Recombinant expression constructs that enhance protein expression from synthetic ribonucleic acid (RNA)

Abstract

The present invention relates to improving protein expression in cells using translation-enhancing elements in the 5' and / or 3' untranslated regions (UTRs) of synthetic mRNAs. The 5'UTR comprises a promoter, a mini-enhancer sequence, and a Kozak sequence, and the 3'UTR comprises a spacer, a stem-loop structure, and optionally a polyadenine tail. The synthetic 5'UTR and 3'UTR of the present invention improve protein expression, and in some examples, do not have modified nucleotides, microRNA sites, or immune evasion factors.

Description

[Technical Field]

[0001] Reference to related applications This application claims priority under U.S. Provisional Patent Application No. 63 / 153,877, filed on 25 February 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence listing reference The sequence listing attached to this application is provided in text format instead of on paper, and is included by reference in this specification. The file name of the text file containing this sequence listing is P183-0010PCT_ST25.txt. The text file is 26.9KB in size, was created on February 24, 2022, and was submitted electronically via EFS-Web.

[0003] This disclosure provides recombinant expression constructs having artificial 5' and / or 3' untranslated regions (UTRs) adjacent to the coding sequence. The 5' UTR comprises a promoter, a mini-enhancer sequence, and a Kozak sequence, and the 3' UTR comprises a spacer, a stem-loop structure, and optionally a polyadenine tail. The artificial 5' and 3' UTRs enhance protein expression, and in some embodiments, modification Nucleoside It is composed without microRNA sites or immune evasion factors. [Background technology]

[0004] Conventional methods for influencing protein expression have several challenges. For example, while the introduction of foreign deoxyribonucleic acid (DNA) into the host cell's genomic DNA can be performed with some frequency, this introduction can cause mutation and / or damage to the host cell's genomic DNA. Alternatively, such foreign DNA introduced into a cell (regardless of whether the foreign DNA is introduced into a chromosome) can be passed on to daughter or progeny cells. Furthermore, even if delivery is performed correctly and no damage or introduction occurs to the host genome, multiple steps are required before the protein encoded by the DNA strand is produced. Once inside the cell, the DNA is taken up into the cell nucleus, where it is transcribed into RNA. Then, the RNA transcribed from the DNA enters the cytoplasm, where it is translated into protein. Because of these multiple processing steps from transfected DNA to the produced protein, there is a delay in the actual production of the functional protein, and errors or damage to the cell can occur at each step. In addition, the transfected DNA may not be expressed, or may not be expressed at the reasonable efficiency or concentration required for the desired application, making it difficult to achieve the desired level of protein expression in the cell. This can be particularly problematic when introducing DNA into primary cells or recombinant cell lines.

[0005] Furthermore, messenger RNA (mRNA) is being studied for its potential to enable rapid recombination of cells. Advantages of using mRNA as a reversible gene therapy include transient expression and non-transformation properties. Since mRNA does not need to enter the nucleus for expression and is not introduced into the host genome, there is no risk of tumor formation. The transfection efficiency achievable with mRNA is relatively high, exceeding 90% for many cell types, eliminating the need for selection of transfected cells.

[0006] While significant advancements have been made in recent years in the fields of mRNA therapeutics and mRNA vaccines, further research is still needed in these areas to improve the expression levels of mRNA-based proteins. [Overview of the project] [Problems that the invention aims to solve]

[0007] The purpose of this disclosure is to provide recombinant ribonucleic acid (e.g., mRNA) that enhances the expression level of encoded proteins.

[0008] Another object of this disclosure is to provide a minimal sequence that enhances the expression level of the encoded protein. [Means for solving the problem]

[0009] To achieve the above objectives, this disclosure provides specific recombinant expression constructs (Engineered Expression Constructs: EECs) that enhance the expression levels of encoded proteins. The EECs of the present invention have artificial 5' and / or 3' untranslated regions (UTRs) adjacent to the coding sequence. The 5'UTR comprises a promoter, a mini-enhancer sequence (CAUACUCA, as herein) and a Kozak sequence, and the 3'UTR comprises a spacer, a stem-loop structure and optionally a polyadenine tail (Poly-A tail). In some embodiments, the 5'UTR is configured to be operably ligated to a start codon to form a functional segment. In some embodiments described herein, the 3'UTR is described as a configuration comprising a stop codon.

[0010] With respect to the recombinant 5'UTR, in some embodiments, the promoter is derived from the bacteriophage T7 promoter and has the sequence GGGAGA. In some embodiments, the Kozak sequence comprises GCCRCC, where R is A or G. The Kozak sequence may also be operably ligated to a start codon to form the sequence GCCRCC-start (e.g., GCCRCCAUG).

[0011] A particular embodiment of the 5'UTR includes a mini-T7 promoter, a mini-enhancer sequence (CAUACUCA, as herein) and a Kozak sequence between 5' and 3', resulting in GGGAGACAUACUCAGCCACC (SEQ ID NO: 2) or GGGAGACAUACUCAGCCGCC (SEQ ID NO: 3). Adding a start codon results in GGGAGACAUACUCAGCCACCAUG (SEQ ID NO: 38) and GGGAGACAUACUCAGCCGCCAUG (SEQ ID NO: 39).

[0012] In some embodiments, the number of nucleotides in these minimum 5'UTR is less than 30. In some embodiments, the number of nucleotides in these minimum 5'UTR is 20 or 23.

[0013] With respect to the recombinant 3'UTR, in some embodiments, the spacer is [N 1-3 ]AUA or [N 1-3 It has AAA. In more specific examples, the spacer has UGCAUA or UGCAAA. Exemplary stem-loop structures are formed by hybridizing sequences such as CCUC and GAGG. In some examples, the loop structure formed between hybridization sequences has a configuration of 7 to 15 nucleotides. The sequence of the loop segment has, for example, UAACGGUCUU (SEQ ID NO: 34). When included as part of a 3'UTR sequence, examples of stop codons include UAA, UAG, and UGA.

[0014] In some examples, the number of nucleotides in these minimum 3'UTR is less than 30.

[0015] Regarding the stem-loop, regardless of the order of the stem-loop sequence, improved protein expression has been observed in the EEC disclosed herein, indicating that the secondary structure is important and not necessarily the 5'-to-3' sequence. The recombinant 3'UTR may further contain a poly(A) tail.

[0016] In one aspect, the present disclosure provides an EEC comprising in vitro synthesized RNA comprising a coding sequence encoding a protein to be translated in mammalian cells, wherein the in vitro synthesized RNA has a 5'UTR having the sequence CAUACUCA.

[0017] In one aspect, the present disclosure provides an EEC comprising in vitro synthesized RNA comprising a coding sequence encoding a protein to be translated in mammalian cells, wherein the in vitro synthesized RNA has a 5'UTR having SEQ ID NO: 2 or SEQ ID NO: 3. Further, the 5'UTR may have a start codon and be SEQ ID NO: 38 or SEQ ID NO: 39.

[0018] In one aspect, the present disclosure provides an EEC comprising a 3'UTR having SEQ ID NO: 4, 5, 6, 7, 8, or 9.

[0019] In one aspect, the present disclosure provides an EEC comprising a 3'UTR having SEQ ID NO: 10, 11, or 12.

[0020] In one aspect, the present disclosure provides an EEC comprising a 3'UTR having SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20 or 21.

[0021] Each of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 may be configured to include a polyA tail.

[0022] In one aspect, the present disclosure provides an EEC comprising in vitro synthesized RNA comprising a coding sequence encoding a protein to be translated in mammalian cells, wherein the in vitro synthesized RNA is configured to include a 5'UTR having the sequence CAUACUCA and a 3'UTR having SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and / or 21.

[0023] In one embodiment, the Disclosure provides an EEC comprising an in vitro synthetic RNA comprising a coding sequence that encodes a protein to be translated in a mammalian cell, wherein the in vitro synthetic RNA comprises a 5'UTR having SEQ ID NO: 2 or SEQ ID NO: 3 and a 3'UTR having SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and / or 21.

[0024] In one embodiment, the Disclosure provides an EEC comprising an in vitro synthetic RNA comprising a coding sequence that encodes a protein to be translated in a mammalian cell, wherein the in vitro synthetic RNA comprises a 5'UTR having SEQ ID NO: 38 or SEQ ID NO: 39 and a 3'UTR having SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and / or 21.

[0025] In some specific embodiments, the EEC disclosed herein is modified Nucleoside It does not include. In some specific embodiments, the EEC disclosed herein does not include a microRNA binding site. In some specific embodiments, the EEC disclosed herein is modified Nucleoside It does not contain microRNA binding sites.

[0026] In the disclosed EEC, the recombinant 5' and 3'UTRs are adjacent to the coding sequence in the open reading frame. The data provided in this disclosure demonstrate that the expression of green fluorescent protein (GFP), interleukin-2 (IL-2), and POU5F1 (OCT3 / 4) was enhanced in various cell types (e.g., lymphoid cells, adherent and suspension embryonic kidney cells) regardless of the transfection morphology.

[0027] The EECs disclosed herein can be used to enhance the expression of various proteins for a number of diverse purposes. Examples of such purposes include use in therapeutics and vaccines. [Brief explanation of the drawing]

[0028] Some drawings may be easier to understand if they were in color. The applicant considers the color versions of the drawings to have been submitted as part of the original application and reserves the right to submit color versions in subsequent proceedings.

[0029] [Figure 1] Figure 1 shows a schematic of an EEC designed to enhance protein expression in vivo. The EEC contains several modules to enhance protein expression. The module located within the 5'UTR is divided into three modules: module 1 ("M1"), which represents the promoter (e.g., T7 promoter hexamer); module 2 ("M2"), which represents the translation enhancer of this application (CAUACUCA, described later); and module 3 ("M3"), which is the Kozak consensus sequence. The 3'UTR, as illustrated here, is also divided into three segments, including a stop codon, a spacer, and a stem-loop segment. It may also include a poly(A) tail.

[0030] [Figure 2] Figure 2 is a flow cytometry histogram, with GFP intensity (FL1-H) on the x-axis and cell number on the y-axis. EXPI293 cells were transfected by gradually increasing the amount of EEC containing the 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) sequences related to the present invention, and having the GFP coding sequence within its open reading frame (see SEQ ID NOs: 55 and 56), as described later. As shown in the figure, the GFP signal reached saturation at 0.4 pmol (100 ng) 24 hours after transfection.

[0031] [Figure 3] Figures 3A and 3B are flow cytometry histograms, showing GFP intensity (FL1-H) on the x-axis and cell number on the y-axis for GFP-coding EEC 5'UTR mutants 3 hours after transfection (Figure 3A) and 24 hours after transfection (Figure 3B). The results of the flow cytometry experiment are also shown as bar graphs. EXPI293 cells were transfected with equimolar amounts of GFP-coding EEC 5'UTR mutants, including RNA-free (negative symmetric), mRNA without 5'UTR, M1 and M3 modules with only 5'UTR, and M1, M2, and M3 modules with 5'UTR (SEQ ID NO: 2).

[0032] [Figure 4] Figures 4A and 4B are flow cytometry histograms, showing GFP intensity (FL1-H) on the x-axis and cell number on the y-axis for GFP-encoding EEC 5'UTR and 3'UTR mutants 3 hours after transfection (Figure 4A) and 24 hours after transfection (Figure 4B). The results of the flow cytometry experiments are also shown as bar graphs. EXPI293 cells were transfected with equimolar amounts of GFP-encoding EEC 5'UTR mutants as described below. (i) No RNA (negative symmetry); (ii) mRNA having only a 3'UTR (UGCAUACCUCUAACGGUCUUGAGG (SEQ ID NO: 10) or UAAUGCAUACCUCUAACGGUCUUGAGG (SEQ ID NO: 13) which has the basic form of the UTR that enables transcription, but lacks a 5'UTR and has a 3'UTR); (iii) Only M3 having a 3'UTR in addition to the 5'UTR (UAAUGCAUACCUCUAACGGUCUUGAGG (SEQ ID NO: 13) as the 5'UTR and GGGAGAGCCACCAUG (ORF) (SEQ ID NO: 63) as the 3'UTR); and (iv) M1, M2, and M3 (SEQ ID NO: 10) having a 5'UTR (SEQ ID NO: 2) and a 3'UTR. Cells treated with EEC having full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) show the highest GFP intensity.

[0033] [Figure 5] Figure 5 shows three proteins expressed using the EEC disclosed herein, illustrating the target protein expression in the cytoplasm, organelles (i.e., nuclear compartment), and extracellular compartment (i.e., secondary proteins).

[0034] [Figure 6]Figures 6A to 6C show HEK293 cells expressing the GFP protein. Figure 6A is a flow cytometry graph showing GFP (x axis), illustrating the changes in detection that occurred with the gradual increase in the amount of GFP-coded EEC transfected into HEK293 cells (the panel labeled "no mRNA" contains no mRNA of GFP-coded EEC, while the other panels, as indicated by their respective panel titles, contain GFP-coded EEC mRNA at 0.2 pmol, 0.4 pmol, 1.0 pmol, 2.0 pmol, and 4.0 pmol respectively). Figure 6B is a chart showing the results from the flow cytometry graph in Figure 6A, representing the increase in the percentage of GFP-positive cells with respect to the gradual increase in the dose of GFP-coded EEC. In the chart in Figure 6B, the percentage of GFP-positive HEK293 cells is shown on the y-axis, and the amount of GFP-coding EEC mRNA is shown on the x-axis. Thus, Figure 6B shows the increase in the percentage of GFP-positive cells in relation to the increase in GFP-coding EEC mRNA. Figure 6C is a chart showing the results from the flow cytometry graph in Figure 6A, and is shown in the form of the increase in median GFP intensity proportional to the increase in the amount of GFP-coding EEC transfection in cells positive for GFP expression. In Figure 6C, the median GFP intensity from GFP-expressing cells is shown on the y-axis (FLI-H), and the amount of GFP-coding EEC mRNA is shown on the x-axis. Thus, Figure 6C shows the increase in median GFP intensity proportional to the increase in GFP-coding EEC mRNA.

[0035] [Figure 7]Figures 7A and 7C show HEK293 cells expressing GFP protein after transfection with GFP-coding EEC mutants. Figure 7A is a histogram of cells transfected with 0.4 pmol of GFP-coding EEC mutants, with GFP intensity (x axis) superimposed. Figure 7B is similar to Figure 7A, showing the case where cells were transfected with 1 pmol of GFP-coding EEC mutants. Figure 7C is a bar graph of median GFP intensity of HEK293 cells transfected with equimolar amounts of GFP-coding EEC mutants (n=2). The mutants used were 5'UTR only (SEQ ID NO: 2), 3'UTR only (described in detail in the explanation of Figure 4B), 5' UTR and 3'UTR (SEQ ID NO: 2 and SEQ ID NO: 10, described in detail in the explanation of Figure 4B), Kozak only, and Kozak and 3'UTR (SEQ ID NO: 10).

