrophoresis on a 4% Polyacrylamide-7 M Urea denature PAGE (SYBR Gold Nucleic Acid Stain, Thermo Fisher Scientific) at 50 W for 2 hours.

[0169] The results for IGS conditions 1 to 16 are shown in Figure 22a. Under IGS conditions 14, 15, and 16, the relative band intensity was high, and it was found that the self-cyclization efficiency was highest for IGS sequence 16.

[0170] The results for the remaining IGS conditions (17 to 32) are shown in Figure 22b, with high relative band intensities for IGS sequences 17, 18, 25, and 28 to 32. Considering the intensity and consistency of the circular RNA bands across repeated tests, it was found that the AU-rich IGS sequence (composed of two A bases and three U bases) had a relatively high self-circularization efficiency, with IGS sequence 16 showing the highest self-circularization efficiency, followed by IGS sequence 28.

[0171]

[0172] Example 9. GOI reconstruction

[0173] Furthermore, to prepare circRNAs consisting only of a GOI, the inventors selected a GOI region capable of forming wobble base pairs with the IGS as the target site to form the circRNA. However, if a target site is selected within the GOI and the circRNA is formed as is, the GOI downstream of the target site is cleaved by the ribozyme and not included in the circRNA. Therefore, it is necessary to reconstruct the GOI located upstream and downstream of the target site (Figure 23a). Hereinafter, the GOI region downstream of the target site is referred to as the 3' region of GOI, and the GOI region excluding the 3' region of GOI is referred to as the 5' region of GOI.

[0174] Specifically, the inventors selected a U base within the GOI, capable of forming a wobble base pair, and five bases upstream of it as the target site. They then reconstructed the GOI so that the 3' region of the GOI downstream of the U base was positioned above the 5' region of the GOI, thereby designing the circRNA to be composed of the complete GOI.

[0175] On the other hand, the GOI may contain two or more U bases capable of forming wobble base pairs. When several candidate target regions exist within the GOI, the target site is selected so that the five consecutive bases upstream of the U base have a high A and U content, as confirmed in Example 8-2 for efficient circRNA preparation. In the figures, regions with a high A and U content are indicated as "AU rich."

[0176] Figure 23b illustrates an example of target site selection within a GOI and the resulting GOI reconstruction.

[0177] The inventors then attempted to confirm whether the intended circRNAs could be prepared through the above-described target site selection and GOI reconstitution. Specifically, target sites were selected within the GOI using CVB3 in the IRES and the GOIs containing sGFP, RLuc M185V / Q253A, or FLuc genes in the transgene. The GOI was then reconstituted to prepare circular RNA precursor expression vectors and perform IVT. For comparison, circular RNA precursors with target sites in the spacer region were prepared, and the target sites located in the spacer region are referred to as spacer targets. Specific circular RNA precursors are described with reference to the figures. Using the IVT results for each circular RNA precursor, circular RNA production and yield were confirmed via PAGE and HPLC.

[0178]

[0179] 9-1.GOI:CVB3 IRES-sGFP

[0180] A target gene was designed to express sGFP via the CVB3 IRES. Two candidate AU-rich target regions (AU11 and AU19) were selected for the CVB3 IRES. Figure 23c shows the target gene sequence and the two AU-rich target regions within it. A spacer target was used as a control. The initially designed spacer region (AC40 spacer) did not contain any candidate target regions. However, previous studies have confirmed that the length of the spacer region does not affect circularization efficiency. Here, the spacer target was prepared by separately designing an AC108 spacer to be included in the circular RNA precursor.

[0181] The GOI was reconstructed upstream and downstream of the target site of the target gene, AU11 or AU19, and IVT was performed to confirm circRNA formation. Figure 23d shows the DNA template regions for circular RNA precursor expression when AU11 was selected as the target site. When AU19 was selected as the target site, the GOI was reconstructed to design DNA templates, similar to when AU11 was selected as the target site. Each template was prepared in a vector according to the method described in Example 1, and then in vitro transcription (IVT) was performed as described in Example 2-1.

[0182] After IVT, PAGE was performed to confirm circRNA generation (Figure 23e). As a result, we confirmed that circular RNA precursors targeting AU11 and AU19 formed circRNAs via the STS reaction, with the AU11 target circular RNA precursor exhibiting higher circularization efficiency.

