Recombinant expression constructs for improving 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 to U.S. Provisional Patent Application No. 63 / 153,877, filed February 25, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference The sequence listing accompanying this application is provided in text format in lieu of paper and is hereby incorporated by reference. The filename of the text file containing this sequence listing is P183-0010PCT_ST25.txt. The text file is 26.9 KB, was created on Feb. 24, 2022, and was submitted electronically via EFS-Web.

[0003] The present disclosure provides recombinant expression constructs having artificial 5' and / or 3' untranslated regions (UTRs) adjacent to a 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 are configured without modified nucleotides, microRNA sites, or immune evasion factors. [Background technology]

[0004] There are several problems with the existing methods for influencing protein expression. For example, although the introduction of foreign deoxyribonucleic acid (DNA) into the genomic DNA of a host cell can occur at some frequency, this introduction can result in mutations and / or damage to the genomic DNA of the host cell. Alternatively, such foreign DNA introduced into a cell can be inherited by daughter and descendant cells (whether the foreign DNA is chromosomally introduced or not). Also, even if the delivery is done properly and no damage or introduction occurs to the host genome, multiple steps are required for the DNA strand to produce the encoded protein. Once inside the cell, the DNA is taken up into the cell nucleus where it is transcribed into RNA. The RNA transcribed from the DNA then enters the cytoplasm where it is translated into protein. These multiple processing steps from transfected DNA to produced protein lead to a lag time before a functional protein is actually produced, and at each step there is the possibility of errors or damage to the cell. Also, it can be difficult to achieve the desired amount of protein expression in cells, as the transfected DNA may not be expressed or may not be expressed with a reasonable efficiency or concentration required for the desired application. This can be a particular problem when introducing DNA into primary cells or recombinant cell lines.

[0005] Messenger RNA (mRNA) is also being investigated for its potential to achieve short-term cellular modification. Advantages of using mRNA as a reversible gene therapy include transient expression and non-transforming properties. mRNA does not need to enter the nucleus for expression, and it is not introduced into the host genome, so there is no risk of tumor formation. The transfection efficiency achievable with mRNA is relatively high, exceeding 90% in many cell types, so no selection is required for transfected cells.

[0006] Although there have been great developments in recent years in the field of mRNA therapeutics and in the field of mRNA vaccines, further research is still needed in these fields to improve protein expression using mRNA. Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present disclosure to provide recombinant ribonucleic acids (eg, mRNAs) that enhance the expression level of an encoded protein.

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

[0009] To achieve the above object, the present disclosure provides a specific recombinant expression construct (Engineered Expression Construct: EEC) that enhances the expression of an encoded protein. The EEC of the present invention has an artificial 5' and / or 3' untranslated region (UTR) adjacent to a coding sequence. The 5'UTR includes a promoter, a mini-enhancer sequence (herein, CAUACUCA), and a Kozak sequence, and the 3'UTR includes a spacer, a stem-loop structure, and optionally a polyadenine tail (polyA tail). In some embodiments, the 5'UTR is operably linked to a start codon to form a functional segment. In some embodiments described herein, the 3'UTR is described as including a stop codon.

[0010] With respect to the recombinant 5'UTR, in some embodiments, the promoter is derived from a 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 linked to a start codon to form the sequence GCCRCC-start (e.g., GCCRCCAUG).

[0011] Particular embodiments of the 5'UTR include, between 5' and 3', a mini-T7 promoter, a mini-enhancer sequence (herein, CAUACUCA), and a Kozak sequence, resulting in GGGGACAUACUCAGCCACC (SEQ ID NO: 2) or GGGGACAUACUCAGCCGCC (SEQ ID NO: 3). With the addition of a start codon, the sequence becomes GGGGACAUACUCAGCCACCAUG (SEQ ID NO: 38) and GGGGACAUACUCAGCCGCCAUG (SEQ ID NO: 39).

[0012] In some embodiments, the number of nucleotides in these minimal 5'UTRs is less than 30. In some embodiments, the number of nucleotides in these minimal 5'UTRs 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 ]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 the hybridization sequences has 7 to 15 nucleotides. The sequence of the loop segment is, for example, UAACGGUCUU (SEQ ID NO: 34). When included as part of the 3'UTR sequence, examples of stop codons include UAA, UAG, and UGA.

[0014] In some embodiments, these minimal 3'UTRs have fewer than 30 nucleotides.

[0015] Regarding the stem-loop, improved protein expression was observed in the EECs disclosed herein regardless of the order of the stem-loop sequence, indicating that it is the secondary structure that is important, and not necessarily the 5' to 3' sequence. The recombinant 3'UTR may further comprise a polyA tail.

