Internal ribosome entry sites (IRES), plasmid vectors and circular mRNA for enhanced protein expression

Abstract

The present invention relates to a plasmid vector for generating circular mRNA encoding an open reading frame (circular mRNA encoding an ORF) with a small backbone, optimized homology arms, and an internal ribosome entry site (IRES) encoding an efficient open reading frame (ORF) resulting in highly efficient protein expression. The IRES was also developed to facilitate translation of the protein of interest, thereby improving protein expression. The plasmid vector and circular mRNA encoding an ORF of the present invention, due to its optimized construct and small size, can overcome previous technical challenges, resulting in ease of delivery, stability in cells, and significantly higher translation efficiency and in vivo protein expression. When applied in medicines or pharmaceutical preparations, the circular mRNA described in the present invention can potentially reduce the frequency of administration and / or the required dose, reducing unwanted side effects and improving patients' access to the medicine.

Description

[Technical field]

[0001] The present invention relates to biotechnology, and more specifically to internal ribosome entry sites (IRES), plasmid vectors and circular mRNA for enhancing protein expression. [Background technology]

[0002] Genetic engineering has become a valuable tool for developing targeted prevention and treatment approaches. This technique involves using genetic material in an organism's cells to control protein synthesis specific to a disease condition. One area of ​​focus is messenger ribonucleic acid (mRNA), a single-stranded molecule complementary to the deoxyribonucleic acid (DNA) of a template gene. mRNA is composed of nucleotides arranged in a linear sequence. During protein synthesis, ribosomes bind to mRNA and read the nucleotide sequence, which is then translated into a protein. The advantages of mRNA in medicine are significant. It is relatively easy to synthesize mRNA in vitro in a short period of time, and it can activate a rapid response when administered to an organism, allowing targeted delivery and reducing the risk of unwanted side effects. mRNA can be easily synthesized in the laboratory in a short period of time, and it can activate a rapid response when administered to an organism. Furthermore, unlike plasmid vectors, mRNA does not raise any concerns about integration into the genome of an organism when applied for medical purposes. Although mRNA technology in medicine offers many advantages, it has the disadvantage that mRNA is inherently unstable and prone to degradation by cellular machinery when administered to living organisms. As a result, mRNA is only suitable for limited periods of use in activating rapid cellular responses. To overcome these technical challenges, researchers are exploring ways to extend the activity of mRNA to enhance protein expression efficiency and stability.

[0003] US Patent Publication No. 20200080106 disclosed a circular RNA developed to increase the efficiency of disease treatment. A vector for constructing a circular RNA, comprising various elements linked together and arranged in the following order: a 5' homology arm linked to a 3' group I intron fragment, a 5' spacer, an internal ribosome entry site (IRES), a protein coding region, a 5' group I intron fragment, and a 3' homology arm. The circular RNA can be translated into protein inside a eukaryotic cell or can be biologically active and can be delivered to cells by transfection. This disclosure showed that the circular RNA can cause higher protein expression in cells for a longer period of time, leading to higher therapeutic efficiency. However, the RNA circularization and the IRES of the disclosed circular RNA are not optimized. Thus, the disclosed circular RNA results in lower protein expression. Furthermore, the expression of the various IRESs used by the disclosed circular RNA was not tested in mice.

[0004] European Patent No. 2996697 disclosed highly stable circular RNAs that efficiently translate proteins of interest in eukaryotic cells, making them useful for therapy. It has been established that circular RNA molecules have a much longer half-life than their linear counterparts. This document suggested increased RNA half-life and stability by RNA circularization, which reduces exonuclease excretion when delivered to cells. This disclosure revealed that the circular RNA half-life is approximately 40 hours in vivo, higher than the linear mRNA half-life of only 6-8 hours. Again, expression of the disclosed circular RNAs using different IRESs was not tested in mice.

[0005] International Patent Application No. 2020237227 disclosed a highly stable circular RNA for controlling gene expression in an organism, which may be applied to disease treatment such as gene therapy or vaccines. This document also disclosed internal ribosome entry site (IRES) modification using viral components such as Sarivirus A SZ1, Sarivirus A BN2, and Coxsackievirus type B3 (CVB3). It was found that modifying the IRES with viral components such as Sarivirus A SZ1 and Sarivirus A BN2 provided the circular RNA with high functional stability. However, according to this disclosure, the RNA stability is not ideal, as the protein expression level decreases over time. As shown by the above disclosure, despite the advantages of circular RNA, its size and limited IRES activity can prevent efficient translation in vivo. Circularization can also be further improved.

[0006] Biotechnology Journal, 2014, 9(9), 1164-1174, disclosed that co-transfection of a plasmid vector encoding a fusion protein with a plasmid vector encoding a Bcl-xL protein into CHO cells significantly increased fusion protein expression and provided cell stability for more than 6 days. However, this co-transfection process used a DNA plasmid vector. Therefore, the present invention develops a plasmid vector for generating an open reading frame-coding circular mRNA (ORF-encoding circular mRNA) with a small backbone with assisted homology arms to facilitate RNA circularization. An internal ribosome entry site (IRES) was also developed to enhance translation of the protein of interest, thereby improving protein expression. The plasmid vector and ORF-encoding circular mRNA of the present invention, due to their optimized constructs and small sizes, can overcome previous technical challenges, resulting in significantly higher translation efficiency and in vivo protein expression. Summary of the Invention