[0036] [Figure 8]Figures 8A and 8B show Jurkat(T) lymphocytes expressing GFP protein after transfection with GFP-coding EEC variants. Figure 8A is a flow cytometry graph showing the amount of GFP-positive Jurkat cells in each cell population transfected with various GFP-coding EECs (full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10)). Figure 8B is a flow cytometry histogram showing the amount of GFP-positive Jurkat cells in each cell population transfected with various EEC constructs of the GFP-coding EECs 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10). The results of the flow cytometry graph in Figure 8A are shown as a line graph, showing the percentage of GFP-positive Jurkat cells transfected with gradually increasing amounts of GFP-coding EECs containing the full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10). Figure 8B shows the results of the flow cytometry experiment as a bar graph, illustrating the GFP intensity and percentage of Jurkat cells transfected with 8 pmol of GFP-coding EEC mutant (n=2). Data are available for mutants with no UTR, Kozak only, 5' only, 3' only, 5' and 3', and Kozak and 3'. For details of the sequences used in these mutants, please refer to the descriptions in Figures 4B and 7C.

[0037] [Figure 9]Figures 9A and 9C show the GFP expression levels in Jurkat(T) lymphocytes 24 hours after electroporation with GFP-coding EECs containing UTR variants. Figure 9A shows a typical histogram, displaying the GFP intensity and percentage of Jurkat cells transfected with 4 pmol of each GFP-coding EEC variant. Figure 9B also shows a typical histogram, displaying the GFP intensity and percentage of Jurkat cells transfected with 8 pmol of each GFP-coding EEC variant. Figure 9C is a bar graph showing the median GFP intensity (experimental count = 2) of lymphocytes containing 4 and 8 pmol of the GFP-coding EEC variants shown in Figures 9A and 9B.

[0038] [Figure 10] Figures 10A to 10C show typical experiments in which GFP-expressing Raji lymphocytes were analyzed by flow cytometry 24 hours after electroporation of lymphocytes with a GFP-coding EEC containing a UTR mutant. Figure 10A is a graph of GFP intensity and percentage for Raji lymphocytes transfected with 4 pmol of the GFP-coding EEC mutant. Figure 10B is a graph of GFP intensity and percentage for Raji lymphocytes transfected with 8 pmol of the GFP-coding EEC mutant. Figure 10C is a bar graph showing the median GFP intensity for the experiments shown in Figures 10A and 10B (number of experiments = 2), showing the GFP intensity for Raji lymphocytes transfected with 4 pmol and 8 pmol of each GFP-coding EEC mutant. The data includes variants with no UTR, Kozak only, 5' only (SEQ ID NO: 2), 3' only (SEQ ID NO: 10), 5' and 3' ((SEQ ID NO: 2) and (SEQ ID NO: 10)), and Kozak and 3' (SEQ ID NO: 10).

[0039] [Figure 11]Figures 11A and 11B show the expression of hOCT3 / 4 protein in HEK293 cells 24 hours after transfection with hOCT3 / 4-coded EECs having the 5' and 3'UTR variants disclosed herein. Figure 11A shows the results of a typical flow cytometry experiment showing the intensity and percentage of hOCT3 / 4 in hOCT3 / 4-positive HEK293 cells transfected with an incrementing dose of hOCT3 / 4-coded EEC (having full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10)). Figure 11B shows the results of a typical flow cytometry experiment showing the intensity and percentage of hOCT3 / 4 in hOCT3 / 4-positive cells transfected with 1.2 pmol of hOCT3 / 4 having the UTR variants. The results of the flow cytometry experiment in Figure 11A are shown as a line graph. The bar graph shown in Figure 11B represents the median value of the results from an experiment (number of experiments = 2) that showed the amount of hOCT3 / 4 with UTR mutants compared to the case without RNA.

[0040] [Figure 12] Figures 12A and 12B show the expression of hIL2 protein (measured by ELISA) from HEK293 cells 24 hours after transfection with hIL2-coding EECs having the 5' and 3' UTR variants disclosed herein. Figure 12A is a graph showing the increase in absorbance (@450 nm) of the ELISA signal with increasing dose escalation of hIL2-coding EECs (having full-length 5' UTR (SEQ ID NO: 2) and 3' UTR (SEQ ID NO: 10)). Figure 12B is a bar graph showing the amount of hIL2 from HEK293 cells transfected with 0.5 pmol of hIL2-coding EECs having each UTR variant ((SEQ ID NO: 2) and (SEQ ID NO: 10)).

[0041] [Figure 13]Figures 13A–13C show the expression of GFP protein in HEK293 cells after transfection with GFP-coding EECs containing the full-length 5'UTR (SEQ ID NO: 2) and 3'UTR variants (SEQ ID NOs: 10, 11, and 12). Figure 13A shows the sequence of the 3'UTR. Figure 13B shows the GFP-positive HEK293 cells (based on a flow cytometry event count / cell count of 10,000) 24 hours after transfection with 1–2 pmol of each EEC. Figure 13C is a bar graph showing the median GFP intensity (from two separate experiments) of HEK293 cells transfected with 1–2 pmol of GFP-coding EECs containing 3'UTR variants A, B, and C shown in Figure 13A.

[0042] [Figure 14] Figure 14 shows Oct4 expression in human foreskin fibroblasts after transfection with recombinant Oct4 mRNA constructs (UO is unmodified mRNA Oct4, UMD is unmodified mRNA MyoD-Oct4, PUO is modified mRNA Oct4, and PUMD is modified mRNA MyoD-Oct4).

[0043] [Figure 15] Figure 15 shows other sequences (SEQ ID NOs. 55-60) supporting this disclosure, including cDNA constructs for producing in vitro synthetic RNA and the synthetic RNA constructs obtained thereby, for EGFP, Oct4, and IL2. [Modes for carrying out the invention]

[0044] Significant scientific progress has been made in the use of RNA as a therapeutic agent, vaccine, and / or in the recombination of protein expression in cells both in vitro and in vivo. One major challenge to be overcome is achieving sufficiently high protein expression levels to obtain the desired results. Several attempts have been made to overcome this challenge and improve the expression of target proteins so that mRNA can be used in various clinical applications. Such research includes, for example, U.S. Patent US20060247195 (filed June 8, 2006, assignee: Ribostem Limited, status: abandoned), U.S. Patent 10,772,975 (filed May 12, 2011, assignee: Moderna, status: issued), PCT application PCT / EP2008 / 01059 (filed December 12, 2008, publication number: WO2009077134, assignee: BioNTech AG), and PCT application PCT / EP2008 / 03033 (filed April 16, 2008, publication number: WO2009127230, assignee: Curevac). It is disclosed in GMBH, PCT application PCT / US2016 / 069079 (filing date: December 29, 2016, assignee: Cellular Reprogramming, Inc.), PCT application PCT / US2019 / 037069 (filing date: June 13, 2019, assignee: Cellular Reprogramming, Inc.), and others.

[0045] The untranslated region (UTR) of a gene is a region that is transcribed but not translated. Generally, the 5' UTR is the region that extends from the transcription start site to the start codon, but does not include the start codon. The 3' UTR, on the other hand, begins after the stop codon and extends to the transcription termination signal. Messenger RNA (mRNA) contains UTRs that are known to recruit ribosomes, initiate translation, and thereby improve protein expression. As mentioned above, start and stop codons are not generally considered part of the UTR, but in this disclosure, these segments may be included within sequences designated as UTRs that form a functional segment.

[0046] There is growing evidence that UTRs play a regulatory role in the stability of nucleic acid molecules and, consequently, in translation and protein expression. The sequences within UTRs differ between prokaryotes and eukaryotes. For example, the Shine-Dalgano consensus sequence (5'-AGGAGGU-3') in bacteria recruits ribosomes, while the RNA Kozak consensus sequence (5'-GCCRCCRUGG-3') in mammalian cells contains a start codon (AUG) and facilitates the translation initiation event.

[0047] In the Kozak consensus sequence, "R" indicates adenosine or guanosine. The -3 position in the Kozak consensus sequence promotes translation initiation, and overall, the Kozak sequence appears to halt the translation initiation complex to the correct recognition site of the start codon. While the Kozak consensus sequence itself can promote ribosome scanning and translation initiation, other UTRs associated with large amounts of proteins in the human transcriptome were analyzed. Studies have shown that these relatively large amounts of proteins are involved in genetic information processing, such as proteins associated with chromosomes and ribosomes (Beck et al., The quantitative proteome of a human cell line. Mol. Syst. Biol. (2011), doi:10.1038 / msb.2011.82; Liebermeister et al., Visual account of protein investment in cellular functions. Proc. Natl. Acad. Sci. USA (2014), doi:10.1073 / pnas.1314810111).For example, alignment of the 5'UTR of highly expressed ribosome-associated proteins (RPLs / RPSs) has shown the appearance of a 5'-terminal oligopyrimidine track (i.e., 5'TOP) or C / U (Lavallee-Adam et al., Functional 5' UTR motif discovery with LESMoN: Local enrichment of sequence motifs in biological networks. Nucleic Acids Res. (2017), doi:10.1093 / nar / gkx751; Yoshihama et al., The human ribosomal protein genes: Sequencing and comparative analysis of 73 genes. Genome Res. (2002), doi:10.1101 / gr.214202; Cardinali et al., La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes. J. Biol. Chem. (2003), doi:10.1074 / jbc.M300722200; Pichon (et al., RNA Binding Protein / RNA Element Interactions and the Control of Translation. Curr. Protein Pept. Sci. (2012), doi:10.2174 / 13892031280161947). Generally, the 5'TOP sequence is located near the start codon and is important for transcription (i.e., RNA synthesis) and transcription translation.

[0048] By genetically modifying genes that exhibit characteristics typically found in highly expressed genes of specific target organs, the expression of coding sequences can be improved. For example, introducing the 5'UTR of liver-expressed mRNA such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, α-fetoprotein, erythropoietin, or factor VIII can improve the expression of coding sequences in hepatocyte lines or the liver. Similarly, it is possible to enhance expression in another tissue using 5'UTRs from tissue-specific mRNAs, including muscle (MyoD, myosin, myoglobin, myogenin, herculin), endothelial cells (Tie-1, CD36), myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (SP-A / B / C / D).

[0049] However, UTR can be as long as hundreds or thousands of nucleotides (nts). When mRNA is used in therapeutics or vaccines, finding the minimum / optimal UTR is preferable for improving the expression of the desired protein in cells. In some examples, the recombinant expression constructs (EECs) disclosed herein are designed to have a minimum UTR (minUT). That is, the EEC is designed to have the minimum 5' and / or 3' UTR that can achieve high expression levels in the intended application.

[0050] Therefore, in certain embodiments, the Disclosure provides minimum UTRs that significantly improve protein expression. In some examples, the Disclosure provides 5' UTRs having 20 to 23 nucleotides. In some examples, the Disclosure provides 3' UTRs having 27 or 67 nucleotides, depending on whether or not they contain an optional poly-A tail. When combined, some examples of 5' and 3' UTR combinations contain 47 to 50 nucleotides, or 87 to 90 nucleotides. These size characteristics are particularly beneficial in therapeutic and / or vaccine applications.

[0051] In some embodiments, to construct minUTs, the Disclosure includes components necessary for in vitro mRNA production and in vivo protein production. For in vitro mRNA production, the Disclosure employs the use of RNA polymerase from T7 bacteriophage (T7P). T7P is a specific DNA double helix sequence (5'-TAATACGACTCACTATA G It binds to -3' (SEQ ID NO: 45) and initiates RNA synthesis by introducing guanosine (the last G in the promoter, underlined) as the first ribonucleic acid. This binding sequence is generally followed by a pentamer (5'-GGAGA-3'). The pentamer stabilizes the transcription complex and promotes T7P clearance and RNA polymer elongation.

[0052] In certain embodiments, the 5'UTR includes a promoter, a mini-enhancer sequence (here, CAUACUCA), and a Kozak sequence such as a cleavage-type Kozak sequence (GCCRCC). In certain embodiments, the 5'UTR is also described as optionally operably concatenated to a start codon to form a functional segment.

[0053] In certain embodiments, minimal promoters are selected for use within the 5'UTR. Minimal promoters do not have gene expression-promoting activity on their own, but can be activated and promote gene expression when ligated to a proximal promoter element. Examples of minimal promoters include minBglobin, minCMV, minCMV with the Sacl restriction site removed, minRho, minRho with the Sacl restriction site removed, and the Hsp68 minimal promoter (proHSP68). In certain embodiments, minimal promoters include the miniT7 promoter (miniT7 promoter).

[0054] An example of a 5'UTR disclosed is the mini-enhancer sequence (CAUACUCA) of this application. The mini-enhancer sequence may be positioned between a minimal promoter (e.g., T7) and a Kozak consensus sequence to produce a minimal 5'UTR having 20 to 23 nucleotides (depending on whether the start codon is designated as part of the UTR). Generally, eukaryotic translation is initiated by the AUG codon, but other start codons may be included. Mammalian cells can also initiate translation with the amino acid leucine using leucyl-tRNA that decodes the CUG codon, and the mitochondrial genome uses AUA or AUU in humans. These components and exemplary 5'UTRs are shown in Table 1. [Table 1]

[0055] In some embodiments, the 5'UTR is capped. For example, eukaryotic mRNA is guanylylated by adding an inverted 7-methylguanosine to the 5' tripphosphate (i.e., m7GpppN (where N is the first base of the mRNA)). The m7GpppN, or 5' cap structure of the mRNA, is associated with nuclear export and binds to mRNA cap-binding proteins (CBPs) related to mRNA translation ability.

[0056] Furthermore, the ribose sugars at the 1st and 2nd nucleotides of mRNA may optionally be methylated at the 2'-oxygen (2'O) position (i.e., the addition of a CH3 group). mRNA in which the 1st and 2nd nucleotides are not methylated is called Cap0 (i.e., m7GpppN), and methylation at 2'O at the 1st and 2nd nucleotides is called Cap1 (i.e., m7GpppNm) and Cap2 (i.e., m7GpppNmNm), respectively. In addition, if the 5' nucleotide at the 1st position is adenosine, it may also be further methylated at the 6th nitrogen (6N) position (i.e., m7Gpppm6A), or modified Cap0 (m7Gpppm6A), modified Cap1 (i.e., m7Gpppm6Am), or modified Cap2 (i.e., m7Gpppm6AmNm).