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

[0017] In one aspect, the disclosure provides an EEC comprising an in vitro synthesized RNA comprising a coding sequence encoding a protein to be translated in a mammalian cell, the in vitro synthesized RNA comprising a 5'UTR having SEQ ID NO: 2 or SEQ ID NO: 3. The 5'UTR may also comprise a start codon resulting in SEQ ID NO: 38 or SEQ ID NO: 39.

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

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

[0020] In one embodiment, the 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 NOs: 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 disclosure provides an EEC comprising an in vitro synthesized RNA comprising a coding sequence that encodes a protein to be translated in a mammalian cell, wherein the in vitro synthesized RNA is configured to comprise 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 aspect, the disclosure provides an EEC comprising an in vitro synthesized RNA comprising a coding sequence encoding a protein to be translated in a mammalian cell, wherein the in vitro synthesized RNA is configured to comprise a 5'UTR having SEQ ID NO: 2 or SEQ ID NO: 3 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.

[0024] In one aspect, the disclosure provides an EEC comprising an in vitro synthesized RNA comprising a coding sequence encoding a protein to be translated in a mammalian cell, wherein the in vitro synthesized RNA is configured to comprise a 5'UTR having SEQ ID NO: 38 or SEQ ID NO: 39 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.

[0025] In some particular embodiments, the EECs disclosed herein do not include modified nucleotides. In some particular embodiments, the EECs disclosed herein do not include a microRNA binding site. In some particular embodiments, the EECs disclosed herein do not include modified nucleotides and do not include a microRNA binding site.

[0026] In the disclosed EEC, the recombinant 5' and 3' UTRs flank the coding sequence in an open reading frame. The data provided in this disclosure show enhanced expression of green fluorescent protein (GFP), interleukin 2 (IL-2), and POU5F1 (OCT3 / 4) in a variety of cell types, including lymphoid cells, adherent and suspension fetal kidney cells, regardless of the form of transfection.

[0027] The EECs disclosed herein can be used to enhance expression of a variety of proteins for a number of different purposes, including use in therapeutics and vaccines. [Brief description of the drawings]

[0028] Some of the drawings may be easier to understand if they are in color, and applicants reserve the right to submit color versions of the figures as part of the original application.

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

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

[0031] [Diagram 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-encoding EEC 5'UTR mutants 3 hours after transfection (Figure 3A) and 24 hours after transfection (Figure 3B). 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, including no RNA (negative symmetric), mRNA without 5'UTR, M1 and M3 modules with only 5'UTR, and M1, M2, 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 at 3 hours post-transfection (Figure 4A) and 24 hours post-transfection (Figure 4B). The results of the flow cytometry experiments are also shown as bar graphs. EXPI293 cells were transfected with equimolar amounts of the GFP-encoding EEC 5'UTR mutants as follows: (i) no RNA (negative control); (ii) mRNA with only 3'UTR (UGCAUACCUCUAACGGUCUUGAGG (SEQ ID NO: 10) or UAAUGCAUACCUCUAACGGUCUUGAGG (SEQ ID NO: 13) with 3'UTR but no 5'UTR); (iii) M3 only with 5'UTR and additional 3'UTR (UAAUGCAUACCUCUAACGGUCUUGAGG (SEQ ID NO: 13) as 5'UTR and GGGAGAGCCACCAUG (ORF) (SEQ ID NO: 63) as 3'UTR); and (iv) M1, M2, and M3 (SEQ ID NO: 10) with 5'UTR (SEQ ID NO: 2) and 3'UTR. Cells treated with EEC with full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) show the highest GFP intensity.

[0033] [Diagram 5] FIG. 5 is a diagram showing three proteins expressed using the EECs disclosed herein, demonstrating targeted protein expression in the cytoplasm, organelles (i.e., nuclear compartment) and extracellular compartments (i.e., secondary proteins).