[0007] The present invention relates to a plasmid vector for generating an open reading frame-encoding circular mRNA (ORF-encoding circular mRNA), which has a small backbone, optimized homology arms encoding an efficient open reading frame (ORF), resulting in highly efficient protein expression. An internal ribosome entry site (IRES) has also been developed to enhance translation of the protein of interest, thereby improving in vivo protein expression. The plasmid vector and ORF-encoding circular mRNA of the present invention, due to its optimized construct and small size, can overcome previous technical challenges, resulting in significantly higher translation efficiency and in vivo protein expression. When applied in medicines or pharmaceutical preparations, the circular mRNA described in the present invention can potentially reduce the frequency of administration and / or the required dose, reducing unwanted side effects and improving the patient's access to the medicine. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows an example of a circular mRNA construct of the present invention. [Diagram 2]FIG. 1 shows flow cytometry histograms of HEK293T cells approximately 48 hours after transfection with circular GFP mRNA (CVB3 IRES) using various commercially available delivery systems. [Diagram 3] FIG. 1 shows the fluorescence intensity observed in HEK293T cells approximately 48 hours after transfection with circular GFP mRNA (CVB3 IRES) using various commercially available delivery systems. [Figure 4] FIG. 1 shows luciferase activity observed in the supernatant of HEK293T cells approximately 24 hours after transfection with circular luciferase mRNA (CVB3 IRES) by using various commercially available delivery systems. [Diagram 5] FIG. 1 shows luciferase activity observed in the supernatant of HEK293T cells approximately 48 hours after transfection with circular luciferase mRNA (CVB3 IRES) by using various commercially available delivery systems. [Figure 6] FIG. 1 shows flow cytometry histograms of HEK293T cells approximately 48 hours after transfection with circular GFP mRNA containing different types of IRES. [Figure 7] FIG. 1 shows the percentage of HEK293T cells showing positive GFP approximately 48 hours after transfection with circular GFP mRNA containing different types of IRES. [Figure 8] FIG. 13 shows luminescence images in mice (BALB / c strain) approximately 24 or 48 hours after injection of circular luciferase mRNA (CVB3 IRES) via the delivery system of Sample B by intramuscular (IM) and intravenous (IV) injection. [Figure 9] FIG. 1 shows flow cytometry histograms of HEK293T cells approximately 48 hours after transfection with circular SARS-CoV-2 spike protein mRNA. [Figure 10]FIG. 1 shows the results of Western blot analysis to detect the expression of SARS-CoV-2 viral spike protein in supernatants obtained from HEK293T cells after transfection with circular SARS-CoV-2 spike protein mRNA. [Figure 11] FIG. 1 shows the analysis of immunoglobulin G (IgG) expression specific for the spike protein of the SARS-CoV-2 virus Omicron species (B.1.1.529) in mouse serum approximately 2 weeks after injection of circular mRNA expressing the spike protein (CVB3 IRES) compared to control mice using enzyme-linked immunosorbent assay (ELISA). [Figure 12] Figure 1 shows the ability of mouse serum to inhibit pseudovirion entry of SARS-CoV-2 virus Omicron species (B.1.1.529) measured using a neutralizing antibody assay approximately two weeks after injection of circular mRNA expressing the spike protein (CVB3 IRES) compared to control mice. [Figure 13] FIG. 13 shows fluorescence images of HEK293T cells approximately 48 hours after transfection with circular cancer antigen protein mRNA H3K27M using immunofluorescence assay. [Figure 14] FIG. 1 shows fluorescence images of HEK293T cells approximately 48 hours after transfection with circular cancer antigen protein mRNA PSCA using immunofluorescence assay. [Figure 15] FIG. 1 shows fluorescence images of HEK293T cells approximately 48 hours after transfection with cyclic cancer antigen protein mRNA TROP2 using immunofluorescence assay. [Figure 16] FIG. 13 shows fluorescence images of HEK293T cells approximately 48 hours after transfection with the circular reprogramming factor mRNA OSCK, using immunofluorescence assay by Gram staining with an antibody specific for the OCT4 protein. [Figure 17]FIG. 13 shows fluorescence images of HEK293T cells approximately 48 hours after transfection with the circular reprogramming factor mRNA OSCK, using immunofluorescence assay by Gram staining with an antibody specific for SOX2 protein. [Figure 18] FIG. 2 shows fluorescence images of HEK293T cells approximately 48 hours after transfection with circular CAR mRNA CD19 using immunofluorescence. [Figure 19] A comparison of in vitro transcription (IVT), circularized products (IC), and RNase R-treated IC between circular RNA-eHA-1 and circular RNA-eHA-2 using agarose gel electrophoresis. The white arrow indicates the circular form of RNA. [Figure 20] Figure 1 shows luciferase activity compared between two different designed external homology arms, cyclic Fluc-eHA-1-CVB3 and cyclic Fluc-eHA-2-CVB3, in HEK293T cells. Statistical significance was determined by unpaired t-test. Mean ± SEM; n=2; *p<0.05. [Figure 21] Figure 1 shows the secondary structures of the IRES sequences predicted using the online tool RNAfold. Only the secondary structures of the IRES from CVB3, ECH20, Chimera 3(20)3, and Chimera 20(3)20 are shown here. [Figure 22] Figure 1 shows the mean fluorescence intensity (MFI) and the difference in mean fluorescence intensity (ΔMFI) of GFP in imHC cells among different IRES. Statistical significance was determined by unpaired t-test. Mean ± SEM; n=3; *p<0.05. [Diagram 23] FIG. 1 shows the mean fluorescence intensity (MFI) and mean fluorescence intensity difference (ΔMFI) of GFP in BHK-21 cells between different IRES in BHK-21 cells. [Figure 24]Figure 1 shows in vivo bioluminescence images taken (A) 6 h and (B) 48 h after intramuscular (IM) injection of LNP-encapsulated cyclic Fluc-eHA-2-CVB3 or cyclic Fluc-eHA-2-3(20)3 in BALB / c mice. The negative control group received PBS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention relates to an ORF-encoding circular mRNA with a small backbone, optimized homology arms and an IRES for highly efficient protein expression in vivo. According to the present invention, an IRES has been developed to enhance the translation of the protein of interest, thus improving protein expression. As a result, the plasmid vector and ORF-encoding circular mRNA of the present invention, due to its small size and optimized construct, can overcome previous technical challenges, leading to ease of delivery, stability in cells, and significantly higher translation efficiency and in vivo protein expression. Therefore, when the circular mRNA of the present invention is applied in medicines or pharmaceutical preparations, it can reduce the frequency of administration and / or the required dose, potentially reducing unwanted side effects and increasing the chances of patients taking advantage of the medicine.

[0010] As will be understood by those skilled in the art, the following aspects of the invention may, and preferably include, one or more or all of the preferred optional features of the invention disclosed herein, as appropriate. Furthermore, those skilled in the art will recognize that the features and / or elements referred to in this disclosure may be modified by other features and / or elements, even if not expressly stated herein, without departing from the scope of the disclosure. For example, ORFs encoding other proteins could be used instead of the ORFs referred to in this disclosure to generate ORFs encoding circular mRNAs corresponding to the appropriate functions. Thus, the scope of the disclosure should not be limited to any particular type of ORF.

[0011] definition The present invention includes a sequence listing, which is provided in XML file format and submitted via the electronic filing system. Technical or scientific terms used herein have definitions that are understood by those of ordinary skill in the art unless stated otherwise. Any instruments, devices, methods, or chemicals referred to herein are those commonly operated or used by those of ordinary skill in the art, unless expressly stated otherwise to be instruments, devices, methods, or chemicals used in the present invention.

[0012] The use of the singular or plural nouns with the term "comprising" in the claims or specification should be interpreted as "one" as well as "one or more," "at least one," and "one or more than one." All compositions and / or processes disclosed and claimed are intended to encompass aspects of the invention involving any operation, manipulation, modification, or change of parameters that, even if not specifically mentioned in the claims, would be obtained by one of ordinary skill in the art with similar objects with the same utility and results as described in the present invention without significantly departing from the experiments performed, the examples described, or the data presented in the present invention. Accordingly, any permutation or similar object of the present invention, including minor modifications or changes that are obvious to one of ordinary skill in the art, should be considered to be within the scope, spirit, and concept of the present invention, as defined by the appended claims. Throughout this specification, the term "about" is used to indicate that any value presented herein can potentially vary or deviate due to a variety of factors, such as calculation errors, variations in the device or method, or differences between individual operators practicing the device or method.