[0057] The guanylylation of RNA, i.e., the addition of Cap0 (i.e., m7GpppN), may be performed enzymatically in vitro (i.e., after RNA synthesis) using vaccinia virus capping enzyme (VCE). Furthermore, the formation of Cap1 and Cap2 structures may be performed enzymatically via the addition of mRNA 2'-O-methyltransferase and S-adenosylmethionine (SAM). Alternatively, the Cap structure may be added in vitro by co-transcription using an anti-reverse cap analog (ARCA). ARCA is methylated at the 3'-oxygen (3'O) of the cap (m73'OmGpppN) to ensure that the cap structure is introduced in the correct orientation. In this application, any of the above cap structures may be used for the final EEC mRNA product.

[0058] In certain embodiments, the 5'UTR is operably ligated to the coding sequence. In this specification, “operably ligated” means a functional linkage formed between a nucleotide expression regulatory sequence (e.g., a promoter sequence or UTR) and another nucleotide sequence, thereby enabling and allowing the transcription and / or translation of the other nucleotide sequence by the regulatory sequence.

[0059] Furthermore, this disclosure provides a 3'UTR that can be optionally used in conjunction with the disclosed 5'UTR. As shown herein, combining the disclosed 5'UTR and the disclosed 3'UTR yields an EEC with significantly improved protein expression compared to using the disclosed 5'UTR alone or the disclosed 3'UTR alone.

[0060] The 3'UTR is known to contain embedded adenosine and uridine sequences. These AU-rich signatures are particularly common in genes with high turnover rates. Based on their sequence features and functional properties, AU-rich elements (AREs) can be classified into three classes. Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more duplicated UUAUUUA(U / A)(U / A) denatures.

[0061] As previously stated, the disclosed 3'UTR comprises a spacer, a stem-loop structure, and optionally a polyadenine tail (poly-A tail). In some of the embodiments disclosed herein, the 3'UTR is optionally linked to a stop codon.

[0062] Examples of stop codons include UAA, UGA, and UAG.

[0063] Examples of spacers include [N 1-3 ]AUA or [N 1-3 Examples include ]AAA (e.g., UGCAUA, UGCAAA, UGAAA, GCAUA, UAAA, and GAUA) (wherein N is any nucleotide such as A, G, C, T, or U). The subscript number indicates the number of nucleotides. For example, [N 1-3 ] contains one, two, or three nucleotides, and means N, NN, or NNN.

[0064] A stem-loop (SL), also known as a hairpin or hairpin loop, is a characteristic of transcripts with high expression levels within the 3'UTR. An SL is a specific secondary structure in which complementary nucleotides pair up as a double helix (i.e., a stem) and are sometimes interposed by other sequences to form a loop. A specific example of a secondary structure represented by an SL is a continuous nucleic acid sequence containing a stem and a (terminal) loop, also called a hairpin loop. The stem is formed by two adjacent, whole or partially complementary sequence elements, and these adjacent complementary sequence elements are interposed by short sequences (e.g., 3 to 10 nucleotides) to form the SL structure loop. Two adjacent, whole or partially complementary sequence elements may be referred to as, for example, SL element stem 1 and stem 2. An SL consists of two adjacent, whole or partially inversely complementary sequence elements (e.g., SL element stem 1 and stem 2) that form base pairs with each other to constitute a double-stranded nucleic acid sequence. However, at the ends, they are not paired, and a short sequence interposed between SL element stem 1 and stem 2 forms a loop. Therefore, one SL contains two stems (stem 1 and stem 2). At the secondary structure level of the nucleic acid molecule, these two stems form base pairs with each other, while at the primary structure level, they are interposed by a short sequence that is not part of stem 1 or stem 2. In diagrams, the secondary representation of an SL resembles a lollipop structure. To form a stem-loop structure, sequences are needed that can fold in half to form a paired double helix, and this double helix is ​​formed by stem 1 and stem 2. The stability of a paired SL element is typically determined by its length, i.e., the number of nucleotides in stem 1 that cannot form base pairs with the nucleotides in stem 2 (mismatches or bulges) compared to the number of nucleotides in stem 1 that can form base pairs with the nucleotides in stem 2 (preferably standard base pairs, more preferably Watson-Crick base pairs).According to the present invention, the optimal loop length is 3 to 10 nucleotides, more preferably 4 to 7 nucleotides, for example, 4 nucleotides, 5 nucleotides, 6 nucleotides, or 7 nucleotides. If a nucleic acid sequence forms an SL, then the corresponding complementary nucleic acid sequence also typically forms an SL. SLs are typically formed by single-stranded RNA molecules. In certain embodiments, the SL length is at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides.

[0065] The SL is located within the 3'UTR of highly expressed transcripts (e.g., those encoding large amounts of cellular proteins such as histones), where it promotes translation regardless of polyadenine tails (Gallie et al., The histone 3′-terminal stem-loop is necessary for translation in Chinese hamster ovary cells (Nucleic Acids Res. (1996), doi:10.1093 / nar / 24.10.1954)). The histone 3'UTR stem consensus is characterized by having six base pairs: two GC pairs, three pyrimidine-purine (YR) pairs, and one AU pair. The loop has four ribonucleic acids: two uridine (U), one purine (Y), and one ribonucleotide (N). (Gallie et al., The histone 3'-terminal stem-loop is necessary for translation in Chinese hamster ovary cells. (Nucleic Acids Res. (1996), doi:10.1093 / nar / 24.10.1954; Tan et al., Structure of histone mRNA stem-loop, human stem-loop binding protein, and 3'hExo teRNAry complex. Science (80). (2013), doi:10.1126 / science.1228705; Battle & Doudna, The stem-loop binding protein forms a highly stable and specific complex with the 3′ stem-loop of histone mRNAs. RNA (2001), doi:10.1017 / S1355838201001820)).

[0066] SL is associated with the stem-loop binding protein (SLBP) in replication-dependent mRNA stability / processing / metabolism / translation. Structural evidence suggests that SLBP is in direct contact with SL at the guanosine nucleic acid (G7) base of SL (Tan et al., Structure of histone mRNA stem-loop, human stem-loop binding protein, and 3′hExo teRNAry complex. Science (80). (2013), doi:10.1126 / science.1228705; Battle & Doudna, The stem-loop binding protein forms a highly stable and specific complex with the 3′ stem-loop of histone mRNAs. RNA (2001), doi:10.1017 / S1355838201001820). Furthermore, adenosine adjacent to the stem, more specifically upstream AAA, influences the binding and function of SLBP (Battle & Doudna, The stem-loop binding protein forms a highly stable and specific complex with the 3′ stem-loop of histone mRNAs. RNA (2001), doi:10.1017 / S1355838201001820; William & Marzluff, The sequence of the stem and flanking sequences at the 3′ end of histone mRNA are critical determinants for the binding of the stem-loop binding protein. Nucleic Acids Res. (1995), doi:10.1093 / nar / 23.4.654). In addition, the 3'UTR enhances translational capacity while also stabilizing protein-coding transcripts.According to some embodiments, the disclosure provides a design for synthetic SLs that introduces an SL configuration of three groups of GC pairs interposed by a sequence having multiple adjacent spacer sequences such as adenosine (UAACGGUCUU (SEQ ID NO: 34)), thereby improving SLBP binding and mRNA translation.

[0067] In particular, it is important that the stem-loop used in EEC is independent of the orientation of the sequences and may be a) a configuration including CCUC and GAGG, b) a configuration including GAGG and CCUC, c) a configuration including AAACCUC and GAGG, and d) a configuration including AAAGAGG and CCUC. Furthermore, the distance between the two arms of the stem (where CCUC and GAGG form a base pair) must be long enough to form a loop. In certain embodiments, the stem-loop may be a configuration including complementary sequences such as a) RRRR and YYYY, b) RYRR and YRYY, c) RRYR and YYRY, d) RRRY and YYYR, e) RYYR and YRRY, f) RRYY and YYRR, g) YYRR and RRYY, h) YYYR and RRRY, or i) RYYY and YRRR (where R is a purine (A or G) and Y is a pyrimidine (e.g., U or C)).

[0068] According to one embodiment, the number of nucleotides between the two arms may be 7, 8, 9, 10, or more. A preferred embodiment of the length between the two arms of the stem loop is 7 or more nucleotides. In some embodiments, the loop segment of the SL includes UAACGGUCUU (SEQ ID NO: 34). According to a particular embodiment, the SL sequence includes GAUGCCCCAUUCACGAGUAGUGGGUAUU (SEQ ID NO: 64), GGCACCCUGCGCAGGUGAUGCAGGUGCC (SEQ ID NO: 65), GUUCGCUCGGUCAGGAGAGCUGACGGAC (SEQ ID NO: 66), UCUUACAGUGGCAUGUGACCGUUUAAGG (SEQ ID NO: 67), CGCGGCGCAUGCACGUGACAUGCCUGCG (SEQ ID NO: 68), CGGUCCCGUGGCAAGAGUCUAUGGAUUG (SEQ ID NO: 69), AUGUUCGGCUCCAAGAGCGAGUUGAUAU (SEQ ID NO: 70), CGAUUCGGGCACAUGUGCUGUCUGAUUG (SEQ ID NO: 71), GUAUUCUGAUGCACGUGCCAUCAAGUAC (SEQ ID NO: 72), or UUGAGCAGGAUCAAGUGCAUUCUUUCAA (SEQ ID NO: 73). According to a particular embodiment, the SL sequence is RRYRYYYYYRYYYRYRRRYRRYRRRYRYY (SEQ ID NO: 74), RRYRYYYYRYRYRRRYRRYRYRRRYRYY (SEQ ID NO: 75) containing RYYYRYYYRRYYRRRRRRRYYRRYRRRY (SEQ ID NO: 76), YYYYRYRRYRRYRYRYRRYYRYYYRRRR (SEQ ID NO: 77), YRYRRYRYRYRYRYRYRYRYRYRYYYRYR (SEQ ID NO: 78), YRRYYYYRYRRYRRRRRYYYRYRRRYYR (SEQ ID NO: 79), RYRYYYRRYYYYRRRRRRRYRRRYYRRYRY (SEQ ID NO: 80), YRRYYYRRRYRYRYRYRYYYYRRYYR (SEQ ID NO: 81), RYRYYYYRRYRYRYRYRYYRYYRRRYRY (SEQ ID NO: 82), or YYRRRYRRRRYYRRRYRYRYYYYYYYRR (SEQ ID NO: 83) (wherein R is a purine (A or G) and Y is a pyrimidine (e.g., U or C)).For other examples of SL motifs, see Gorodkin et al. (Nucleic Acids Research 29(10):2135-2144, 2001).

[0069] Any Poly A Tail In natural RNA processing, long chains of adenine nucleotides (poly-A tails) can be added to polynucleotides such as mRNA molecules to improve their stability. Immediately after transcription, the 3' end of the transcript is cleaved, releasing the 3' hydroxyl group. Then, poly-A polymerase adds the adenine nucleotide chain to the RNA. This process is called polyadenylation, and it adds a poly-A tail that is, for example, 100 to 250 residues long. In in vitro RNA synthesis, a DNA template encoded with poly-A tails is introduced into the in vitro transcription process.

[0070] In some embodiments, the poly-A tail has a nucleotide length ranging from 0 to 500 (e.g., 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotide lengths). In some embodiments, a 40-nucleotide poly-A tail is used. The length can also be expressed as a unit of poly-A binding protein binding or as a function thereof. In such embodiments, the poly-A tail is long enough to bind to four monomers of the poly-A binding protein, three monomers of the poly-A binding protein, two monomers of the poly-A binding protein, or one monomer of the poly-A binding protein. The poly-A binding protein monomers bind to a sequence of 38 nucleotides.

[0071] Based on the foregoing description of 3'UTR, the 3'UTR structures disclosed herein include one or more spacers (e.g., [N 1-3 ]AUA, [N 1-3]AAA, UGCAUA or UGCAAA, etc.), stem-loop hybridization sequence (e.g., CCUC or GAGG), stem-loop loop segment (e.g., [N 7-15 ]、 It contains UAACGGUCUU (SEQ ID NO: 34), and / or optionally a poly-A tail. When the stop codon is considered part of the 3'UTR, examples of stop codons include UAA, UGA, and UAG. Examples of 3'UTR constructs based on these components are shown in Table 2.

[0072] [Table 2]

[0073] In certain embodiments, the 5'UTR (or 3'UTR) may be modified to include other non-UTR sequences. For example, an intron sequence or a portion of an intron sequence may be modified to include these regions. The introduction of an intron sequence may further enhance protein expression and mRNA levels.

[0074] Disclosed EEC structure using 5' and 3' UTR The genetically modified 5'UTR and 3'UTR sequences disclosed herein, when used adjacent to any coding sequence, can be used to form an EEC useful for enhancing the protein expression of various proteins. Examples of these proteins include green fluorescent protein (GFP), interleukin-2 (IL-2), and POU5F1 (OCT3 / 4), as disclosed herein. Furthermore, the 5'UTR and 3'UTR sequences relating to these inventions demonstrate similar action in various cell types, such as lymphoid cells and adherent and suspension embryonic kidney cells, regardless of the mode of transfection.

[0075] Figure 1 shows a typical EEC of the present disclosure. In some embodiments, “EEC” refers to a polynucleic acid transcript having a coding sequence that codes for one or more proteins, and having a 5' and / or 3' UTR adjacent to the coding sequence that maintains sufficient structural and / or chemical configuration to enable translation of the proteins encoded therein.

[0076] As shown in Figure 1, the illustrated EEC contains a coding sequence consisting of linked nucleotides within an open reading frame adjacent to the first and second flanking regions. This coding sequence contains an RNA sequence that codes for a protein. This protein may also contain one or more signal sequences encoded by a signal sequence region at its 5' end. The first flanking region may contain a region consisting of linked nucleotides containing one or more complete or incomplete 5'UTR sequences. The first flanking region may also contain a 5' end cap. The first functional segment connects the 5' end of the coding sequence to the first flanking region. Conventionally, this functional segment contains a start codon. Alternatively, the functional segment may contain any translation start sequence or signal containing a start codon.

[0077] The first flanking region may consist of modules located within the 5'UTR. This first flanking region may be divided into three modules: module 1 ("M1") representing a minimal promoter (e.g., a T7 promoter hexamer), module 2 ("M2") representing the translation enhancer of the present invention (CAUACUCA, described later), and module 3 ("M3") being the Kozak consensus sequence. The T7 promoter hexamer is part of the T7 polymerase promoter, that is, part of a T7 class III promoter, which is a specific type of promoter known in the art that is associated with and induces the transcription of a specific promoter of the T7 bacteriophage. More specifically, the T7 promoter hexamer has a complete sequence (5'-TAATACGACTCACTATAGGGAGA-3' (SEQ ID NO: 31)) and initiates RNA synthesis by introducing guanosine as the ribonucleic acid at position 1. The Kossack consensus sequence is defined as the Kossack consensus sequence (5'-GCCRCCATGG-3' (SEQ ID NO: 30), where "R" represents adenosine or guanosine).