[0034] [Figure 6]6A-C show HEK293 cells expressing GFP protein. FIG. 6A is a flow cytometry graph showing GFP (x-axis) and the change in detection that occurred with increasing amounts of GFP-encoded EEC transfected into HEK293 cells (the panel labeled "No mRNA" shows no GFP-encoded EEC mRNA, the remaining panels show 0.2 pmol GFP-encoded EEC mRNA, 0.4 pmol GFP-encoded EEC mRNA, 1.0 pmol GFP-encoded EEC mRNA, 2.0 pmol GFP-encoded EEC mRNA, and 4.0 pmol GFP-encoded EEC mRNA, as indicated in the title of each panel). FIG. 6B shows the results from the flow cytometry graph of FIG. 6A as a chart showing the increase in the percentage of GFP-positive cells for increasing doses of GFP-encoded EEC. In the chart of FIG. 6B, the percentage of HEK293 cells that are GFP positive is shown on the y-axis, and the amount of GFP-encoded EEC mRNA is shown on the x-axis. Thus, FIG. 6B shows an increase in the percentage of GFP positive cells with increasing GFP-encoded EEC mRNA. FIG. 6C shows a chart of the results from the flow cytometry graph of FIG. 6A, in the form of an increase in median GFP intensity proportional to increasing amounts of GFP-encoded EEC transfection in cells positive for GFP expression. In FIG. 6C, the median GFP intensity from cells that expressed GFP is shown on the y-axis (FLI-H), and the amount of GFP-encoded EEC mRNA is shown on the x-axis. Thus, FIG. 6C shows an increase in median GFP intensity proportional to increasing GFP-encoded EEC mRNA.

[0035] [Figure 7]7A-7C show HEK293 cells expressing GFP protein after transfection with GFP-encoding EEC mutants. FIG. 7A shows a histogram overlaid with GFP intensity (x-axis) for cells transfected with 0.4 pmol of GFP-encoding EEC mutants. FIG. 7B shows a similar view to FIG. 7A, but cells were transfected with 1 pmol of GFP-encoding EEC mutants. FIG. 7C shows a bar graph of median GFP intensity for HEK293 cells transfected with equimolar amounts of GFP-encoding EEC mutants (n=2). The mutants used were 5'UTR only (SEQ ID NO: 2), 3'UTR only (described in detail in the legend to FIG. 4B), 5' UTR and 3'UTR (SEQ ID NO: 2 and SEQ ID NO: 10, described in detail in the legend to FIG. 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-encoding EEC mutants. Figure 8A is a flow cytometry graph showing the amount of GFP-positive Jurkat cells in each group of cells transfected with various GFP-encoding 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 group of cells transfected with various GFP-encoding EEC 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10) EEC constructs. 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 increasing amounts of GFP-encoding EECs with full-length 5'UTR (SEQ ID NO: 2) and 3'UTR (SEQ ID NO: 10). The results of the flow cytometry experiment in Figure 8B are shown as bar graphs, showing GFP intensity and percentage of Jurkat cells transfected with 8 pmol of GFP-encoding EEC mutants (n=2). Data are for no UTR, Kozak only, 5' only, 3' only, 5' and 3', and Kozak and 3' mutants. For details of the sequences used in these mutants, see legends to Figures 4B and 7C.

[0037] [Figure 9]Figures 9A and 9C show the amount of GFP expression in Jurkat (T) lymphocytes 24 hours after electroporation of GFP-encoding EECs with UTR mutants. Figure 9A shows a representative example of a histogram showing the GFP intensity and percentage of Jurkat cells transfected with 4 pmol of each GFP-encoding EEC mutant. Figure 9B shows a representative example of a histogram showing the GFP intensity and percentage of Jurkat cells transfected with 8 pmol of each GFP-encoding EEC mutant. Figure 9C is a bar graph showing the median GFP intensity (number of experiments=2) of lymphocytes with 4 and 8 pmol of the GFP-encoding EEC mutants shown in Figures 9A and 9B.

[0038] [Figure 10] Figures 10A-C show a typical experiment in which Raji lymphocytes expressing GFP protein were analyzed by flow cytometry 24 hours after electroporation of lymphocytes with GFP-encoding EECs carrying UTR mutants. Figure 10A is a graph of GFP intensity versus percentage of Raji lymphocytes transfected with 4 pmol of GFP-encoding EEC mutants. Figure 10B is a graph of GFP intensity versus percentage of Raji lymphocytes transfected with 8 pmol of GFP-encoding EEC mutants. Figure 10C is a bar graph showing the median GFP intensity of the experiment shown in Figures 10A and 10B (number of experiments=2), showing the GFP intensity of Raji lymphocytes transfected with 4 pmol and 8 pmol of each GFP-encoding EEC mutant. Data is for 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-encoding EECs with 5' and 3' UTR mutants disclosed herein. Figure 11A shows the results of a typical flow cytometry experiment showing the hOCT3 / 4 intensity and percentage of hOCT3 / 4-positive HEK293 cells transfected with increasing amounts of hOCT3 / 4-encoding EECs (with 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 hOCT3 / 4 intensity and percentage of hOCT3 / 4-positive cells transfected with 1.2 pmol of hOCT3 / 4 with UTR mutants. The results of the flow cytometry experiment in Figure 11A are shown as lines. The bar graph shown in FIG. 11B is a median bar graph of the results of experiments (number of experiments=2) showing the amount of hOCT3 / 4 with UTR mutants compared to no RNA.