[0013] According to a first embodiment, the present invention relates to an internal ribosome entry site (IRES) comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26 or a combination thereof. In a preferred exemplary embodiment, the IRES comprises the nucleotide sequence set forth in SEQ ID NO:21. In a second embodiment, the present invention provides a method for the preparation of a medicament comprising the steps of: - RNA polymerase promoter, -5'spacer 1, - 5' external homology arm, -3'PIE (permuted intron-exon), - 5' internal homology arm, -5'spacer 2, - internal ribosome entry site (IRES), - open reading frame (ORF), -3'spacer 1, - 3' internal homology arm, -5'PIE (intron-exon permuted), -3'spacer 2, and -3' external homology arm The present invention relates to a plasmid vector for producing an open reading frame-encoding circular mRNA (ORF-encoding circular mRNA), comprising elements arranged as follows:

[0014] In an exemplary embodiment of the invention, the plasmid vector for generating an ORF encoding circular mRNA comprises an RNA polymerase promoter having a length ranging from 15 to 25 nucleotides. In an exemplary embodiment of the invention, the RNA polymerase promoter is selected from the group consisting of a T7 viral RNA polymerase promoter, a SP6 viral RNA polymerase promoter, or a T3 viral RNA polymerase. In a preferred exemplary embodiment of the invention, the RNA polymerase promoter is a T7 viral RNA polymerase promoter.

[0015] In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 5'spacer 1 having a length ranging from 5 to 15 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 5' external homology arm having a length ranging from 15 to 30 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 5' external homology arm having a length in the range of 15-20 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating the ORF coding mRNA comprises a 5' external homology arm having a length in the range of 20-30 nucleotides.

[0016] In a preferred exemplary embodiment of the invention, the 5' external homology arm is the sequence of SEQ ID NO:4. In an exemplary embodiment of the invention, the plasmid vector for generating an ORF encoding circular mRNA comprises a 3' PIE (intron-exon permuted) having a length ranging from 100 to 250 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating an ORF encoding circular mRNA contains a 3' PIE (intron-exon permuted) derived from the Cyanobacterium anabaena pre-tRNA group I intron gene. In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 5' internal homology arm having a length ranging from 15 to 25 nucleotides.

[0017] In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 5' spacer 2 having a length in the range of 50-100 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating an ORF encoding circular mRNA comprises an IRES having a length of 190-900 nucleotides. In exemplary embodiments of the invention, the IRES is selected from the group consisting of an echovirus 33 IRES, an echovirus 20 IRES, an echovirus 29 IRES, a coxsackievirus B1 IRES, a coxsackievirus B3 IRES, a coxsackievirus A12 IRES, an enterovirus 80 IRES, an Aphis glycines virus 1 IRES, a halastavi arva virus IRES, or a pegivirus J IRES, or a combination thereof.

[0018] In a preferred exemplary embodiment of the invention, the IRES is a Coxsackievirus type B3 IRES. In an exemplary embodiment of the invention, the IRES is selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, or a combination thereof. In a preferred exemplary embodiment of the invention, the IRES is the sequence of SEQ ID NO:21. In an exemplary embodiment of the present invention, in a plasmid vector for generating an ORF-encoding circular mRNA, the ORF is selected from the group consisting of an ORF encoding a viral spike protein, an ORF encoding a cancer antigen protein, an ORF encoding a reprogramming factor protein, or an ORF encoding a chimeric antigen receptor protein (CAR protein), or a combination thereof.

[0019] In a preferred exemplary embodiment of the present invention, the ORF encoding the viral spike protein is an ORF encoding the SARS-CoV-2 viral spike protein. In a preferred exemplary embodiment of the present invention, the ORF encoding a cancer antigen protein is an ORF encoding a cancer antigen protein H3K27M, an ORF encoding a cancer antigen protein PSCA, or an ORF encoding a cancer antigen protein TROP2. In a preferred exemplary embodiment of the present invention, the ORF encoding the reprogramming factor protein is an ORF encoding the reprogramming factor protein OSCK. In a preferred exemplary embodiment of the invention, the ORF encoding a chimeric antigen receptor protein (CAR protein) is an ORF encoding the CAR protein CD19.

[0020] In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 3' internal homology arm having a length ranging from 15 to 25 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating an ORF encoding circular mRNA comprises a 5' PIE (intron-exon permuted) having a length in the range of 100-150 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating an ORF encoding circular mRNA contains a 5' PIE (permuted intron-exon) derived from the Cyanobacterium anabaena pre-tRNA group I intron gene.

[0021] In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 3' spacer 2 having a length ranging from 5 to 15 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 3' external homology arm having a length ranging from 15 to 30 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 3' external homology arm having a length ranging from 15 to 25 nucleotides. In an exemplary embodiment of the invention, the plasmid vector for generating the ORF encoding circular mRNA comprises a 3' external homology arm having a length in the range of 25-30 nucleotides.

[0022] In a preferred exemplary embodiment of the invention, the 3' external homology arm is the sequence of SEQ ID NO:32. In an exemplary embodiment of the invention, the ORF encoding circular mRNA is obtained from a plasmid vector according to any one of the above-mentioned embodiments of the invention. According to a third embodiment, the present invention provides a method for the preparation of a compound having a structure comprising the steps of: -3' exon -Internal ribosome entry site (IRES) -Open reading frame (ORF) -5' exon The present invention relates to an open reading frame encoding circular mRNA (ORF encoding circular mRNA) obtained from a plasmid vector containing elements arranged in the above-mentioned order. In an exemplary embodiment of the invention, the ORF encoding circular mRNA comprises an IRES having a length in the range of 190-900 nucleotides.

[0023] In exemplary embodiments of the invention, the IRES is selected from the group consisting of an Echovirus 33 IRES, an Echovirus 20 IRES, an Echovirus 29 IRES, a Coxsackievirus B1 IRES, a Coxsackievirus B3 IRES, a Coxsackievirus A12 IRES, an Enterovirus 80 IRES, a Soybean Aphidvirus 1 IRES, a Halastavir albavirus IRES, or a Pegivirus J IRES, or a combination thereof. In a preferred exemplary embodiment of the invention, the IRES is a Coxsackievirus type B3 IRES. In an exemplary embodiment of the invention, the IRES is selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, or a combination thereof. In a preferred exemplary embodiment of the invention, the IRES is the sequence of SEQ ID NO:21.

[0024] In an exemplary embodiment of the present invention, the ORF of the ORF encoding circular mRNA is selected from the group consisting of an ORF encoding a viral spike protein, an ORF encoding a cancer antigen protein, an ORF encoding a reprogramming factor protein, or an ORF encoding a chimeric antigen receptor protein (CAR protein), or a combination thereof. In a preferred exemplary embodiment of the present invention, the ORF encoding the viral spike protein is an ORF encoding the SARS-CoV-2 viral spike protein. In a preferred exemplary embodiment of the present invention, the ORF encoding a cancer antigen protein is an ORF encoding a cancer antigen protein H3K27M, an ORF encoding a cancer antigen protein PSCA, or an ORF encoding a cancer antigen protein TROP2. In a preferred exemplary embodiment of the present invention, the ORF encoding the reprogramming factor protein is an ORF encoding the reprogramming factor protein OSCK. In a preferred exemplary embodiment of the invention, the ORF encoding a chimeric antigen receptor protein (CAR protein) is an ORF encoding the CAR protein CD19.

[0025] Nucleotide sequence In an exemplary embodiment of the invention, the T7 promoter is a T7 promoter comprising the nucleotide sequence of SEQ ID NO:1. In an exemplary embodiment of the invention, 5'spacer 1 is 5'spacer 1 comprising the nucleotide sequence of SEQ ID NO:2. In an exemplary embodiment of the invention, the 5' external homology arm is a 5' external homology arm comprising the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4. In a preferred exemplary embodiment of the invention, the 5' external homology arm is the nucleotide sequence of SEQ ID NO:4.