[0078] The second flanking region may consist of a region of linked nucleotides containing one or more complete or incomplete 3'UTRs. The flanking region may also consist of a 3' tailing sequence (e.g., a poly-A tail). The 3'UTR may also consist of three segments, such as a stop codon, a spacer, and a stem-loop segment.

[0079] The second functional segment connects the 3' end of the code sequence to the second flanking region. Conventionally, this functional segment includes a stop codon. Alternatively, the functional segment may include any translation start sequence or signal containing a stop codon. According to this disclosure, it may also be a configuration using multiple consecutive stop codons.

[0080] Generally, the shortest length of the EEC coding sequence may be a nucleic acid sequence length sufficient to code a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In other embodiments, this length may be sufficient to code a peptide of 2 to 30 amino acids, for example, a peptide of 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 20 amino acids. This length may be sufficient to code a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or a peptide consisting of 40 or fewer amino acids, for example, a peptide consisting of 35 or fewer, 30 or fewer, 25 or fewer, 20 or fewer, 17 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, or 10 or fewer amino acids. Examples of dipeptides that can be encoded by polynucleotide sequences include carnosine and anserine.

[0081] Generally, the length of a code sequence is greater than 30 nucleotides (for example, at least 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, (nucleotide lengths of 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or more, or nucleotide lengths of 100,000 or less).

[0082] In some embodiments, the EEC contains 30 to 100,000 nucleotides (for example, 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000) 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000 1, 500 to 50,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000 (0, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000).

[0083] According to this disclosure, the first and second flanking regions may each independently range in length from 5 to 100 nucleotides (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 4 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 sets (nucleotide length).

[0084] According to this disclosure, the cap region may be a cap or a configuration including a series of nucleotides forming a cap. In this embodiment, the cap region may be of a length having 1 to 10 nucleotides, for example, 2 to 9, 3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 2, or 10 or fewer nucleotides. In some embodiments, no cap is provided.

[0085] According to this disclosure, the first or second functional segment may have a nucleotide length in the range of 3 to 40, for example, 5 to 30, 10 to 20, 15, or at least 4, or 30 or fewer nucleotide lengths, and may include one or more signal sequences and / or restriction sequences in addition to start and / or stop codons.

[0086] Various modifications have been attempted to stabilize transfected IVT-RNA in order to achieve high expression and prolonged expression of IVT-RNA. While RNA transfection-based strategies have been successful in the expression of peptides and proteins in cells, problems remain regarding RNA stability, maintenance of the expression of encoded peptides and proteins, and RNA cytotoxicity. For example, exogenous single-stranded RNA is known to activate defense mechanisms in mammalian cells.

[0087] According to several research groups, in order to achieve sufficiently high protein expression levels from transfected IVT-RNA in cells due to the activation of defense mechanisms, the mRNA transcript needs to contain modified nucleotides (see, for example, U.S. Patent No. 9,750,824 (filed August 4, 2012, assignee: University of Pennsylvania)) or other reagents in the form of proteins or IVT-RNA containing immune evasion factors (see, for example, U.S. Patent No. 10,207,009 (filed May 7, 2015, assignee: BioNTech)). These immune evasion factors include viral genes encoding proteins that weaken the cellular immune response. These immune evasion proteins weaken the cellular immune response by, for example, interfering with the binding of extracellular IFN receptors by extracellular IFN (e.g., B18R from vaccinia virus), inhibiting intracellular IFN signaling (e.g., both E3 and K3 from vaccinia virus), or performing both functions (e.g., NS1 from influenza) (Liu et al., Sci Rep 9: 11972, 2019). In certain embodiments, the immune evasion protein includes B18R, E3, K3, NS1, or ORF8 (from SARS-CoV-2).

[0088] Aspects of this disclosure are designed to overcome activated defense mechanisms by introducing secondary and tertiary structures into mRNA transcripts, rather than by using modified nucleotides, microRNAs, or immune evasion factors. According to other embodiments, certain embodiments enhance protein expression without using modified nucleotides or microRNAs. Even further embodiments do not use modified nucleotides or microRNAs for purposes such as extending translation from IVT-RNA transfected into cells or for other purposes.

[0089] In some embodiments, the EEC does not contain microRNA binding sites and / or modified NTPs in the 5'UTR, in the 3'UTR, in both the 5'UTR and 3'UTR, or in the EEC as a whole.

[0090] MicroRNAs (miRNAs) are non-coding RNAs with a length of 19 to 25 nucleotides that bind to the 3'UTR of nucleic acid molecules, suppressing gene expression by reducing the stability of the nucleic acid molecule or inhibiting translation. In some examples, the EEC (Enhanced Ecosystem) is composed of elements that do not include known microRNA target sequences, microRNA sequences, or microRNA seeds.

[0091] A microRNA seed is a sequence located in the region between positions 2 and 8 of a mature microRNA, and it is a sequence that exhibits complete Watson-Crick complementarity to the miRNA target sequence.

[0092] In some embodiments, the EECs of this disclosure are designed without specifically including modified NTPs. A modified NTP is one whose chemical structure is modified by having an additional chemical group bound to it. Examples of these modified NTPs include pseudouridine, methylpseudridine, N1-methylpseudridine, methyluridine (m5U), 5-methoxyuridine (mo5U), and 2-thiouridine (s2U). The 5' cap does not modify the NTP.

[0093] In some embodiments, the EEC includes messenger RNA (mRNA). In this specification, “messenger RNA” (mRNA) means any polynucleotide that codes for a protein and can be translated to produce a coding protein in vitro, in vivo, in situ, or ex vivo.

[0094] EEC codes for proteins or fragments thereof. “Protein” refers to a polymer of amino acid residues (natural or unnatural) linked together, often by peptide bonds. The term encompasses polypeptides and peptides of all sizes, structures, or functions. Encoding proteins may be smaller than 50 amino acids; such proteins are referred to as peptides. If a protein is a peptide, it has at least two linked amino acids. Proteins include naturally occurring proteins, synthetic proteins, homologs, orthologues, paralogs, fragments, recombinant proteins, fusion proteins, and other equivalents, variants, and analogues thereof. A protein may be a single protein or a multimolecular complex such as a dimer, trimer, or tetramer. It also includes single-chain proteins and multi-chain proteins, such as antibodies and insulin, which may associate or link together. Disulfide bonds are typically found in multi-chain proteins. The term protein also applies to amino acid polymers, which are artificial chemical analogues of corresponding naturally occurring amino acids, with one or more amino acid residues.

[0095] The term "protein variant" refers to a protein whose amino acid sequence differs from that of the native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at specific positions in the amino acid sequence compared to the native or reference sequence. Generally, variants have at least 50% sequence identity with respect to the native or reference sequence, preferably at least 80% sequence identity, and more preferably at least 90% sequence identity.

[0096] EEC may encode a protein selected from one of several target categories, which include proteins or peptides for biologics, antibodies, vaccines, therapeutics, cell-permeable peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane-bound proteins, nucleoproteins, human disease-related proteins, target sites, or proteins encoded by the human genome that are useful in the research and discovery of therapeutic uses that have not yet been identified. A specific protein may fall into one or more of these categories.

[0097] In some embodiments, specific sequences encoding particular proteins are used. Examples of these specific proteins include green fluorescent protein (GFP), interleukin 2 (IL-2), and POU5F1, i.e., OCT3 / 4 (see Figure 15). GFP is a protein that emits bright green fluorescence when exposed to light. Human POU5F1, i.e., OCT3 / 4 (here, hOCT4), is a major nuclear transcription factor important for stem cell reprogramming and maintenance. IL-2 is an interleukin, a type of cytokine signaling molecule in the immune system. It is a 15.5 to 16 kDa protein that regulates the activity of white blood cells. IL-2 is part of the body's natural response to bacterial infection, distinguishing between foreign substances ("non-self") and "self." IL-2 exerts its effect by binding to IL-2 receptors expressed on lymphocytes. The main sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells.

[0098] The EECs disclosed herein may encode one or more biologics. “Biologics” include proteins used in the treatment, cure, alleviation, prevention, or diagnosis of diseases or conditions. Examples of biologics include allergen extracts (e.g., for allergy injections and tests), blood components, gene therapy products, human tissue or cell products for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytics, immunomodulators, and others.

[0099] antibody The EECs disclosed herein may encode one or more antibodies or fragments thereof. The term “antibody” includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), polyepitope-specific antibody compositions, polyspecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments. The term “immunoglobulin (Ig)” is used herein as synonymous with “antibody.” In this specification, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, i.e., an individual antibody from a population that is identical except for spontaneously occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), although these may be present in small amounts. Monoclonal antibodies are highly specific and directed toward a single antigen site.

[0100] Examples of monoclonal antibodies used herein include "chimeric" antibodies (immunoglobulins) and fragments of such antibodies, insofar as they exhibit the desired biological activity. A "chimeric" antibody (immunoglobulin) is an antibody in which part of its heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the rest of its heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass. Examples of chimeric antibodies used herein include "primatized" antibodies that contain a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0101] Examples of "antibody fragments" include a portion of an intact antibody, preferably the antigen-binding and / or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; nanobodies; single-chain antibody molecules; and polyspecific antibodies formed from antibody fragments.

[0102] The five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, may all be encoded by coding sequences that include heavy chains, represented as α, δ, ε, γ, and μ, respectively. Polynucleotide sequences encoding subclasses such as γ and μ are also examples. Therefore, any of the antibody subclasses may be partially or entirely encoded, with examples including subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0103] In certain embodiments, the EEC disclosed herein may encode a monoclonal antibody and / or a variant thereof. Examples of antibody variants include substitutional variants, conservative amino acid substitutions, insertional variants, deletion variants, and / or covalent derivatives. In certain embodiments, the EEC disclosed herein may encode an immunoglobulin Fc region. In other embodiments, the EEC may encode a variant of the immunoglobulin Fc region. As a non-limiting example, the EEC may encode an antibody having a variant of the immunoglobulin Fc region.

[0104] Specific embodiments may encode anti-SARS-CoV-2 antibodies, anti-SARS antibodies, anti-RSV antibodies, anti-HIV antibodies, anti-dengue virus antibodies, anti-borrhea antibodies, anti-hepatitis C antibodies, anti-influenza virus antibodies, anti-parainfluenza virus antibodies, anti-metapneumovirus (MPV) antibodies, anti-cytomegalovirus antibodies, anti-Epstein-Barr virus antibodies, anti-herpes simplex virus antibodies, anti-Clostridium difficile toxin antibodies, or anti-tumor necrosis factor (TNF) antibodies.

[0105] Examples of known anti-RSV antibodies include palivizumab, described in U.S. Patent No. 9,403,900, AB1128 (MILLIPORE), and ab20745 (ABCAM).

[0106] One example of a known anti-HIV antibody is 10E8, a broad-spectrum neutralizing antibody that binds to gp41. VRC01 is also a broad-spectrum neutralizing antibody that binds to the CD4 binding site of gp120. Other examples of anti-HIV antibodies include ab18633 and 39 / 5.4A (ABCAM) and H81E (THERMOFISHER).

[0107] Examples of anti-dengue virus antibodies include antibody 55 (described in U.S. Patent No. 20170233460), antibody DB2-3 (described in U.S. Patent No. 8,637,035), and ab155042 and ab80914 (both manufactured by ABCAM).

[0108] One example of an anti-borrheumatoid arthritis antibody is described in U.S. Patent No. 9,512,204.

[0109] Examples of anti-hepatitis C antibodies include MAB8694 (MILLIPORE) and C7-50 (ABCAM).

[0110] Anti-influenza virus antibodies are described in U.S. Patent No. 9,469,685, with C102 (manufactured by THERMOPISHER) being an example.

[0111] An example of an anti-MPV antibody is MPE8.

[0112] Examples of anti-cytomegalovirus (CMV) antibodies include MCMV5322A, MCMV3068A, LJP538, and LJP539. See also, for example, Deng et al., Antimicrobial Agents and Chemotherapy 62(2) e01108-17 (Feb. 2018); and Dole et al., Antimicrobial Agents and Chemotherapy 60(5) 2881-2887 (May 2016).

[0113] Examples of anti-herpes simplex virus (HSV) antibodies include HSV8-N and MB66.

[0114] Examples of anti-Clostridium difficile toxin antibodies include actoxumab and bezlotoxumab. See also, for example, Wilcox et al., N Engl J Med 376(4) 305-317 (2017).

[0115] Those skilled in the art will know that numerous other antibody sequences are available and usable within the scope of the teachings of this disclosure. Sequence information for commercially available antibodies may be found in databases such as DrugBank, the CAS Registry, and / or the RSCB protein data bank.

[0116] vaccine The EECs disclosed herein may encode one or more vaccines. In this specification, “vaccine” means a composition that stimulates an immune response to produce acquired immunity to a specific pathogen or infectious agent that causes a disease or infection, and / or to a specific pathogen or infectious agent necessary for the onset of such disease or infection, thereby enhancing immunity to that pathogen or infectious agent. For example, a vaccine is a preparation that produces an immune system that responds to a specific antigen by pre-exposing the immune system to that antigen. The pathogenic antigen may be an intact but non-infectious form of the pathogen (e.g., heat-sterilized). Alternatively, the antigen may be a protein or protein fragment of the pathogen, or a protein or protein fragment expressed by an abnormal cell type (e.g., an infected cell or cancer cell). When the immune system recognizes the antigen after prior exposure, it leads to long-term immunological memory, which enables the immune system to produce an immediate and effective response if the antigen is exposed again.

[0117] For example, the viral vaccine antigen may be derived from adenovirus, arenavirus, bunyavirus, coronavirus, flavivirus, hantavirus, hepadnavirus, herpesvirus, papillomavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, orthomyxovirus, retrovirus, reovirus, rhabdovirus, rotavirus, spongiform virus, or togavirus. In certain embodiments, the vaccine antigen includes peptides expressed by viruses such as CMV, EBV, influenza virus, hepatitis A, B, or C, herpes simplex, HIV, influenza, Japanese encephalitis, measles, polio, rabies, RSV, rubella, smallpox, varicella-zoster virus, West Nile fever, and / or Zika fever.

[0118] Examples of vaccine antigens derived from all pathogens include attenuated poliovirus used in oral polio vaccine (OPV) and germicidal polio vaccine used in inactivated polio vaccine.