[0183] The PAGE results were further confirmed by IP-RP HPLC (Figures 24a-24d). Analysis of samples subjected to self-circularization using the AU11 target site followed by RNase R treatment revealed a high level of circular RNA peak. Conversely, as in the PAGE results, the self-circularized constructs using the AU19 target site, which have a relatively low self-circularization efficiency, exhibited a relatively small circRNA peak when analyzed by HPLC after RNase R treatment. Each peak observed in the IP-RP HPLC results (peaks in Figures 24b and 24d) was subjected to 4% denatured PAGE, confirming the purification of circular RNA (Figure 25).

[0184] Table 7 below shows the self-circularization efficiency and final yield of circular RNA precursors designed by selecting target sites within the GOI. In Table 7, AU16 and AU28 are not shown in Figure 23c, and the target site located in the spacer (5'-ACGGCU-3') is used as a control for comparison.

[0185] [Table 7]

[0186] The results show that significant differences in self-circularization efficiency and final yield occur depending on the exact GOI sequence or the AU-rich target site selected for the P1 construct.

[0187]

[0188] 9-2.GOI:CVB3 IRES-R.Luciferase(M185V / Q235A)

[0189] A gene of interest was designed to express the RLuc mutation (M185V / Q235A) via the CVB3 IRES. An AU11 target site was selected within the gene of interest, and a GOI was reconstructed based on this to design a DNA template. This template was then prepared in a vector as described in Example 1, followed by in vitro transcription (IVT) as described in Example 2-1. A spacer target was used as a control. As in Example 9-1, the circular RNA precursor used as a control was designed and prepared to include an AC108 spacer.

[0190] Figure 26a shows the sequence of the target gene and the AU-rich target site region present within it, and Figure 26b shows the specific sequence and each region of the circular RNA precursor expression DNA template in which the GOI was reconstructed based on the selected AU11 target site region.

[0191] The results of PAGE after IVT are shown in Figure 26c. Circular RNA precursors targeting AU11 were converted to circRNA via STS reaction, which was confirmed by IP-RP HPLC. The HPLC peak was then subjected to 4% denatured PAGE to confirm the purification of circular RNA (Figure 26d).

[0192] When AU11 is used as the target site, the yield of circular RNA is as shown in Table 8 below.

[0193] [Table 8]

[0194] 9-3.GOI:CVB3 IRES-F.Luciferase

[0195] The same test as in Example 9-2 was carried out, except that the transgene was changed to F. luciferase.

[0196] Figure 27a shows the sequence of the gene of interest and the AU-rich and spacer target regions within it. Figure 27b shows the specific sequence and each region of the circular RNA precursor expression DNA template in which the GOI was reconstructed based on the selected AU11 target region. The spacer target was used as a control; all circular RNA precursors in this study were designed and prepared to contain the AC108 spacer.

[0197] The results of PAGE after IVT are shown in Figure 27c. Circular RNA precursors targeting AU11 were converted to circRNA via STS reaction, which was confirmed by IP-RP HPLC. The HPLC peak was then subjected to 4% denatured PAGE to confirm the purification of circular RNA (Figure 27d).

[0198] [Table 9]

[0199] These results indicate that even when a target site is selected within a gene of interest and a GOI is reconstructed using it, a circular RNA containing only the complete GOI is formed from the circular RNA precursor, regardless of the type of transgene.

[0200]

[0201] Although the embodiments of the present invention have been described in detail above with reference to the drawings, those skilled in the art may 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 combined in a different manner than described, and other components or equivalents may be substituted or replaced, and still suitable results may be achieved.

[0202] Accordingly, other implementations, other embodiments, and equivalents of the claims are intended to be within the scope of the following claims.

[0203] [Industrial Applicability]

[0204] The present invention can be used to prepare circRNAs for protein expression in vitro, in cells, and in vivo, and can be used to prepare functional RNAs using circRNAs, such as miRNAs, anti-miRNAs, shRNAs, aptamers, mRNA vaccines, mRNA therapeutics, antibodies, vaccine adjuvants, and CAR-T mRNA.

Claims

1. A self-circularizing RNA construct comprising: The structure has a 5'-IGS (internal guide sequence)-ribozyme-target gene-target site-3' structure, The IGS region forms a guanine (G):uracil (U) wobble base pair with the target site; the wobble base pair-forming guanine is located at the 5' end of the IGS region; the wobble base pair-forming uracil is located at the 3' end of the target site region; The IGS region is a self-circularizing RNA structure that consists of adenine (A) or uracil in addition to the bases that form the wobble base pair.