[0026] In an exemplary embodiment of the invention, the Anabaena 3'PIE (intron-exon) is an Anabaena 3'PIE comprising the nucleotide sequence of SEQ ID NO:5. In an exemplary embodiment of the invention, the 5' internal homology arm is a 5' internal homology arm comprising the nucleotide sequence of SEQ ID NO:6. In an exemplary embodiment of the invention, 5'spacer 2 is a 5'spacer 2 comprising the nucleotide sequence of SEQ ID NO:7. In an exemplary embodiment of the invention, the IRES of the invention is an IRES set forth in Table 1 (SEQ ID NOs: 8-26).

[0027] [Table 1]

[0028] In an exemplary embodiment of the invention, the Age1 restriction enzyme site has the following nucleotide sequence: ACCGGT is an Age1 restriction enzyme site having the following structure: In an exemplary embodiment of the present invention, the ORFs of the present invention are those set forth in Table 2 (SEQ ID NOs: 27-35).

[0029] [Table 2]

[0030] In an exemplary embodiment of the invention, the NotI restriction enzyme site is provided with the following nucleotide sequence: GCGGCCGC is a NotI restriction enzyme site having the following structure: In an exemplary embodiment of the invention, 3'spacer 1 is 3'spacer 1 comprising the nucleotide sequence of SEQ ID NO:36. In an exemplary embodiment of the invention, the 3' internal homology arm is a 3' internal homology arm comprising the nucleotide sequence of SEQ ID NO:37. In an exemplary embodiment of the invention, the Anabaena 5'PIE (intron-exon) is an Anabaena 5'PIE comprising the nucleotide sequence of SEQ ID NO:38. In an exemplary embodiment of the invention, 3'spacer 2 is a 3'spacer 2 comprising the nucleotide sequence of SEQ ID NO:39.

[0031] In an exemplary embodiment of the invention, the 3' external homology arm is a 3' external homology arm comprising the nucleotide sequence of SEQ ID NO:40 or SEQ ID NO:41. In a preferred exemplary embodiment of the invention, the 3' external homology arm is the nucleotide sequence of SEQ ID NO:41.

[0032] In an exemplary embodiment of the invention, a plasmid vector for generating an ORF encoding circular mRNA comprises the nucleotide sequence of SEQ ID NO:42. In an exemplary embodiment of the present invention, in the plasmid vector for generating an ORF encoding circular mRNA, the ORF is an ORF encoding the SARS-CoV-2 viral spike protein (Pan-Hexapro-SPIKE), which is a full-length ORF comprising the nucleotide sequence of SEQ ID NO: 43. In an exemplary embodiment of the present invention, in the plasmid vector for generating an ORF encoding circular mRNA, the ORF is an ORF encoding the SARS-CoV-2 viral spike protein (Pan-VFLIP-SPIKE), which is a full-length ORF comprising the nucleotide sequence of SEQ ID NO:44.

[0033] In an exemplary embodiment of the present invention, in the plasmid vector for generating an ORF encoding circular mRNA, the ORF is an ORF encoding a cancer antigen protein H3K27M, which is a full-length ORF comprising the nucleotide sequence of SEQ ID NO:45. In an exemplary embodiment of the present invention, in the plasmid vector for generating an ORF encoding circular mRNA, the ORF is an ORF encoding the cancer antigen protein PSCA, comprising the nucleotide sequence of SEQ ID NO:46. In an exemplary embodiment of the present invention, in the plasmid vector for generating an ORF encoding circular mRNA, the ORF is an ORF encoding the cancer antigen protein TROP2, comprising the nucleotide sequence of SEQ ID NO:47. In an exemplary embodiment of the present invention, in a plasmid vector for generating an ORF-encoding circular mRNA, the ORF is an ORF encoding the reprogramming factor protein OSCK, comprising the nucleotide sequence of SEQ ID NO:48. In an exemplary embodiment of the present invention, in the plasmid vector for generating an ORF encoding circular mRNA, the ORF is an ORF encoding the chimeric antigen receptor protein CD19 comprising the nucleotide sequence of SEQ ID NO:49.

[0034] According to the present invention, an open reading frame-encoding circular mRNA (ORF-encoding circular mRNA) has been developed with an internal ribosome entry site (IRES) for facilitating ribosome binding and activating translation of a protein of interest. This results in at least a 2- to 10-fold increase in protein expression compared to linear mRNA. Moreover, it has a relatively short backbone, approximately 1,100 to 1,600 nucleotides. In order to better understand the present invention, various examples of the circular mRNA of the present invention are presented.These examples are provided to illustrate the embodiments of the present invention and should not be interpreted as limiting the scope of the present invention.The scope of the present invention is defined by the claims and their equivalents derived from this disclosure. EXAMPLES

[0035] List of sample abbreviations used herein below o "Sample A" refers to Lipofectamine® MessengerMax™ delivery system. "Sample A / circGFP" refers to the Lipofectamine® MessengerMax™ delivery system loaded with circular GFP mRNA (CVB3 IRES) of the present invention. "Sample A / circFluc" refers to the Lipofectamine® MessengerMax™ delivery system loaded with circular Fluc mRNA (CVB3 IRES) of the present invention. "Sample B" refers to a lipid nanoparticle formulation delivery system prepared by the method of International Journal of Pharmaceutics, 2021, 601, 120586. "Sample B / circGFP" refers to a lipid nanoparticle formulation delivery system carrying the circular GFP mRNA (CVB3 IRES) of the present invention. "Sample B / circFluc" refers to the lipid nanoparticle delivery system of the present invention carrying circular Fluc mRNA (CVB3 IRES). "Sample B / pCAG-Fluc" refers to the lipid nanoparticle formulation delivery system loaded with the positive control (pCAG-Fluc DNA). o "Sample C" refers to GenVoy Ionizable Lipid Mix (GenVoy-ILM™) delivery system. o "Sample C / circGFP" refers to the GenVoy Ionizable Lipid Mix (GenVoy-ILM™) delivery system loaded with the circular GFP mRNA (CVB3 IRES) of the present invention. o "Sample C1" refers to the GenVoy Ionizable Lipid Mix (GenVoy-ILM™) delivery system formulated with a ratio between ionizable lipid nitrogen and nucleic acid phosphate of 4:1 and a flow rate of approximately 12 ml / min. o "Sample C1 / circGFP" refers to the GenVoy Ionizable Lipid Mix (GenVoy-ILM™) delivery system loaded with circular GFP mRNA (CVB3 IRES) of the present invention. o "Sample C2" refers to the GenVoy Ionizable Lipid Mix (GenVoy-ILM™) delivery system formulated with a ratio between ionizable lipid nitrogen and nucleic acid phosphate of 6:1 and a flow rate of approximately 6 ml / min. "Sample C2 / circGFP" refers to the GenVoy Ionizable Lipid Mix (GenVoy-ILM™) delivery system loaded with circular GFP mRNA (CVB3 IRES) of the present invention. o "Sample D" refers to the commercially available delivery system Invivofectamine®. "Sample D / circFluc" refers to the commercially available delivery system Invivofectamine® loaded with the circular Fluc mRNA (CVB3 IRES) of the present invention. o "Sample E" refers to the LP3000 delivery system. o "Sample E / pCAG-Fluc" refers to the LP3000 delivery system loaded with the positive control (pCAG-Fluc DNA).