[0119] Other specific examples include: SARS-CoV-02 vaccine antigens such as the spike protein and its fragments (e.g., receptor-binding domain (RBD)); CMV vaccine antigens such as the envelope glycoprotein B and CMV pp65; EBV vaccine antigens such as EBV EBNAI, EBV P18, and EBV P23; hepatitis vaccine antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, HBCAG DELTA, HBV HBE, hepatitis C virus RNA, HCV NS3, and HCV NS4; herpes simplex vaccine antigens such as the initial protein and glycoprotein; and human immunodeficiency virus (HIV) vaccine antigens such as HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, and HIV Examples include gag, pol, and env genes such as GP36, Nef protein, and reverse transcriptase; L1 protein is an example of a human papillomavirus (HPV) viral antigen; hemagglutinin and neuraminidase are examples of influenza virus antigens; proteins E, ME, ME-NS1, NS1, NS1-NS2A, and 80%E are examples of Japanese encephalitis vaccine antigens; plasmodium protein circumsporozoite (CSP), glutamate dehydrogenase, lactate dehydrogenase, and fructose diphosphate aldolase are examples of malaria vaccine antigens; measles virus fusion protein is an example of a measles vaccine antigen; rabies glycoprotein and rabies nucleoprotein are examples of rabies virus antigens; RSV fusion protein and M2 protein are examples of RS vaccine antigens; VP7sc is an example of a rotavirus vaccine antigen; proteins E1 and E2 are examples of rubella vaccine antigens.Examples of varicella-zoster virus antigens include GPL and GPL; examples of Zika vaccine antigens include the premembrane, envelope (E), and domain (III) of protein E, as well as non-structural proteins 1-5.

[0120] Other specific examples of viral antigen sequences include: Nef (66-97): (VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQ ID NO: 48)); Nef (116-145): (HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO: 49)); Gag p17 (17-35): (EKIRLRPGGKKKYKLKHIV (SEQ ID NO: 50)); Gag p17-p24 (253-284): (NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD (SEQ ID NO: 51)); Pol 325-355 (RT 158-188): (AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ ID NO: 52)); CSP central repeat region: (NANPNANPNANPNANPNANP (SEQ ID NO: 53)); and E protein domain III: Examples include (AFTFTKIPAETLHTVTEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITEGTENSKMMLELDPPFGDSYIVIGVGE (Sequence ID 54)). For other examples of viral antigens, see Fundamental Virology, 2nd edition, eds. Fields, BN and Knipe, DM (Raven Press, New York, 1991).

[0121] In certain embodiments, the vaccine antigen is expressed by cells involved in bacterial infection. Examples of bacteria include Bacillus anthrax, Gram-negative bacilli, Chlamydia, Diphtheriae, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis, pertussis toxin, Streptococcus pneumoniae, Rickettsia, Staphylococcus, Streptococcus, and Neisseria tetanus.

[0122] Specific examples of bacterial vaccine antigens include, for example, the anthrax vaccine antigen, the protective antigen of Bacillus anthrax; for example, lipopolysaccharides for Gram-negative bacilli vaccine antigens; for example, membrane polysaccharides for Haemophilus influenzae vaccine antigens; for example, diphtheria toxin for diphtheria vaccine antigens; for example, mycolic acid, heat shock protein 65 (HSP65), 30kDa secretory proteins, and antigen 85A for Mycobacterium tuberculosis vaccine antigens; for example, hemagglutinin, partactin, FIM2, FIM3, and adenylyl cyclase for pertussis toxin vaccine antigens; for example, pneumolysin and pneumococcal membrane polysaccharides for pneumococcal vaccine antigens; for example, rompA for rickettsia vaccine antibodies; for example, M protein for streptococcal vaccine antigens; and for example, tetanus toxin for tetanus vaccine antigens.

[0123] In certain embodiments, the vaccine antigen is derived from a multidrug-resistant "superbug." Examples of superbugs include Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and members of the Enterobacteriaceae family (such as Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp.).

[0124] Furthermore, the vaccine antigen may contain proteins that are specifically and preferentially expressed by cancer cells, and may be configured to activate the immune system that fights cancer. Examples of cancer antibodies include: A33; BAGE; B-cell maturation antigen (BCMA); Bcl-2; β-catenin; CA19-9; CA125; carboxy-anhydrase-IX (CAIX); CD5; CD19; CD20; CD21; CD22; CD24; CD33; CD37; CD45; CD123; CD133; CEA; c-Met; CS-1; cyclin B1; DAGE; EBNA; EGFR; ephrin B2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2 / neu; HPV E6; HPV E7; Ki-67; L1-CAM; LRP; MAGE; MART; mesoserine; MUC; Examples include MUM-1-B; myc; NYESO-1; p53, PRAME; progesterone receptor; PSA; PSCA; PSMA; ras; RORl; Survivin; SV40 T; tenascin; TSTA tyrosinase; VEGF; and WT1.

[0125] The use of RNA vaccines offers an attractive alternative to avoid the potential risks of DNA vaccines. Similar to DNA, RNA can be introduced into cells to induce both cellular and humoral immune responses in vivo. In particular, different strategies for both approaches have been explored in immunotherapy using in vitro transcribed RNA (IVT-RNA), both of which have yielded favorable experimental results in various animal models. RNA can be injected directly into the patient via various immune pathways, or IVT-RNA can be transfected into cells in vitro using known transfection methods, and the transfected cells administered to the patient. For example, the RNA may be translated into proteins expressed on MHC molecules on the cell surface to elicit an immune response.

[0126] Therapeutic proteins are proteins that, when expressed by cells, treat existing symptoms or diseases. "Therapeutic" means that the expression of the protein alleviates the cause of an existing symptom or disease and / or alleviates the side effects of the symptom or disease (e.g., pain, inflammation, congestion, fatigue, fever, chills, etc.).

[0127] Cell permeability proteins The EECs disclosed herein may encode one or more cell-permeable proteins (CCPs, also referred to as cell-permeable peptides). A CCP is a protein that can facilitate the uptake and absorption of molecules by cells. Generally, cell-permeable peptides are (short) peptides that can carry various cargo molecules across the cell membrane, thereby facilitating the uptake of various molecular cargoes (from nano-sized particles to small chemical molecules, large fragments of DNA, etc.) into cells. Typically, the association of cargo and peptides occurs via covalent chemical linkage or non-covalent interactions. While cell-permeable peptides vary in size, amino acid sequence, and charge, all CCPs share the common ability to permeate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm and organelles of cells. Currently, theories regarding the intracellular translocation of CPP suggest that there are three main mechanisms for intracellular translocation: direct permeation across the cell membrane, entry mediated by endocytosis, and translocation via the formation of transient structures (Jafari S, Solmaz MD, Khosro A, 2015, Bioimpacts 5(2): 103-111; Madani F, Lindberg S, Langel LI, Futaki S, Graslund A, 2011, J Biophys: 414729).

[0128] Examples of CCPs include penetratin (Derossi, D., et al., J Biol Chem, 1 994. 269(14): p. 1 0444-50); the minimal domain of TAT required for protein transduction (Vives, E., P. Brodin, and B. Lebleu, J Biol Chem, 1997. 272(25): p. 1 6010-7); viral proteins (e.g., VP22 (Elliott, C. and P. O'Hare, Cell, 1 997. 88(2): p. 223-33) and ZEBRA (Rothe, R. et al., J Biol Chem, 2010. 285(26): p. 20224-33)); and toxin-derived substances (e.g., melittin (Dempsey, CE, Biochim Biophys Acta, 1 990. 1031 (2): p. 143-61), mastoparane (Konno, K. et al., Toxicon, 2000. 38(11): p. 1 505-1 5), maurocalcin (Esteve, E. et al., J Biol Chem, 2005. 280(13): p. 12833-9), crotamine (Nascimento, FD et al., J Biol Chem, 2007. 282(29): p. 21 349-60) or buforine (Kobayashi, S. et al., Biochemistry, 2004. 43(49): p. 1 561 0-6)); or synthetic CPP (e.g., polyarginine (R8, R9, R10 and R12) (Futaki, S. et al., J Biol Chem, Examples include the 2001. 276(8): p. 5836-40) or the Transportan (Pooga, M. et al., FASEB J, 1 998. 1 2(1): p. 67-77).

[0129] The CPP may contain one or more detectable labels. Such proteins may be partially or entirely labeled. The EEC may completely or partially encode a detectable label, or may not encode one at all. The cell-permeable peptide may also contain a signal sequence. In this specification, “signal sequence” means the sequence of amino acid residues attached to the amino terminus of the nascent protein during protein translation. The signal sequence may be used to signal the secretion of the cell-permeable polypeptide.

[0130] The CPP encoded by the EEC may form a complex after translation. This complex may consist of a charged protein linked to a cell-permeable polypeptide.

[0131] In certain embodiments, the CPP may have a configuration having a first domain and a second domain. The first domain may contain an overcharged polypeptide. The second domain may contain a protein-binding partner. Here, the "protein-binding partner" is an antibody or a functional fragment thereof, a scaffolding protein, or a peptide. The CPP may further contain an intracellular binding partner to the protein-binding partner. The CPP may be secretible from cells into which the EEC has been introduced. The CPP may also be able to enter cells into which the EEC has been introduced.

[0132] In another embodiment, the CPP is capable of invading a second cell. The second cell may originate from the same site as the first cell or from a different site. Examples of such sites include tissues and organs. Furthermore, the second cell may be proximal or distal to the first cell.

[0133] In certain embodiments, the EEC may also encode a fusion protein. The fusion protein has at least two domains that do not coexist in naturally occurring proteins. The domains may be directly fused or linked via an intervening linker sequence. In certain examples, the fusion protein includes a charged protein linked to a therapeutic protein. A “charged protein” means a protein that is positively, negatively, or overall neutrally charged. The therapeutic protein is preferably covalently bonded to the charged protein in the formation of the fusion protein. The surface charge may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 of the total amino acids or the ratio of surface amino acids. Other examples of fusion proteins include bispecific antibodies, chimeric antigen receptors, and recombinant T cell receptors (TCRs).

[0134] Secretory Protein Human and other eukaryotic cells are compartmentalized by membranes, dividing them into numerous compartments with different functions. Each membrane-bound compartment, or organelle, contains various proteins essential for its function. Cells use "sorting signals," which are amino acid motifs located within proteins, to target proteins to specific organelles.

[0135] One type of sorting signal, also known as a signal sequence, signal peptide, or leader sequence, directs a certain class of proteins to an organelle called the endoplasmic reticulum (ER). Proteins targeted to the ER by this signal sequence can be released into the extracellular region as secreted proteins. Similarly, proteins present in the cell membrane can also be secreted into the extracellular region through proteolytic cleavage of "linkers" that hold proteins in the cell membrane.

[0136] In some embodiments, EEC can be used to mass-produce human gene products.

[0137] In some embodiments, EEC can be used to express plasma membrane proteins.

[0138] In some embodiments, EECs can be used to express cytoplasmic proteins or cytoskeletal proteins.

[0139] In some embodiments, EECs can be used to express intracellular membrane-bound proteins.

[0140] In some embodiments, EEC can be used to express nuclear proteins.

[0141] In some embodiments, EECs can be used to express proteins associated with human diseases.

[0142] In some embodiments, EECs can be used to express proteins with therapeutic functions not currently known.

[0143] In some cases, the EEC encodes one or more proteins that are currently commercially available or under development. By introducing a polynucleic acid encoding a currently commercially available or under development protein into the EEC, it is possible to improve protein expression as disclosed herein.

[0144] EECs can encode one or more proteins by containing a coding sequence for a self-cleaving peptide in their coding sequence, or by containing a ribosome skipping element.

[0145] Proteins encoded by EEC may be used to treat symptoms and diseases in many therapeutic fields, including hematology, cardiology, CNS, toxicology (antitoxin, etc.), dermatology, endocrinology, gastroenterology, medical imaging, musculoskeletal system, oncology, immunology, respiratory medicine, sensory organs, and anti-infective medicine.

[0146] When used for the treatment of the target, EEC may be formulated for administration.

[0147] The preparation of EEC formulations can be carried out by any method known or to be developed in the field of pharmacology. Generally, such preparation methods include the steps of mixing the EEC with excipients and / or one or more other adjuncts, and then, if necessary and / or desirable, dividing, shaping, and / or packaging the formulation into desired single or multi-dose units.

[0148] The relative amounts of EEC, pharmaceutically acceptable excipients, and / or any other adjuncts in the formulations described herein will vary depending on the attributes of the subject, body size, and / or condition, and further, on the route through which the formulation is administered. For example, the formulations may contain the active ingredient in a range of 0.1% to 100%, e.g., 0.5% to 50%, 1% to 30%, 5% to 80%, or at least 80% (w / w).

[0149] EEC formulations, by containing one or more excipients, can: (1) improve stability; (2) improve cell transfection; (3) prolong or delay release (e.g., from depot formulations); (4) alter in vivo distribution (e.g., alteration to target specific tissues or specific cell types); and / or (5) alter in vivo the release profile of the encoded protein. Examples of excipients include lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with EEC (e.g., for transplantation to a target), hyaluronidases, nanoparticle mimetic compounds, and combinations thereof.

[0150] EEC in vitro synthesis

[0151] The mRNA production process may consist of in vitro transcription, removal of a cDNA template, RNA cleanup, mRNA capping, and / or tailing reactions.

[0152] In in vitro transcription, cDNA from a desired construct is produced by methods known in the art. Such arbitrary cDNA may be transcribed using an in vitro transcription (IVT) system. Such IVT makes it possible to obtain in vitro synthesized mRNA of the disclosed EEC. This system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), a ribonuclease (RNAse) inhibitor, and a polymerase. The NTPs may be produced in-house, selected from a supplier's product, or synthesized by methods known in the art. The NTPs are selected from naturally occurring NTPs. The polymerases may be selected from mutant polymerases such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and polymerases capable of introducing modified nucleic acids.

[0153] Transfection of EEC into mammalian cells EECs designed and synthesized as described herein can be transfected into various cell types, and their encoded proteins within their open reading frames can be translated into desired proteins. Transfection may be carried out using any method known in the art, such as electroporation or lipofection. Examples of cell types include any mammalian cells known or to be known in the art. Examples of usable mammalian cells include Jurkat cells, Raji cells, HEK293, primary fibroblasts, primary hematopoietic cells (various leukocytes, etc.), primary renal cells, primary hepatocytes, primary pancreatic cells, and primary nerve cells.