2. The self-circularizing RNA construct of claim 1 , wherein the target site region overlaps with a region of a gene of interest.

3. The self-circularizing RNA construct according to claim 1 , wherein the base sequence of the IGS region is reverse-complementary to the base sequence of the target site region, excluding the guanine.

4. The self-circularizing RNA construct of claim 1 , wherein the ribozyme is a group I intron ribozyme.

5. The self-circularizing RNA structure of claim 1 , wherein the ribozyme comprises the base sequence of SEQ ID NO:

6.

6. The self-circularizing RNA construct of claim 1 , wherein the construct comprises nucleotides extending in the 5′ direction of the IGS region to form a P10 helix.

7. The self-circularizing RNA construct of claim 1 , wherein the construct comprises nucleotides extending in the 5′ direction of the IGS region and in the 3′ direction of the target site to form a P1 helix.

8. The self-circularizing RNA construct of claim 7 , wherein the construct does not form a P10 helix.

9. The structure includes an AS (antisense sequence) region in the 5' direction of the IGS region, an ABS (antisense binding sequence) region capable of binding complementarily to the AS region in the 3' direction of the target site; The self-circularizing RNA construct of claim 1 or 6, further comprising:

10. The self-circularizing RNA construct of claim 9, wherein the length of the AS region is 50 to 400 nt.

11. The self-circularizing RNA construct according to claim 1 , wherein the target gene region comprises an IRES (internal ribosome entry site) region at the 5′ end.

12. The self-circularizing RNA construct according to claim 1 , wherein the ribozyme region and the target gene region are linked to a spacer region consisting of a random base sequence.

13. The self-circularizing RNA construct according to claim 1 , wherein the construct comprises a target gene region and a target site region linked to a spacer region consisting of a random base sequence.

14. A vector that expresses the self-circularizing RNA construct of claim 1.

15. The vector of claim 14 , wherein the vector comprises a promoter operably linked to a gene encoding the self-circular RNA.

16. A self-circularizing RNA construct comprising: The construct has a structure of 5'-IGS (internal guide sequence)-ribozyme-reconstituted target gene-3', The reconstituted gene of interest is a gene of interest with a different sequence, the target gene contains a uracil (U) base, The reconstructed gene of interest has a 3' region of the gene of interest linked in the 3' direction of the uracil base linked to the 5' end of the gene of interest, so that the uracil base is located at the 3' end; The IGS region is a self-circularizing RNA structure that includes a guanine (G) at the 5' end that can form a wobble base pair with a uracil base within the target gene, and includes a sequence that is reverse complementary to the sequence of five consecutive bases linked in the 5' direction of the uracil base in the target gene.

17. The self-circularizing RNA construct according to claim 16, wherein the sequence of five consecutive bases linked in the 5' direction of the uracil base at the 3' end of the reconstituted target gene contains 1 to 5 adenine (A) and / or uracil bases.

18. 17. The self-circularizing RNA construct of claim 16, wherein the ribozyme is a group I intron ribozyme.

19. The self-circularizing RNA structure of claim 16 , wherein the ribozyme comprises the base sequence of SEQ ID NO:

6.

20. The self-circularizing RNA construct of claim 16, wherein the construct comprises nucleotides extending in the 5' direction of the IGS region, the extended nucleotides comprising a sequence reverse-complementary to the 5' end of the reconstituted target gene and forming a P10 helix.

21. The construct comprises nucleotides extending in the 5' direction of the IGS region, containing nucleotides extending in the 3' direction of the reconstructed gene of interest, 17. The self-circularizing RNA construct of claim 16, wherein each extended nucleotide is a reverse complementary sequence.

22. The structure includes an AS (antisense sequence) region in the 5' direction of the IGS region, The self-circularizing RNA construct according to claim 16 or 20, further comprising an ABS (antisense binding sequence) region capable of binding complementarily to the AS region in the 3' direction of the reconstituted target gene.

23. The self-circularizing RNA construct of claim 22, wherein the length of the AS region is 50 to 400 nt.

24. The self-circularizing RNA construct according to claim 16, wherein the ribozyme region and the reconstituted target gene region are linked to a spacer region consisting of a random base sequence.

25. A vector that expresses the self-circularizing RNA construct of claim 16.

26. 26. The vector of claim 25, wherein the vector comprises a promoter operably linked to a gene encoding the self-circular RNA.

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