[0036] Example 1 Preparation of circular mRNA In vitro transcription reactions were performed with the HiScribe™ T7 High Yield RNA Synthesis Kit using a DNA template containing the ORF and a circular RNA element. The reaction was incubated at approximately 37° C. for approximately 2 hours, after which the DNA template was removed by adding deoxyribonuclease (DNase) with DNase buffer. The synthesized RNA was then purified using phenol / chloroform extraction and alcohol precipitation, and the resulting RNA precipitate was dissolved in ultrapure water. The RNA concentration was measured using a spectrophotometer, and the approximate size of the RNA was determined using agarose gel electrophoresis.

[0037] For RNA circularization, the RNA solutions containing the nucleotide sequence of the circular RNA element were first heated at approximately 65°C for about 5 minutes. Then, they were immediately transferred onto ice and cooled for about 3 minutes. The resulting RNA solutions were diluted with guanosine-5'-triphosphate (GTP) and Mg 2+ The mixture was mixed with an ion-containing buffer (e.g., T4 RNA ligase reaction buffer, NEB) and incubated at approximately 55°C for approximately 15 minutes. The circularized product was then purified by alcohol precipitation, after which the RNA precipitate was dissolved in ultrapure water. The concentration of the circular RNA was measured using a spectrophotometer, and the approximate size of the circular RNA was determined using agarose gel electrophoresis.

[0038] Example 2 Study of the efficiency of circular mRNA transfection into HEK293T cells using different delivery systems Samples were prepared using various commercially available delivery systems: HEK293T cells were transfected with approximately 1 microgram (μg) of circular mRNA with green fluorescent protein (GFP) (CVB3 IRES) (circular GFP mRNA).

[0039] The preparation details are as follows: 1. Sample A / circGFP Sample A reagent was diluted in Opti-MEM™ medium and incubated at room temperature for about 10 minutes. The diluted circular GFP mRNA in Opti-MEM™ medium was then mixed with the prepared Sample A and incubated at room temperature for about 5 minutes to obtain Sample A / circGFP. 2. Sample B / circGFP Circular GFP mRNA was mixed with sample B using a NanoAssemblr® Benchtop™ with a ratio between ionized lipid nitrogen and nucleic acid phosphate (N:P) of approximately 6:1 and a flow rate of approximately 6 ml / min. The prepared solution was then dialyzed against phosphate buffered saline (PBS) to obtain sample B / circGFP. 3. Sample C / circGFP Circular GFP mRNA was mixed with sample C using NanoAssemblr® Benchtop™ at different ratios between ionized lipid nitrogen and nucleic acid phosphate (N:P) and different flow rates. The prepared solution was then dialyzed against phosphate buffered saline (PBS) to obtain sample C1 / circGFP and sample C2 / circGFP.

[0040] After 48 hours of transfection, the transfection efficiency and mean fluorescence intensity (MFI) of HEK293T cells were analyzed using flow cytometry. Analysis of transfection efficiency and mean fluorescence intensity showed that sample A / circGFP transfection into HEK293T cells produced the best efficiency and highest expression of GFP compared to other samples. The highest transfection efficiency obtained from sample A / circGFP was about 92.3%. Sample A / circGFP transfected HEK293T cells also produced the highest mean fluorescence intensity of about 250,000 MFI compared to other samples, as shown in Figures 2 and 3.

[0041] Example 3 Study of the expression and stability of circular mRNA in HEK293T cells HEK293T cells were transfected with approximately 1 μg of luciferase-expressing circular mRNA (CVB3 IRES) (circular luciferase mRNA) using various commercially available delivery systems (samples A and D). Sample D / circFluc was prepared similarly to sample A / circFluc. After approximately 24 and 48 hours of transfection, the transfected HEK293T cells were lysed, and the lysed cells were analyzed to measure luciferase activity using a luciferase assay. Analysis of luciferase activity showed that Sample A / circFluc and Sample D / circFluc produced higher luciferase expression compared to the transfected positive controls, Sample B / pCAG-Fluc and Sample E / pCAG-Fluc. Sample E was prepared similarly to Sample A. Sample A / circFluc and Sample D / circFluc produced luciferase expression in the range of about 10,000,000-100,000,000 RLU (relative light units) and about 1,000,000-100,000,000 RLU at about 24 hours and about 48 hours, as shown in Figures 4 and 5, respectively. Furthermore, after transfection, analysis of luciferase activity showed that Sample A / circFluc and Sample D / circFluc remained stable in HEK293T cells for up to 48 h, with no significant difference in luciferase activity at 24 and 48 h, as shown in Figures 4 and 5.

[0042] Example 4 Study of expression and stability of circular mRNAs with different types of IRES in HEK293T cells Each of the IRES sequences, namely Coxsackievirus type B3 IRES (CVB3) (SEQ ID NO: 14), Soybean Aphidvirus type 1 IRES (A5I) (SEQ ID NO: 15), Halastavir albavirus IRES (H5I, HCI, HII) (SEQ ID NO: 16, 17, 18, respectively), Pegivirus type J IRES (Peg) (SEQ ID NO: 19), and combination of Pegivirus type J and Halastavir albavirus IRES (CPH) (SEQ ID NO: 20), were cloned into circular GFP vectors. These circular GFP vectors were used to generate circular mRNA. Approximately 2 μg of circular mRNA was then transfected into HEK293T cells using Sample A as a delivery system. Approximately 48 hours after transfection, HEK293T cells were analyzed using flow cytometry. Analysis of GFP expression and stability of circular mRNAs with various IRES in HEK293T cells showed that circular mRNA with CVB3 IRES produced the highest expression with positive results up to about 89% compared to cells transfected with circular mRNAs with other IRES. The flow cytometer results also showed that circular mRNA with CVB3 IRES was also stable in HEK293T cells for about 48 hours, as shown in Figures 6 and 7.

[0043] Example 5 Study of the expression and stability of circular mRNA in animals Sample B / circFluc was administered to mice (BALB / c strain, approximately 7 weeks old, female) via intramuscular (IM) and intravenous (IV) routes at doses of approximately 1 μg and approximately 10 μg. Approximately 24 and 48 hours after injection, the mice were anesthetized and their fluorescence values ​​were measured using an IVIS® Spectrum in vivo imaging system. Analysis of luminescence images showed that compared with the positive control (sample B / pCAG-Fluc) under the same conditions, sample B / circFluc had the highest expression and long stability in mice for about 48 hours, regardless of the injection route (intramuscular or intravenous injection) or dose (about 1 μg or about 10 μg) (Figure 8).