[0154] This disclosure provides an EEC containing an in vitro synthesized RNA with a coding sequence within an open reading frame for translation in mammalian cells. This protein can be selected from a wide range of proteins, including those present in the cytoplasm, those transported to organelles, and those secreted. Such an EEC may consist of a 5'UTR containing one of the sequences CAUACUCA, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 38, or SEQ ID NO: 39, or a 3'UTR containing SEQ ID NOs: 4, 5, 6, or 7. The 5'UTR may also contain a T7 polymerase promoter, a mini-enhancer sequence (CAUACUCA), or a Kozak sequence. Furthermore, the bacteriophage T7 promoter may be selected from a T7 class III promoter in a recombinant sequence (SEQ ID NO: 2).

[0155] Furthermore, this disclosure provides an EEC for in vitro synthetic RNA containing a coding sequence within an open reading frame for translation in mammalian cells, and the EEC may further include a 3'UTR containing sequence numbers 4, 5, 6, or 7 along with any of the stop codons (UAA / UAG / UGA). Furthermore, the in vitro synthetic mRNA may also include a 3'UTR containing a) CCUC and GAGG, or b) GAGG and CCUC. Each of the sequence sets a) CCUC and GAGG, or b) GAGG and CCUC, in the 3'UTR sequence may be interposed by seven or more nucleotides, or more than seven nucleotides. Preferably, the total number of nucleotides in the 3'UTR sequence is 50 or less.

[0156] This disclosure provides an EEC and method for recombinant in vitro synthetic mRNA, the recombinant in vitro synthetic mRNA may comprise a 5'UTR containing one of the mini-enhancer sequence (CAUACUCA), SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 38, and SEQ ID NO: 39, and a 3'UTR containing one of SEQ ID NO: 4, 5, 6, or 7. Furthermore, the recombinant in vitro synthetic mRNA may comprise a 5'UTR containing one of the mini-enhancer sequence (CAUACUCA), SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 38, or SEQ ID NO: 39, and a 3'UTR containing one of SEQ ID NO: 4, 5, 6, or 7. In addition, the recombinant in vitro synthetic mRNA may comprise one of the mini-enhancer sequence (CAUACUCA), SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 38, or SEQ ID NO: 39, and one of SEQ ID NO: 4, 5, 6, or 7. Furthermore, the EEC of this disclosure may comprise a recombinant mRNA having a coding sequence encoding one of the following: green fluorescent protein (GFP), interleukin-2 (IL-2), and human POU5F1 (i.e., OCT3 / 4). The disclosure also provides an EEC in which an in vitro synthesized RNA enhances the expression level of the encoded protein. Furthermore, in one embodiment, the EEC of this disclosure is modified... Nucleoside It does not contain and / or microRNA binding sites. In other embodiments, the EEC of this disclosure is modified Nucleoside It does not contain microRNA binding sites, nor does it contain immune evasion factors.

[0157] As described herein, EEC enhances protein expression. This enhancement may be relative to the innate expression level of the protein, compared to coding sequences that do not include the mini-enhancer sequence at the 5'UTR, compared to coding sequences that do not include the stem-loop sequence at the 3'UTR, compared to coding sequences that do not include the mini-enhancer sequence at the 5'UTR and do not include the stem-loop sequence at the 3'UTR, compared to coding sequences that include modified nucleotides but do not include the EEC disclosed herein, and / or relative to the level to which the protein was previously or conventionally expressed. In some embodiments, improved protein expression may be at least 10% improved, at least 20% improved, at least 30% improved, at least 40% improved, at least 50% improved, at least 60% improved, at least 70% improved, at least 80% improved, at least 90% improved, at least 100% improved, at least 200% improved, or at least 300% improved compared to the relevant control system or control state.

[0158] To illustrate specific embodiments of the Disclosure, exemplary embodiments and examples are provided. Those skilled in the art will understand by reference to this Disclosure that many modifications can be made to the embodiments disclosed herein, and that similar or equivalent results can still be obtained without departing from the spirit and scope of the Disclosure.

[0159] Exemplary Embodiments 1. A recombinant expression construct (EEC) having a 5' untranslated region (UTR) operably ligated to a coding sequence, wherein the 5'UTR has a sequence shown as CAUACUCA between the minimal promoter and the Kozak sequence. 2. The EEC according to Embodiment 1, wherein the minimum promoter is the T7 promoter. 3. The EEC according to Embodiment 2, wherein the T7 promoter has a sequence represented as GGGAGA. 4. The EEC according to any one of Embodiments 1 to 3, wherein the Kozak sequence has a sequence represented as GCCRCCAUG, where R is A or G. 5. The aforementioned 5'UTR is (i) The sequence described in Sequence ID No. 2 operably concatenated to the start codon, or (ii) An EEC according to any one of embodiments 1 to 4, having the sequence described in Sequence ID No. 3 operably concatenated to a start codon. 6. The EEC according to Embodiment 5, wherein the sequence described in Sequence ID No. 2, operably linked to the start codon, has the sequence described in Sequence ID No. 38. 7. The EEC according to Embodiment 5, wherein the sequence described in Sequence ID No. 3, operably linked to the start codon, has the sequence described in Sequence ID No. 39. 8. The EEC according to any one of Embodiments 1 to 7, wherein the 5'UTR has fewer than 30 nucleotides. The EEC according to any one of Embodiments 1 to 8, further comprising 9.3'UTR. 10. The EEC according to Embodiment 9, wherein the 3'UTR includes a spacer and stem-loop structure operably connected to a stop codon. 11. The EEC according to Embodiment 10, wherein the stop codon has the sequence UAA, UGA, or UAG. 12. The EEC according to embodiment 10 or 11, wherein the spacer has the arrangement [N1-3]AUA or [N1-3]AAA. 13. The EEC according to embodiment 10 or 11, wherein the spacer has the array UGCAUA or UGCAAA. 14. The EEC according to any one of embodiments 10 to 13, wherein the stem-loop structure has hybridization sequences represented as CCUC and GAGG. 15. The EEC according to any one of embodiments 10 to 13, wherein the stem-loop structure has a hybridization sequence represented as AAACCUC and GAGG, or as AAAGAGG and CCUC. 16. The EEC according to any one of embodiments 10 to 15, wherein the stem-loop structure has a loop segment having at least seven nucleotides. 17. The EEC according to any one of embodiments 10 to 16, wherein the stem-loop structure has a loop segment having 7 to 15 nucleotides. 18. The EEC according to any one of embodiments 10 to 17, wherein the stem-loop structure has loop segments having the sequence indicated as UAACGGUCUU (Sequence ID 34). 19. The EEC according to any one of embodiments 9 to 18, wherein the 3'UTR has fewer than 30 nucleotides. 20. The EEC according to any one of embodiments 9 to 19, wherein the 3'UTR further comprises a polyadenine (poly-A) tail. 21. The EEC according to Embodiment 20, wherein the poly(A) tail has 60 or fewer residues. 22. The EEC according to Embodiment 20 or 21, wherein the poly(A) tail has 40 residues. 23. The EEC according to any one of embodiments 9 to 22, wherein the 3'UTR has the sequence described in sequence number 4, 5, 6, 7, 8, or 9. 24. The EEC according to any one of embodiments 9 to 22, wherein the 3'UTR has the sequence described in sequence number 10, 11, or 12. 25. The EEC according to any one of embodiments 9 to 24, wherein the 3'UTR has the sequence described in sequence number 13, 14, 15, 16, 17, 18, 19, 20, or 21. 26. An EEC according to any one of Embodiments 1 to 25, comprising in vitro synthesized messenger RNA (mRNA). 27. An EEC according to any one of Embodiments 1 to 26, wherein the coding sequence codes for green fluorescent protein (GFP), human interleukin-2 (IL-2), or human POU5F1 (i.e., OCT3 / 4). 28. An EEC according to any one of Embodiments 1 to 27, having the sequence described in Sequence ID No. 56, 58, or 60. 29. The EEC according to any one of Embodiments 1 to 26, wherein the coding sequence codes for a therapeutic protein. 30. The EEC according to Embodiment 29, wherein the therapeutic protein comprises an antibody or a bound fragment thereof. 31. The EEC according to Embodiment 30, wherein the antibody or its conjugated fragment comprises an anti-SARS-CoV-2 antibody or its conjugated fragment, an anti-SARS antibody or its conjugated fragment, an anti-RSV antibody or its conjugated fragment, an anti-HIV antibody or its conjugated fragment, an anti-dengue virus antibody or its conjugated fragment, an anti-borrhea antibody or its conjugated fragment, an anti-hepatitis C antibody or its conjugated fragment, an anti-influenza virus antibody or its conjugated fragment, an anti-metanupneumovirus (MPV) antibody or its conjugated fragment, an anti-cytomegalovirus antibody or its conjugated fragment, an anti-Epstein-Barr virus antibody, an anti-herpes simplex virus antibody or its conjugated fragment, an anti-Clostridium difficile toxin antibody or its conjugated fragment, or a tumor necrosis factor (TNF) antibody or its conjugated fragment. 32. The EEC according to any one of Embodiments 1 to 26, wherein the coding sequence codes for a vaccine antigen. 33. The EEC according to Embodiment 32, wherein the vaccine antigen includes SARS-CoV-02 vaccine antigen, CMV vaccine antigen, EBV vaccine antigen, hepatitis vaccine antigen, herpes simplex virus vaccine antigen, human immunodeficiency virus (HIV) vaccine antigen, human papillomavirus (HPV) virus antigen, influenza vaccine antigen, Japanese encephalitis vaccine antigen, malaria vaccine antigen, measles vaccine antigen, rabies vaccine antigen, RS vaccine antigen, rotavirus vaccine antigen, varicella-zoster vaccine antigen, or Zika fever vaccine antigen. 34. The EEC according to any one of Embodiments 1 to 26, wherein the coding sequence codes for a cytokine. 35. The EEC according to any one of Embodiments 1 to 26, wherein the coding sequence encodes a cell-permeable protein. 36. The EEC according to Embodiment 35, wherein the cell-permeable protein comprises penetratin, the minimal domain of TAT, VP22, ZEBRA, melittin, mastoparan, maurocalcin, crotamine, buforin, polyarginine, or transportan. 37. Qualification Nucleoside An EEC according to any one of embodiments 1 to 36, which does not include the EEC. 38. An EEC according to any one of Embodiments 1 to 37, which does not contain a microRNA binding site. 39. An EEC according to any one of embodiments 1 to 38, wherein the coding sequence encodes an immune evasion factor. 40. The EEC according to Embodiment 40, wherein the immune evasion factor comprises B18R, E3, K3, NS1, or ORF8. 41. An EEC according to any one of Embodiments 1 to 38, which does not contain an immune evasion factor. 42. An EEC according to any one of Embodiments 1 to 41, formulated for administration to a subject. 43. A recombinant expression construct (EEC) having a coding sequence operably concatenated to a 5' untranslated region (UTR) containing the sequence described in SEQ ID NO: 38 and a 3' UTR containing the sequence described in SEQ ID NO: 13, 14, or 15. 44. An enhancer sequence containing the sequence indicated as CAUACUCA. 45. A recombinant expression (EEC) construct having 1, 2, 3, 4, or 5 copies of the sequence represented as CAUACUCA. 46. ​​The EEC according to embodiment 44, wherein the enhancer array is operably connected to a promoter. 47. The EEC according to embodiment 46, wherein the promoter is the minimum promoter. 48. Recombinant expression construct comprising an in vitro synthetic RNA containing a coding sequence within an open reading frame encoding a protein translated in mammalian cells, wherein the in vitro synthetic RNA further comprises one of a 5' untranslated region containing CAUACUCA and a 3' untranslated region containing any of SEQ ID NOs: 4, 5, 6, or 7. 49. Recombinant expression construct comprising an in vitro synthetic RNA containing a coding sequence within an open reading frame encoding a protein translated in mammalian cells, wherein the in vitro synthetic RNA further comprises any of the following: a 5' untranslated region containing SEQ ID NO: 2 or SEQ ID NO: 3 and a 3' untranslated region containing SEQ ID NO: 4, 5, 6, or 7. 50. Recombinant expression construct comprising in vitro synthetic RNA comprising an open reading frame encoding a protein translated in mammalian cells, wherein the in vitro synthetic RNA further comprises a 5' untranslated region comprising a T7 polymerase promoter, a sequence indicated as CAUACUCA, and a Kozak sequence. 51. A recombinant expression construct according to any one of Embodiments 1 to 42, wherein the T7 promoter is selected from T7 class III promoters. 52. A recombinant expression construct comprising an in vitro synthetic RNA comprising an open reading frame encoding a protein translated in mammalian cells, wherein the in vitro synthetic RNA comprises a 3' untranslated region including SEQ ID NOs. 4, 5, 6, or 7 and a stop codon. 53. The recombinant expression construct according to Embodiment 52, wherein the stop codon is UAA, UAG, or UGA. 54. A recombinant expression construct comprising in vitro synthetic RNA containing an open reading frame encoding a protein translated in mammalian cells, wherein the in vitro synthetic RNA comprises a 3' untranslated region containing either a) CCUC and GAGG, or b) GAGG and CCUC, and the sequence of the 3' untranslated region is divided into seven or more nucleotides between each sequence. 55. A recombinant expression construct (EEC) comprising in vitro synthetic RNA comprising an open reading frame encoding a protein to be translated in mammalian cells, wherein the in vitro synthetic RNA comprises a 3' untranslated region comprising either a) AAACCUC and GAGG, or b) AAAGAGG and CCUC, and the sequence of the 3' untranslated region is divided into seven or more nucleotides between each sequence. 56. The 5' untranslated region (UTR) containing the sequence indicated as CAUACUCA, located between the minimal promoter and the Kozak sequence. 57. The 5'UTR according to embodiment 56, wherein the minimum promoter is the T7 promoter. 58. The 5'UTR according to Embodiment 57, wherein the T7 promoter has a sequence shown as GGGAGA. 59. The 5'UTR according to any one of embodiments 56 to 58, wherein the Kozak sequence has a sequence represented as GCCRCCAUG (wherein R is adenosine or guanine). 60. The 5'UTR according to any one of embodiments 56 to 59, comprising the sequence described in Sequence ID No. 2 operably concatenated to the start codon. 61. A 5'UTR according to any one of embodiments 56 to 60, comprising the sequence described in Sequence ID No. 3 operably concatenated to a start codon. 62. The 5'UTR according to Embodiment 60, wherein the sequence described in Sequence ID No. 2, operably linked to the start codon, has the sequence described in Sequence ID No. 38. 63. The 5'UTR according to Embodiment 61, wherein the sequence described in Sequence ID No. 3, operably linked to the start codon, has the sequence described in Sequence ID No. 39. 64. The 5'UTR according to any one of embodiments 56 to 63, wherein the 5'UTR has fewer than 30 nucleotides. 65. A 3'UTR comprising a spacer and stem-loop structure operably connected to a stop codon, wherein the stop codon has the sequence UAA, UGA, or UAG, and the spacer has the sequence [N1-3]AUA or [N1-3]AAA. 66. The 3'UTR according to embodiment 65, wherein the spacer has the sequence UGCAUA or UGCAAA. 67. The 3'UTR according to embodiment 65 or 66, wherein the stem-loop structure includes hybridization sequences represented as CCUC and GAGG. 68. The 3'UTR according to any one of embodiments 65 to 67, wherein the stem-loop structure includes a hybridization sequence represented as AAACCUC and GAGG, or as AAAGAGG and CCUC. 69. The 3'UTR according to any one of embodiments 65 to 68, wherein the stem-loop structure has a loop segment having at least 7 nucleotides. 70. The 3'UTR according to any one of embodiments 65 to 69, wherein the stem-loop structure has a loop segment having 7 to 15 nucleotides. 71. The 3'UTR according to any one of embodiments 65 to 70, wherein the stem-loop structure has a loop segment having the sequence indicated as UAACGGUCUU (Sequence ID 34). 72. The 3'UTR according to any one of embodiments 65 to 71, wherein the number of nucleotides is less than 30. 73. A 3'UTR according to any one of embodiments 65 to 72, further comprising a polyadenine (poly-A) tail. 74. The 3'UTR according to embodiment 73, wherein the poly(A) tail has 60 or fewer residues. 75. The 3'UTR according to embodiment 73 or 74, wherein the poly(A) tail has 40 residues. 76. A 3'UTR according to any one of embodiments 65 to 75, having the sequence described in sequence numbers 4, 5, 6, 7, 8, or 9. 77. A 3'UTR according to any one of embodiments 65 to 75, having the sequence described in sequence number 10, 11, or 12. 78. A 3'UTR according to any one of embodiments 65 to 77, having the sequence described in sequence numbers 13, 14, 15, 16, 17, 18, 19, 20, or 21. 79. A 3'UTR according to any one of embodiments 65 to 78, which is operably linked to a code sequence. 80. The 3'UTR according to Embodiment 79, wherein the coding sequence codes for a therapeutic protein, a vaccine antigen, a cytokine, or a fluorescent protein. 81. The 3'UTR according to embodiment 79 or 80, wherein the coding sequence encodes an immune evasion factor. 82. The 3'UTR according to Embodiment 81, wherein the immune evasion factor comprises B18R, E3, K3, NS1, or ORF8. 83. The 3'UTR according to any one of embodiments 79 to 80, wherein the coding sequence does not contain an immune evasion factor. 84. The 3'UTR according to any one of embodiments 79 to 83, wherein the coding sequence encodes a cell-permeable protein. 85. The 3'UTR according to Embodiment 84, wherein the cell-permeable protein comprises penetratin, the minimal domain of TAT, VP22, ZEBRA, melittin, mastoparan, maurocalcin, crotamine, buforin, polyarginine, or transportan. 86. 5'UTR and / or 3'UTR sequences disclosed herein. 87. 5'UTR and / or 3'UTR sequences as disclosed herein, operably concatenated to a code sequence.

[0160] Examples from experiments Example 1: Materials and Method UTR design and structural prediction The minimum transcriptional and translational elements, each 4 to 10 nucleotides in length (e.g., the 5’UTR enhancer, T7 hexamer, and Kozak sequence of the present application disclosed herein), are assembled to construct the UTRs of the present disclosure. Based on the characteristics of the above stem-loop, a synthetic 3’ sequence for testing was assembled. The RNA secondary structure prediction web server (rna.urmc.rochester.edu / RNAstructureWeb / ) was used with default parameters to verify the possibility of stem-loop secondary structures formed from various UTR sequences.

[0161]

Table 3

[0162] mRNA synthesis Polymerase chain reaction (PCR) was performed using DNA fragments obtained from Integrated DNA Technologies Inc. (IDT) to construct the T7 promoter, UTR, and polyadenosine (40 adenosines) sequences with the oligonucleotides described in Table 1. Using these templates, a transcription reaction was carried out with T7 RNA polymerase and an anti-reverse cap analog (ARCA) to synthesize mRNA (HiScribe TM T7 ARCA mRNA Kit, NEB). After treatment with DNase I, the mRNA was quantified and stored appropriately.

[0163] Cell culture HEK293 (ATCC® CRL-1573 TM ) cells, Jurkat cells, clone E6-1 (ATCC TIB-152 TM ) cells, and Raji (ATCC CCL-86 TM ) cells were obtained from the American Type Culture Collection (ATCC). Each cell was maintained at 37°C under 5% CO2. The medium for HEK293 was Eagle's Minimum Essential Medium (EMEM) (ATCC 30-2003) supplemented with 10% fetal bovine serum (FBS)TM ) contains. Jurkat cells and Raji cells are cultured in RPMI-1640 medium supplemented with 10% FBS (ATCC 30-2001 TM ) was maintained using Expi293. TM The expression system kit (ThermoFisher) was used according to the manufacturer's instructions.

[0164] Transfection and electroporation To determine optimal transfection parameters, cells were transfected with incremental EECs containing full-length UTR. As a control, HEK293 cells were transfected with 0.4 to 1 pmol of mRNA supplemented with MessengerMax lipofectamine (Thermo Fisher Scientific). Jurkat and Raji cells were transfected with 0 to 16 pmol of GFP-coded EECs supplemented with jetMessenger (PolyPlus). For electroporation, a Neon Transfection System (Thermo Fisher Scientific) was used according to the manufacturer's manual. Optimal electroporation parameters (voltage, time, and pulse count) were determined by referring to databases.

[0165] Flow cytometry Cells were fixed by treating them with 4% paraformaldehyde for 30 to 60 minutes and stored in phosphate-buffered saline (PBS). For hOCT3 / 4 staining, cells were permeabilized, cultured with antibody (BioLegend), washed twice with PBS, and stored in PBS until use. Cell analysis was performed using a FACSCalibur flow cytometer (BD) and a CytoFlex flow cytometer (Beckman Coulter), and analyzed with FlowJo software.

[0166] ELISA 24 hours after transfection, the cell culture medium was collected, centrifuged, and diluted. Expression levels were quantified using a human IL2 ELISA (BioLegend) according to the manufacturer's protocol. Briefly, plates (Costar) were coated with capture antibody, and then cultured with the diluted cell culture medium, detection antibody, and avidin HRP. Absorbance (450 nm) was measured and analyzed.

[0167] Example 2: Improvement of protein expression by EEC containing the 5'UTR sequence of the present application as disclosed herein, compared to the absence of the 5'UTR sequence. To test the protein expression-enhancing ability of EEC sequences, EXPI293 suspension cells (Thermo Fisher Scientific) were transfected with EECs containing the modified 5'UTR and 3'UTR sequences described above, using GFP as the reporter protein. EXPI293 suspension cells derived from the HEK293 cell line were used first, as they are designed for high protein expression. To determine the appropriate amount of mRNA to use in the experiment, EXPI293 cells were transfected with gradually increasing amounts of GFP-coded EECs ranging from 0 to 2 pmol (0 to 500 ng) using the EXPIfectamine transfection reagent. After 24 hours, flow cytometry of the cells was performed as described above. In this experiment, the GFP fluorescence signal is assumed to be proportional to the amount of protein in the cell. As shown in Figure 2, according to the flow cytometry data, the GFP median intensity was 6.0 × 10⁶ cells. 5 Saturation occurs when the amount of mRNA per individual reaches 0.4 pmol (100 ng).

[0168] Next, cells were transfected with equimolar mRNA containing various 5'UTRs, and GFP expression was analyzed at 3 and 24 hours after transfection. The various 5'UTR sequences included those with a Kozak sequence (M3) (M1)(T7 promoter); (M1, M2 ((CAUACUCA), and M3)); or those without other 5'UTRs beyond the 5' methyl cap and T7 hexamer. Each construct contained a 5' methyl cap and a T7 hexamer. Figures 3A and 3B show the results of this experiment. Here, Figures 3A and 3B are flow cytometry graphs showing GFP intensity (FL1-H) on the x-axis and cell number on the y-axis at 3 hours after transfection (Figure 3A) and 24 hours after transfection (Figure 3B) for GFP-coded EEC 5'UTR variants, and The bar graphs illustrating this data are shown. As predicted, the median intensity of the GFP signal shown in cells transfected with a UTR-free transcript (a construct containing only the T7 hexamer GGGAGA) was the lowest at all post-transfection time points (Figures 3A, 3B). Adding a Kozak consensus sequence with A at the R position resulted in a 200% increase in protein expression, and further addition of the present translational enhancer (CAUACUCA) resulted in GFP expression at 3 hours and 24 hours, respectively, compared to the GFP signal detected in cells not treated with the 5'UTR transcript, resulting in 635% and 240% increases.

[0169] Therefore, this experiment demonstrated that the recombinant 5'UTR of the present invention, in particular the 5'UTR having the translational enhancer of the present invention, is important for significantly improving protein expression.

[0170] Example 3: Improvement of protein expression by including the 3'UTR sequence of the present invention in EECs having various 5'UTR sequences. Next, we investigated the effect of adding the 3'UTR of this invention on GFP expression. The results of this experiment are shown in Figures 4A and 4B. Figures 4A and 4B show flow cytometry graphs with GFP intensity (FL1-H) on the x-axis and cell number on the y-axis at 3 hours after transfection (Figure 4A) and 24 hours after transfection (Figure 4B) with 5'UTR and 3'UTR mutants of the GFP-encoding EEC, and bar graphs showing the data. EXPI293 cells were transfected with equimolar amounts of RNA-free (negative control), mRNA with only 3'UTR (no 5'UTR), M3 only with 5'UTR and additional 3'UTR, and 5'UTR and 3'UTR mutants of the GFP-encoding EEC such as M1, M2, and M3 with 5'UTR and additional 3'UTR. As shown in Figures 4A and 4B, cells transfected with transcripts containing only 3'UTR showed low GFP expression levels at all measurement time points. Adding the Kozak consensus sequence along with the 3'UTR slightly improved GFP median intensity. However, adding the full-length 5'UTR (SEQ ID NO: 2) along with the 3'UTR (SEQ ID NO: 10) increased GFP expression by 660% and 925% at 3 and 24 hours, respectively. Compared to cells treated with transcripts containing only the full-length 5'UTR (SEQ ID NO: 2), adding the 3'UTR (SEQ ID NO: 10) further improved the GFP signal by 137%. In particular, cells treated with mRNA containing both the full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) showed the highest GFP intensity.

[0171] Therefore, this experiment demonstrated that including the 3' stem loop of the present invention in mRNA is important for further improving protein expression levels when desired.

[0172] Example 4: Improvement of protein expression in various protein types using EEC having the 5'UTR sequence and 3'UTR sequence of the present invention. To confirm the usefulness of this recombinant UTR in enhancing the expression of various proteins, we demonstrated its superiority in coding multiple proteins with different properties. Figure 5 illustrates the targeted expression of three different types of proteins tested on the EEC disclosed herein, namely, cytoplasm (GFP), organelles (nuclear compartment, in this example human POU5F1 or OCT3 / 4), and extracellular compartment (secreted protein, in this example IL2). For experiments on the effects of the 5'UTR and 3'UTR sequences of this application on cytoplasmic protein (GFP) expression, please refer to Examples 2 and 3 and Figures 3A to 4B.

[0173] The effect of the UTR of the present invention on the expression of organelle-bound proteins was investigated using human POU5F1 or OCT3 / 4 (hOCT4 in this example), which are major nuclear transcription factors in stem cell reprogramming (Yu et al., Induced pluripotent stem cell lines derived from human somatic cells. Science (80). (2007), doi:10.1126 / science.1151526). Similar to GFP, treatment of HEK293 with gradually increasing doses (0-4.8 pmol) of hOCT4-coding mRNA resulted in increased intracellular protein levels after 24 hours (Figures 11A and 11C). The percentage of hOCT4-positive cells was 2.0 × 10⁶. 5 The maximum (75%) was reached when the mRNA concentration in each cell was 1.2 pmol. When HEK293 cells were transfected with equal amounts of hOCT4 transcripts containing 5' and 3' mutants, only those with full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) showed a significant percentage of hOCT4-positive cells (40%). Furthermore, median (experimental count = 2) hOCT4 intensity was four-fold higher in cells treated with hOCT4 mRNA containing full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) (Figures 11A and 11B).

[0174] Finally, we investigated the effect of the UTR of the present invention on the expression of the secreted protein human interleukin-2 (hIL2). hIL2 activates T lymphocytes and is currently a target in the treatment of autoimmune diseases and cancer (Spolski, Li, & Leonard, Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat. Rev. Immunol. (2018), doi:10.1038 / s41577-018-0046-y). Similar to the above experiment, HEK293 was transfected with gradually increasing amounts of hIL2-coding mRNA (including full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10)). ELISA 24 hours after transfection showed a proportional increase in hIL2 protein levels (Figure 11A). Furthermore, when UTR mutants were transfected with hIL2 mRNA, the highest expression levels of the hIL2 protein were observed when both the full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) were present (Figure 11B).

[0175] Therefore, this experiment demonstrated that mRNA possessing both recombinant UTRs of the present invention is useful in improving protein expression using various protein types.

[0176] Example 5: Improvement of protein expression in various protein types by EEC having the 5'UTR sequence and 3'UTR sequence of the present invention.

[0177] To verify whether the improved protein expression could be replicated in other cell types, the above experiment was repeated using the following cell lines: HEK293 (ATCC CRL1573™) lymphocytes, Jurkat (clone E6-1; ATCC TIB-152) lymphocytes, and Raji (ATCC CCL-86) lymphocytes. Adherent HEK293 cells were derived from human embryonic kidney cells transformed with sheared fragments of adenovirus type 5 DNA (Graham et al., Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. (1977), doi:10.1099 / 0022-1317-36-1-59). Seeded HEK293 cells were treated with escalating doses of a new lot of GFP-coded EEC containing 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10). In this experiment, mRNA transfection was performed using MessengerMax Lipofectamine reagent (ThermoFisher) as described in Example 1. As shown in Figures 6A, 6B, and 6C, approximately 90% of the cells showed a GFP signal that saturated at 1 pmol (250 ng) of GFP-coding EEC. Increasing the amount of mRNA used to treat the cells only slightly increased the percentage of GFP-positive cells. The median GFP intensity, which indicates protein expression level, reached saturation at 2 pmol (500 ng).

[0178] Next, HEK293 cells were treated with various EECs ranging from 0.4 to 1 pmol, including the 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) disclosed herein (Figures 7A and 7B). All mRAN mutants produced GFP expression in 60% to 80% of cells, but only cells treated with full-length UTRs showed a 5-fold higher GFP median intensity compared to the others (Figure 7C). Median intensity was calculated by performing the experiment twice.