[0044] Example 6 Study of the expression and stability of circular SARS-CoV-2 spike protein mRNA in HEK293T cells Human embryonic kidney 293T (HEK293T) cells were transfected with circular mRNA (CVB3 IRES) expressing the SARS-CoV-2 spike protein in two forms: Pan-Hexapro-SPIKE (HexaPro-VI) and Pan-VFLIP-SPIKE (VFLIP-VI) at a dose of approximately 1 μg. The positive control was approximately 1 μg of SARS-CoV-2 spike protein DNA (pCMV3-S DNA). Approximately 48 hours after transfection, HEK293T cells were stained with SARS-CoV-2 (2019-nCoV) spike RBD antibody (rabbit PAb, antigen affinity purified (40592-T62, SinoBiological)) and goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor 488 (A11034, Invitrogen)). Flow cytometry was then used to analyze the efficiency of expression.

[0045] Flow cytometry analysis showed that compared to the positive control (pCMV3-S DNA), the circular SARS-CoV-2 spike protein mRNA (CVB3 IRES) resulted in high expression of spike protein in both Pan-Hexapro-SPIKE and Pan-VFLIP-SPIKE formats (Figure 9). We also transfected HEK293T cells with circular SARS-CoV-2 spike protein mRNA (CVB3 IRES), Pan-Hexapro-SPIKE (HexaPro-VI) and Pan-VFLIP-SPIKE (VFLIP-VI). Circular SARS-CoV-2 spike protein mRNA was lysed and lysed cells were analyzed by Western blot to determine the expression of SARS-CoV-2 spike protein in the supernatant. Cells were stained with SARS-CoV-2 spike RBD antibody (rabbit PAb), affinity purified (40592-T62, SinoBiological) and donkey anti-rabbit IgG-HRP (sc-2077, SANTA CRUZ BIOTECHNOLOGY,INC). The internal control protein was β-actin protein.

[0046] From the Western blot, it was found that both the Pan-Hexapro-SPIKE and Pan-VFLIP-SPIKE forms of circular SARS-CoV-2 spike protein mRNA (CVB3 IRES) resulted in high expression of spike protein compared to the positive control (pCMV3-S DNA), as shown in Figure 10.

[0047] Example 7 Study of the expression and stability of circular SARS-CoV-2 spike protein mRNA in experimental animals Using sample B, mice (BALB / c strain, approximately 7 weeks old, female) were injected with approximately 5 μg of circular mRNA (CVB3 IRES) expressing the SARS-CoV-2 spike protein of Pan-Hexapro-SPIKE (HexaPro-VI) and Pan-VFLIP-SPIKE (VFLIP-VI). The route of administration included two intramuscular (IM) injections, with the second dose administered 3 weeks after the first. Two weeks after the second injection, serum samples from the mice were analyzed by determining the expression levels of immunoglobulin G (IgG) specific for the SARS-CoV-2 spike protein omicron variant (B.1.1.529) using enzyme-linked immunosorbent assay (ELISA) compared to a control group of mice that received phosphate-buffered saline (PBS) injections.

[0048] The ELISA results showed that compared with the control group of mice, both forms of circular SARS-CoV-2 spike protein mRNA (CVB3 IRES), Pan-Hexapro-SPIKE (HexaPro-VI) and Pan-VFLIP-SPIKE (VFLIP-VI), were able to induce the expression of IgG specific to SARS-CoV-2 spike protein Omicron species (B.1.1.529) in mouse serum (as shown in Figure 11). We also used a neutralizing antibody assay to determine titers of antibodies specific to the SARS-CoV-2 pseudoviral spike protein Omicron species (B.1.1.529) by examining serum samples obtained from mice that received both forms of circular SARS-CoV-2 spike protein mRNA, Pan-Hexapro-SPIKE (HexaPro-VI) and Pan-VFLIP-SPIKE (VFLIP-VI). This analysis was performed and the results were compared to those obtained from a control group of mice that received injections of phosphate-buffered saline (PBS).

[0049] Neutralizing antibody assay analysis revealed that sera obtained from mice injected with circular mRNA (CVB3 IRES) expressing Pan-VFLIP-SPIKE (VFLIP-VI) spike protein showed the production of antibodies specific to the SARS-CoV-2 pseudoviral spike protein Omicron species (B.1.1.529) compared to the control group of mice (as illustrated in Figure 12).

[0050] Example 8 Study on the expression and stability of circular cancer antigen protein mRNA in HEK293T cells HEK293T cells were transfected with approximately 2 μg of circular cancer antigen protein mRNA containing H3K27M, PSCA, or TROP2. Approximately 48 hours after transfection, HEK293T cells were analyzed for protein expression using immunofluorescence. Cells were subjected to staining with recombinant anti-histone H3 (mutated K27M) antibody ([EPR18340]-ChIP grade (ab190631)) and goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor 594 (A-11037, Thermo Fisher)) to detect H3K27M protein expression. Anti-PSCA antibody (rabbit polyclonal (201684-T32, SinoBiological)) and goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor 594 (A-11037, Thermo Fisher)) were used to stain for PSCA protein expression. In addition, recombinant anti-TROP2 antibody (APC) (rabbit monoclonal (10428-R001-A, SinoBiological)) was used to stain for TROP2 protein expression. Immunofluorescence analysis showed that the circular cancer antigen protein mRNA in all three of its forms (i.e., H3K27M, PSCA, and TROP2) led to the expression of the corresponding cancer antigen protein, as evidenced by the red staining observed in the cells, which was comparable to the expression of the cancer antigen protein in non-transfected cells (as shown in Figures 13-15).

[0051] Example 9 Expression and stability of circular reprogramming factor mRNA in HEK293T cells HEK293T cells were transfected with approximately 2 μg of circular reprogramming factor mRNA OSCK. Approximately 48 hours after transfection, HEK293T cells were analyzed for protein expression using immunofluorescence. Cells were stained with anti-oct-3 / 4 antibody ((C-10) (sc-5279, Santa Cruz)) and goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor 488 (A-11290, Thermo Fisher)) to detect OCT4 protein. Sox2 (D6D9) XP® rabbit mAb (3579, Cell Signaling Technology) and goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor 488 (A-11290, Thermo Fisher)) were used to stain for SOX2 protein expression. Immunofluorescence analysis showed that the circular reprogramming factor mRNA OSCK led to the expression of reprogramming factor protein, as evidenced by the green staining observed in the cells, which was comparable to the expression of cancer antigen protein in non-transfected cells (as shown in Figures 16-17).

[0052] Example 10 Study of the expression and stability of circular chimeric antigen receptor mRNA (circular CAR mRNA) in HEK293T cells HEK293T cells were transfected with approximately 2 μg of circular mRNA expressing the chimeric antigen receptor (CVB3 IRES) (circular CAR mRNA)CD19. Approximately 48 hours after transfection, HEK293T cells were analyzed for protein expression using immunofluorescence. Cells were stained with CD3-zeta polyclonal antibody (PA5-98304, Thermo Fisher) and goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (Alexa Fluor 488 (A-11290, Thermo Fisher)) to detect CD19 protein. Immunofluorescence analysis showed that circular CAR mRNA (CVB3 IRES)CD19 led to expression of the chimeric antigen receptor protein, as evidenced by the red staining observed in the cells, which was comparable to the expression of the chimeric antigen receptor protein in non-transfected cells (as shown in FIG. 18).