[0179] Next, the above experiment was performed using the lymphocyte lines Jurkat cells and Raji cells. Jurkat cells are T lymphocytes established from the peripheral blood of a 14-year-old boy with acute T-cell leukemia (Schneider, Schwenk, & Bornkamm, Characterization of EBV-genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int. J. Cancer (1977), doi:10.1002 / ijc.2910190505). Raji cells are B lymphocytes established from an 11-year-old male patient with Burkitt lymphoma (Osunkoya, The preservation of burkitt tumor cells at moderately low temperature. Br. J. Cancer (1965), doi:10.1038 / bjc.1965.87; Pulvertaft, A Study of Malignant Tumors In Nigeria by Short-Term Tissue Culture. J. Clin. Pathol. (1965), doi:10.1136 / jcp.18.3.261). In this experiment, mRNA transfection was performed using the jetMessenger reagent (Polyplus-transfection®). Unlike HEK293 cells (i.e., GFP-positive cells), which had a transfection efficiency of 90%, only 10% of Jurkat cells showed a GFP signal when the mRNA level was at its maximum (16 pmol) (Figure 7A). Furthermore, while cells treated with equimolar amounts of various constructs showed some GFP signaling, those transfected with transcripts containing full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) showed the highest number of GFP-positive cells (14%, Figure 7B). Similar results were observed in Raji cells (data not shown).

[0180] Therefore, this experiment demonstrated that the recombinant UTR of the present invention is useful in improving protein expression in various cell types.

[0181] Example 6: In the protein expression-enhancing effect of EEC disclosed herein, the transfection method is of low importance. To verify whether the transfection method is important in the expression enhancement effect shown by the EEC disclosed herein, Jurkat cells were electroporated with an increasing dose of EEC containing a coding sequence encoding GFP.

[0182] Electroporation has been shown to improve nucleic acid delivery to lymphocyte lines (Ohtani et al., Electroporation: Application to human lymphoid cell lines for stable introduction of a transactivator gene of human T-cell leukemia virus type I. Nucleic Acids Res. (1989), doi:10.1093 / nar / 17.4.1589). First, to determine the optimal amount of mRNA to use in this experiment, Jurkat cells were electroporated with gradually increasing amounts of GFP-coding EEC using a neon electroporation system (Thermo Fisher Scientific) as shown in Example 1. As a result, the GFP signal increased proportionally. Next, Jurkat cells were electroporated using 4 pmol and 8 pmol of GFP-coding EEC containing the UTR mutant. 6 to 8 × 10 5At 4 pmol (1 μg) of GFP-coded EEC per cell, up to 10% of cells showed GFP positivity, with cells treated with mRNA containing full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) showing the highest percentage of GFP-positive cells (Figure 9A). At 8 pmol (2 μg), approximately 90% of Jurkat cells were GFP-positive, with cells treated with mRNA containing full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) showing the highest median GFP intensity (Figures 9B and 9C). Similar results were obtained with Raji cells, except that the number of GFP-positive cells decreased at the maximum amount of mRNA used (Figures 10A, 10B, and 10C).

[0183] In Example 7, which used the genetically modified EEC of this application, the same amount of protein was observed as in Examples 2, 3, and 4. Therefore, the transfection method does not affect the effect of this EEC.

[0184] Example 7: Reversing the stem-loop sequence of the 3'UTR had no effect on improving protein expression levels. To investigate the effect of 3'UTR sequence configuration on protein expression while maintaining stem-loop pairing, the original 3'UTR sequence (Figure 13A, 3'UTR-A) was edited, replacing CCUC with GAGG (Figure 13A, 3'UTR-B). GFP-coding EECs were constructed to contain either 3'UTR-A or 3'UTR-B, and GFP expression in HEK293 cells was tested (transfected with MessengerMax lipofectamine). Using 1 to 2 pmol, 60 to 70% of cells showed GFP expression (2 to 3 experiments), and no difference was observed between constructs containing either 3'UTR (Figure 13B). GFP median intensity (2 to 3 experiments) was also similar between EECs containing either 3'UTR (Figure 13C). Furthermore, GFP expression was tested using EECs with other 3'UTRs in which a single nucleotide was substituted (from U to A) at the -2 position ahead of GGAG in 3'UTR-B (Figure 13A, 3'UTR-C). This sequence is similar to a histone stem-loop in which the adenosine chain, crucial for stem-loop binding proteins (SLBPs) and translation-related mRNAs, precedes the stem region (Battle & Doudna, The stem-loop binding protein forms a highly stable and specific complex with the 3′ stem-loop of histone mRNAs. RNA (2001), doi:10.1017 / S1355838201001820; William & Marzluff, The sequence of the stem and flanking sequences at the 3′ end of histone mRNA are critical determinants for the binding of the stem-loop binding protein. Nucleic Acids Res. (1995), doi:10.1093 / nar / 23.4.654).Adding 3'UTR-C did not increase the percentage of GFP-positive cells as much as adding 3'UTR, but it did increase the GFP median intensity in transfected cells by 60% (Figures 13B and 13C).

[0185] Therefore, as observed in the above examples, the recombinant 3'UTR containing the stem loop having the flanking sequence of the present invention enhances GFP expression in human cells.

[0186] Example 8: In transfection of fibroblasts, recombinant mRNA containing the 5'UTR sequence of the present invention improves protein expression compared to mRNA using modified nucleotides.

[0187] To compare the amount of protein produced from the disclosed recombinant mRNA with the amount produced by mRNA (N1-methylpseudolidine) using modified nucleotides, several Oct4-expressing mRNA constructs, including unmodified mRNA Oct4 (UO), unmodified mRNA MyoD-Oct4 (UMD), modified mRNA Oct4 (PUO), and modified mRNA MyoD-Oct4 (PUMD), were transfected into human foreskin fibroblasts. As shown in Figure 14, OCT4 expression was highest when using unmodified mRNA (UO). In the unmodified recombinant transcript using 800 ng of mRNA, the percentage of OCT4-positive cells was highest at 50.7%. Also, as shown in Figure 14, compared to the recombinant mRNA disclosed herein, modified mRNA showed the highest percentage of OCT4-positive cells. Nucleoside When pseudouridine was used (PUO and PUMD), the percentage of OCT4-positive cells was significantly lower (36.9% compared to 50.7%). These results demonstrate that the 5'UTR and 3'UTR disclosed herein yield higher protein expression compared to transcripts obtained with modified nucleotides such as N1-methylpseudridine.

[0188] Please refer to Figure 15 for the sequences used in each experimental example.

[0189] Conclusion Each embodiment disclosed herein may be substantially composed of, or constitutes essentially of, certain elements, processes, materials, or components described herein, as will be apparent to those skilled in the art. Therefore, the terms “include” or “including” should be interpreted as “comprising, consist essentially of, or consist of.” In this application, the terms “comprise” or “compose” in the transitional section of a claim mean that it may, and may or may not, include, elements, processes, materials, or components not specified in the claim. The expression “consisting of” in the transitional section means that it does not include elements, processes, materials, or components not specified in the claim. The expression “consisting essentially of” in the transitional section means that the scope of the embodiment is limited to the elements, processes, materials, or components specified in the claim and those that do not significantly affect the embodiment. In this specification, "significant effect" means an effect that may result in a statistically significant reduction in the improvement of protein expression observed by using an EEC containing SEQ ID NO: 2 at the 5'UTR and SEQ ID NO: 10 at the 3'UTR.

[0190] Unless otherwise stated, all numerical values ​​used in this specification and the claims should be interpreted in all cases as being modified by the term "approximately." Therefore, unless otherwise stated, the numerical parameters described in this specification and the attached claims are approximations that may vary depending on the desired properties to be achieved by the present invention. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims of this application, the interpretation of each numerical parameter should be made at a minimum in consideration of the stated number of significant figures and by applying ordinary rounding. Where further clarity is required, the term "approximately" should have the meaning reasonably understood by a person skilled in the art when used with the stated numerical values ​​or numerical ranges. In other words, the term "approximately" indicates a value that is somewhat larger or smaller than the stated value or range, and is within the range of ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the stated value.

[0191] While the numerical ranges and parameters defining the broad scope of this invention are approximations, the numerical values ​​specified in the specific embodiments are described as accurately as possible. However, any numerical value inevitably contains a certain degree of error as a result of the standard deviation in the experimental measurements.

[0192] Furthermore, the variants of the protein and EEC (including the 5'UTR and 3'UTR) disclosed in this specification have sequences that have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the reference sequence.

[0193] "Percent sequence identity" refers to the relationship between two or more sequences determined by comparing them. In this technology, "identity" refers to the degree of sequence relationship between proteins, nucleic acids, or gene sequences, determined by how closely their sequence arrangements match. "Identity" (often also referred to as "similarity") can be easily calculated using known methods, such as those described in literature like Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). The preferred method for determining identity is one designed to best demonstrate the agreement between the sequences being tested. Methods for determining identity and similarity are coded as commercially available computer programs. Sequence alignment and the calculation of identity percentages may also be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin).Furthermore, multiple alignment of sequences can also be performed using the clustering method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989)) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Examples of related programs include the GCG program set (GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin)), BLASTP, BLASTN, and BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990)), DNASTAR (DNASTAR, Inc., Madison, Wisconsin), and the FASTA program including the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), meeting date 1992). 111-20. Editor: Suhai, Sandor. Publisher: Plenum, New York, NY) are some examples. In the contents of this disclosure, if sequence analysis software is used for the analysis, it will be understood that the results of the analysis are based on the “default values” of the referenced program. In this specification, “default values” means any original values ​​or parameters that the software loads when it is first initialized.

[0194] In the descriptions of this invention (particularly in the claims below), “a,” “an,” “the,” and similar reference terms are to be interpreted as including both singular and plural references, unless otherwise stated herein or unless clearly inconsistent with the context. The ranges of values ​​described herein are merely a convenient way of referring to each value within that range individually. Unless otherwise stated herein, each individual value is included in this specification as being described individually. All methods described herein can be performed in any preferred order, unless otherwise stated herein or unless clearly inconsistent with the context. All examples and illustrative expressions (e.g., “etc.”) described herein are used merely to illustrate the invention in a more understandable way and do not limit the scope of the invention as otherwise claimed in these claims. The language in this specification should not be interpreted as indicating any element that is not described in these claims but is essential for carrying out the invention.

[0195] The interchangeable elements and combinations of embodiments of the Invention disclosed herein are not to be construed as limitations. Each element in a combination may be referenced or claimed individually, or may be referenced or claimed in combination with other elements in that combination or with other elements described herein. It will be understood that one or more elements in one combination may be included or omitted in another combination for the sake of clarity and / or for patentability. Even if implemented with any inclusion or omission, this specification is deemed to include the combination in which it was modified and thus satisfies the description requirements for all of the Markushes used in the appended claims.

[0196] Several embodiments of the present invention, including those which, to the best knowledge of the inventors, represent the best mode of implementation of the invention, are described herein. Naturally, any modifications to these embodiments will be apparent to those skilled in the art by reading the above description. The inventors anticipate that those skilled in the art will make such modifications as appropriate, and they also intend that the invention may be implemented in ways different from those specifically described herein. That is, the invention also includes any modifications and equivalents to the claims appended herein, as permitted by applicable law. Furthermore, all possible variations of the combinations of the above elements are included in the scope of the invention unless otherwise specified herein or unless they are clearly inconsistent with the context.

[0197] Furthermore, this specification references numerous publications, patents, and / or patent applications (collectively referred to as “References”). Each Reference is incorporated herein by reference, in particular, with respect to its cited teachings.

[0198] Finally, the embodiments of the present invention disclosed herein should be understood as illustrative examples of the principles of the present invention. Other possible modifications are also within the scope of the invention. Thus, alternative configurations of the present invention may be used in accordance with the teachings herein, as non-limiting examples. In other words, the present invention is not limited to those shown and described herein.

[0199] The specific configurations described herein are for illustrative purposes only and illustrate preferred embodiments of the present invention, and are presented only to provide what is considered most useful and easy to understand in explaining the principles and conceptual aspects of various embodiments of the present invention. In this regard, since it will be obvious to those skilled in the art how some forms of the present invention are carried out by reading the above description with reference to the drawings and / or examples, the details of the configuration of the present invention are not described in more detail than is necessary for a basic understanding of the present invention. The definitions and explanations used in this disclosure are intended to prescribe future interpretations, unless explicitly and unambiguously modified in a particular instance, or if the application of such meaning would render the interpretation meaningless or substantially meaningless. Where interpreting a term would render it meaningless or substantially meaningless, the definitions found in Webster's Dictionary, Third Edition, or in dictionaries known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004), shall be adopted.

Claims

1. In vitro synthetic RNA, (i) A 5' untranslated region (UTR) having the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 38; (ii) 3'UTR having the sequence shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21; and (iii) containing an open reading frame that codes for a protein, It is characterized by not containing modified nucleosides, In vitro synthesized RNA, wherein the open reading frame is located between the 5'UTR and the 3'UTR.

2. The in vitro synthetic RNA according to claim 1, characterized by any one of the following (a) to (d): (a) The 5'UTR has the sequence shown in Sequence ID 2, and the 3'UTR has the sequence shown in Sequence ID 10; (b) The 5'UTR has the sequence shown in Sequence ID 38, and the 3'UTR has the sequence shown in Sequence ID 10; (c) The 5'UTR has the sequence shown in Sequence ID 2, and the 3'UTR has the sequence shown in Sequence ID 13; (d) The 5'UTR has the sequence shown in sequence number 38, and the 3'UTR has the sequence shown in sequence number 13.

3. The in vitro synthetic RNA according to claim 1, formulated for administration to a target.

4. The in vitro synthetic RNA according to claim 1, wherein the protein comprises a vaccine antigen, green fluorescent protein (GFP), human interleukin-2 (IL2), or human POU5F1 (OCT3 / 4).

5. A 5'UTR having the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 38, and A recombinant expression construct (EEC) comprising a 3'UTR having the sequence shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO:

21.

6. The EEC according to claim 5, comprising any one of the following (a) to (d): (a) A 5'UTR having the sequence shown in SEQ ID NO: 2 and a 3'UTR having the sequence shown in SEQ ID NO: 10; (b) A 5'UTR having the sequence shown in SEQ ID NO: 38 and a 3'UTR having the sequence shown in SEQ ID NO: 10; (c) A 5'UTR having the sequence shown in SEQ ID NO: 2 and a 3'UTR having the sequence shown in SEQ ID NO: 13; (d) A 5'UTR having the sequence shown in SEQ ID NO: 38 and a 3'UTR having the sequence shown in SEQ ID NO: 13.

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