[0053] Example 11 Circular RNA preparation To further prepare circular RNA using various types of IRES, DNA templates were designed to contain regions for circular ribonucleic acid. Polymerase chain reaction (PCR) was performed to obtain DNA precursors for in vitro RNA generation. All single-stranded RNAs were synthesized using HiScribe™ T7 High Yield RNA synthesis kit from New England Biolabs (NEB). A 10 μl reaction mixture was prepared with a final 1× IVT reaction buffer, 10 mM NTPs (ATP, UTP, CTP, and GTP), 500 ng DNA precursor, RNase inhibitor, and T7 RNA polymerase mixture. The reaction mixture was then incubated at approximately 37° C. for approximately 2 hours. The DNA precursor was then removed by the addition of DNase I, and the mixture was incubated at approximately 37° C. for approximately 15 minutes. The resulting RNA was purified using Monarch RNA column kit (NEB). Finally, RNA gel electrophoresis was performed to confirm RNA integrity.

[0054] To circularize the RNA, 50 μg of linear RNA from the in vitro transcription (IVT) reaction was heated to about 65° C. for about 5 minutes. It was then quickly transferred to ice and cooled for about 1 minute. The resulting RNA was then ligated to 1000 μl of 2 mM GTP and Mg in 1× T7 RNA ligase buffer (NEB). 2+and circularized at about 55°C for about 15 minutes. The reaction was immediately stopped by transferring the solution onto ice. The circularized product (IC) was then purified using a Monarch RNA column kit (NEB) and the approximate size of the circular RNA was determined by agarose gel electrophoresis. The resulting circularized IVT product is referred to as IC. The resulting RNA species from the IC product was confirmed. Briefly, the sample was treated with the exonuclease RNase R, which specifically digests linear RNA, at about 37° C. for about 2 hours. The entire reaction was then purified using the Monarch RNA column kit (NEB). Agarose gel electrophoresis was used to determine the approximate size of the circular RNA. As shown in Figure 19, circular RNA-eHA-1 and eHA-2 bands, representing the circular RNA obtained after treatment with RNase R, were seen in the IC and RNase R lanes. The homology arm eHA-2 duplex is designed to have a higher free energy than the homology arm eHA-1 duplex.

[0055] Example 12 Study of the expression and stability of circular mRNA in HEK293T cells HEK293T cells were cultured at approximately 50,000 cells / cm 2The cells were plated at a density of 1000 x 1000 and incubated at approximately 37°C in 5% CO2. After 24 hours of culture in Dulbecco's Modified Eagle Medium (DMEM) High Glucose (Cytiva) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), the cells were ready for RNA transfection. Circular RNA with different designs of 5' external homology arms (eHA) (eHA-1 or eHA-2) expressing firefly luciferase (Fluc) was added to the cells using Lipofectamine® MessengerMax™ (Invitrogen). The cells were washed once with 1X PBS and harvested 48 hours after transfection. The cells were counted and calculated to be 500,000 cells per reaction. 1X cell culture lysis reagent was introduced to perform cell lysis. Luciferase assay reagent was added to the luminometer tube, followed by cell lysate at a volume ratio of luciferase assay reagent to cell lysate of 5:1. Luciferase activity was then monitored using a luminometer.

[0056] Analysis of luciferase activity between the two different designed constructs showed that the circular luciferase mRNA with eHA-2 expressed higher luciferase activity than the eHA-1 construct. 24 hours after transfection, the circular luciferase mRNA produced a maximum luciferase expression of about 5,000-7,000 RLU (relative light units) (see FIG. 20). Both gel electrophoresis results (FIG. 19) and luciferase activity measurements (FIG. 20) showed that the circular RNA-eHA-2 construct had higher circularization efficiency and luciferase activity than the circular RNA-eHA-1 construct. Based on these findings, the eHA-2 design of external homology arms was used to construct a circular RNA, and the CVB3 IRES was replaced with other types of IRES, including natural or chimeric sequences.

[0057] (Example 13) Study of the expression and stability of circular mRNAs with different types of IRES in imHC and BHK-21 cells Chimeric sequences were engineered using domain IV replacement. For example, when domain IV of the CVB3 IRES was replaced with that of the ECH20 IRES, the resulting chimeric IRES was called 3(20)3 (Figure 21). CircRNA-eHA-2 engineered with chimeric IRES 3(20)3 is called 3(20)3 in the figures where appropriate. Mesenchymal stem cell-derived hepatocyte-like cells (imHC) or baby hamster kidney fibroblast (BHK-21) cells were cultured at approximately 50,000 cells / cm. 2 The cells were plated at a density of 0.01 mg / mL and incubated at approximately 37°C in 5% CO2. After approximately 24 hours of culture in Dulbecco's Modified Eagle's Medium (DMEM) High Glucose (Cytiva) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), the cells were ready for RNA transfection. Various types of IRES sequences were used, including chimeric IRES 3(20)3 (SEQ ID NO:21), chimeric IRES 20(3)20 (SEQ ID NO:22), chimeric IRES 3(80)3 (SEQ ID NO:23), chimeric IRES 80(3)80 (SEQ ID NO:24), chimeric IRES 12(20)12 (SEQ ID NO:25), and chimeric IRES 20(12)20 (SEQ ID NO:26). Circular RNA-eHA-2 constructs with different IRES expressing green fluorescent protein (GFP) were added to the cells using Lipofectamine® MessengerMax™ (Invitrogen). After 24 hours of transfection, GFP fluorescent signals were visualized using a fluorescent microscope.

[0058] Analysis of fluorescence intensity revealed that circular luciferase mRNAs with different IRESs showed different GFP expression in imHC and BHK-21 cells. In particular, the circular luciferase mRNA with 3(20)3 IRES had the highest mean fluorescence intensity (MFI) and differential mean fluorescence intensity (ΔMFI) of GFP compared with other IRES types (see Figures 22 and 23).

[0059] Example 14 Study of the expression and stability of circular mRNAs with different IRES in experimental animals In this study, Moderna Inc's formulation was used as a guide to select lipid components for the construction of lipid nanoparticles (LNPs) to encapsulate circular RNA. Circular RNA-LNP encapsulation was performed using a NanoAssemblr® Benchtop microfluidic device (Precision Nanosystems). Briefly, the process was summarized as follows: lipid-containing ethanol phase and circular RNA-containing aqueous phase were mixed in a volume ratio of 1:3. The ethanol phase contained a mixture of SM-102, DSPC, cholesterol, and DMG-PEG 2000 in absolute ethanol, all of which were obtained from Sinopeg. The aqueous phase was circular RNA in 25 mM acetate buffer at pH 4.0. After encapsulation, the concentration of circular RNA-LNP was examined using Qubit™ RNA High Sensitivity Assay Kit (Thermo Fisher). The functionality of circular RNA-LNP was confirmed by evaluating in vitro luciferase activity before administration to mice.

[0060] For intramuscular (IM) injection in mice, cyclic FLuc-eHA-2-CVB3 or cyclic FLuc-eHA-2-3(20)3 encapsulated in LNPs was first injected into the right hind leg of female BALB / c mice (n=3) using an insulin syringe at an amount of 5 μg. PBS was administered as a negative control. 6 or 48 hours after administration, mice were anesthetized with 2% isoflurane inhalation in an airtight transparent anesthesia box for 3 to 5 minutes. After anesthesia, mice were intraperitoneally injected with D-luciferin (150 micrograms per kilogram in 200 microliters (μl) of PBS) and placed in the imaging chamber of an in vivo imaging system (IVIS) for imaging. Bioluminescence imaging (BLI) was performed using an IVIS Spectrum, and consecutive images were obtained at 1-minute intervals with a 60-second exposure time.

[0061] Bioluminescence imaging analysis showed that circular luciferase mRNA bearing eHA-2-3(20)3 showed the highest expression and maintained stability in mice at both approximately 6 and 48 hours after intramuscular (IM) injection compared to the negative control (PBS) (see FIG. 24).

[0062] Best mode of invention The best mode of the present invention is as provided in the present specification.

Claims

1. An intrasequence ribosome entry site (IRES) comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, or a combination thereof.

2. The intra-sequence ribosome entry site (IRES) according to claim 1, comprising the nucleotide sequence shown in Sequence ID No.

21.

3. Connect them to each other in the following order: - RNA polymerase promoter, -5' Spacer 1, -5' External homology arm, -3'PIE (reordered intron-exon), -5' Internal homology arm, -5' Spacer 2, - Intra-sequence ribosome entry sites (IRES), - Open Reading Frame (ORF), -3' Spacer 1, -3' Internal homology arm, -5' Reordered intron-exon, -3' Spacer 2, and -3' External homology arm A plasmid vector for constructing an open reading frame coding circular mRNA containing elements arranged in a manner, The plasmid vector wherein IRES is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, or a combination thereof.

4. The plasmid vector according to claim 3, wherein the RNA polymerase promoter has a length of 15 to 25 nucleotides.

5. The plasmid vector according to claim 3 or 4, wherein the RNA polymerase promoter is selected from the group consisting of the T7 virus RNA polymerase promoter, the SP6 virus RNA polymerase promoter, or the T3 virus RNA polymerase.

6. The plasmid vector according to claim 5, wherein the RNA polymerase promoter is the T7 virus RNA polymerase promoter.

7. The plasmid vector according to claim 3, wherein the 5' spacer 1 has a length of 5 to 15 nucleotides.

8. The plasmid vector according to claim 3, wherein the 5' external homology arm has a length of 15 to 30 nucleotides.

9. The plasmid vector according to claim 8, wherein the 5' external homology arm has a length of 15 to 20 nucleotides.

10. The plasmid vector according to claim 8, wherein the 5' external homology arm has a length of 20 to 30 nucleotides.

11. The plasmid vector according to claim 3, wherein the 5' external homology arm is the sequence of sequence number 4.

12. The plasmid vector according to claim 3, wherein the 3'PIE (reordered intron-exon) has a length of 100 to 250 nucleotides.

13. The plasmid vector according to claim 3 or 12, wherein the 3'PIE (reordered intron-exon) is obtained from the cyanobacterium anabaena tRNA precursor group I intron gene.

14. The plasmid vector according to claim 3, wherein the 5' internal homology arm has a length of 15 to 25 nucleotides.

15. The plasmid vector according to claim 3, wherein the 5' spacer 2 has a length of 50 to 100 nucleotides.

16. The plasmid vector according to claim 3, wherein IRES has a length of 190 to 900 nucleotides.

17. The plasmid vector according to claim 3, wherein IRES is the sequence of SEQ ID NO:

21.

18. The plasmid vector according to claim 3, wherein the ORF is selected from the group consisting of an ORF encoding a viral spike protein, an ORF encoding a cancer antigen protein, an ORF encoding a reprogramming factor protein, or an ORF encoding a chimeric antigen receptor protein (CAR protein), or a combination thereof.

19. The plasmid vector according to claim 18, wherein the ORF encoding the viral spike protein is an ORF encoding the SARS-CoV-2 viral spike protein.

20. The plasmid vector according to claim 18, wherein the ORF encoding the cancer antigen protein is selected from an ORF encoding the cancer antigen protein H3K27M, an ORF encoding the cancer antigen protein PSCA, or an ORF encoding the cancer antigen protein TROP2.

21. The plasmid vector according to claim 18, wherein the ORF encoding the reprogramming factor protein is the ORF encoding the reprogramming factor protein OSCK.

22. The plasmid vector according to claim 18, wherein the ORF encoding a chimeric antigen receptor protein (CAR protein) is an ORF encoding the CAR protein CD19.

23. The plasmid vector according to claim 3, wherein the 3' spacer 1 has a length of 15 to 25 nucleotides.

24. The plasmid vector according to claim 3, wherein the 3' internal homology arm has a length of 15 to 25 nucleotides.

25. The plasmid vector according to claim 3, wherein the 5'PIE (reordered intron-exon) has a length of 100 to 250 nucleotides.

26. The plasmid vector according to claim 3 or 25, wherein the 5'PIE (reordered intron-exon) is obtained from the cyanobacterium anabaena tRNA precursor group I intron gene.

27. The plasmid vector according to claim 3, wherein the 3' spacer 2 has a length of 5 to 15 nucleotides.

28. The plasmid vector according to claim 3, wherein the 3' external homology arm has a length of 15 to 30 nucleotides.

29. The plasmid vector according to claim 28, wherein the 3' external homology arm has a length of 15 to 25 nucleotides.

30. The plasmid vector according to claim 28, wherein the 3' external homology arm has a length of 25 to 30 nucleotides.

31. The plasmid vector according to claim 3, wherein the 3' external homology arm is the sequence of SEQ ID NO:

41.

32. ORF-coding circular mRNA obtained from the plasmid vector described in claim 3.

33. Connect them to each other in the following order: -3' Exxon - Intra-sequence ribosome entry sites (IRES) - Open Reading Frame (ORF) -5' Exxon An open reading frame coding circular mRNA (ORF coding circular mRNA) obtained from a plasmid vector containing elements arranged in the following way, The ORF-coding circular mRNA wherein IRES is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, or a combination thereof.

34. The ORF-coding circular mRNA according to claim 33, wherein IRES has a length of 190 to 900 nucleotides.

35. The ORF-coding circular mRNA according to claim 33, wherein IRES is the sequence of SEQ ID NO:

21.

36. The ORF-coding circular mRNA according to claim 33, wherein the ORF is selected from the group consisting of an ORF encoding a viral spike protein, an ORF encoding a cancer antigen protein, an ORF encoding a reprogramming factor protein, or an ORF encoding a chimeric antigen receptor protein (CAR protein), or a combination thereof.

37. The ORF-coding circular mRNA according to claim 36, wherein the ORF encoding the viral spike protein is an ORF encoding the SARS-CoV-2 viral spike protein.

38. The ORF-coding circular mRNA according to claim 36, wherein the ORF encoding the cancer antigen protein is selected from an ORF encoding the cancer antigen protein H3K27M, an ORF encoding the cancer antigen protein PSCA, or an ORF encoding the cancer antigen protein TROP2.

39. The ORF-coding circular mRNA according to claim 36, wherein the ORF encoding the reprogramming factor protein is the ORF encoding the reprogramming factor protein OSCK.

40. The ORF-coding circular mRNA according to claim 36, wherein the ORF encoding the chimeric antigen receptor protein (CAR protein) is the ORF encoding the CAR protein CD19.

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