[0040] [Figure 12] Figures 12A and 12B show hIL2 protein expression (measured by ELISA) from HEK293 cells 24 hours after transfection with hIL2-encoding EECs with the 5' and 3' UTR mutants disclosed herein. Figure 12A is a graph showing the increase in absorbance (@450 nm) of the ELISA signal for increasing doses of hIL2-encoding EECs (with 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-encoding EECs with each UTR mutant ((SEQ ID NO:2) and (SEQ ID NO:10)).

[0041] [Figure 13]Figures 13A-C show GFP protein expression in HEK293 cells after transfection with GFP-encoding EECs with full-length 5'UTR (SEQ ID NO:2) and 3'UTR mutants (SEQ ID NO:10, 11, and 12). Figure 13A shows the sequence of the 3'UTR. Figure 13B shows the GFP-positive HEK293 cells (based on flow cytometric events / 10,000 cells) 24 hours after transfection with 1 to 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 to 2 pmol of GFP-encoding EECs with 3'UTR mutants A, B, and C shown in Figure 13A.

[0042] [Figure 14] FIG. 14 shows expression of Oct4 in human foreskin fibroblasts upon 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] FIG. 15 shows other sequences (SEQ ID NOs: 55 to 60) that support the present disclosure, such as cDNA constructs for producing in vitro synthetic RNA for EGFP, Oct4, and IL2, and the synthetic RNA constructs obtained thereby. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Significant scientific advances have been made in the use of RNA as therapeutics, vaccines, and / or in the engineering of protein expression in cells both in vitro and in vivo. One of the major challenges to overcome is getting the protein expressed at a high enough level to achieve the desired outcome. Several attempts have been made to overcome this challenge and improve the expression of the protein of interest, making mRNA useful in various clinical settings. Such work is described, for example, in U.S. Patent No. US20060247195 (filed June 8, 2006, assigned to Ribostem Limited, currently abandoned), U.S. Patent No. 10,772,975 (filed May 12, 2011, assigned to Moderna, Inc., currently issued), PCT application PCT / EP2008 / 01059 (filed December 12, 2008, published number WO2009077134, assigned to BioNTech AG), PCT application PCT / EP2008 / 03033 (filed April 16, 2008, published number WO2009127230, assigned to Curevac), and U.S. Patent No. 10,772,975 (filed May 12, 2011, assigned to Moderna, Inc., currently issued). GMBH), PCT application No. PCT / US2016 / 069079 (filing date: December 29, 2016, Assignee: Cellular Reprogramming, Inc.), and PCT application No. PCT / US2019 / 037069 (filing date: June 13, 2019, Assignee: Cellular Reprogramming, Inc.).

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

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

[0047] The "R" in the Kozak consensus sequence indicates adenosine or guanosine. The -3 position of the Kozak consensus sequence promotes translation initiation, and overall, the Kozak sequence appears to pause the translation initiation complex for the proper recognition position of the start codon. Although the Kozak consensus sequence itself can promote ribosome scanning and translation initiation, we analyzed other UTRs associated with abundant proteins in the human transcriptome. Studies have shown that the relatively abundant proteins mentioned above 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'UTRs of highly expressed ribosome-associated proteins (RPLs / RPSs) showed that they display 5'-terminal oligopyrimidine tracks (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., (E. et al., RNA Binding Protein / RNA Element Interactions and the Control of Translation. Curr. Protein Pept. Sci. (2012), doi:10.2174 / 13892031280161947). In general, the 5'TOP sequence is located near the start codon and is important in transcription (i.e., synthesis of RNA) and translation of the transcript.

[0048] Protein expression of coding sequences can be improved by genetically modifying features typical of highly expressed genes in a particular target organ, for example, the 5'UTR of liver-expressed mRNAs such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, α-fetoprotein, erythropoietin, or factor VIII can be introduced to improve expression of the coding sequence in hepatic cell lines and liver. Similarly, the 5'UTR from another tissue-specific mRNA can be used to improve expression in that tissue, such as for 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, UTRs can be hundreds or thousands of nucleotides (nts) long. For use of mRNA in therapeutics or vaccines, it is preferable to search for minimal / optimal UTRs to improve expression of the desired protein in cells. In some embodiments, the recombinant expression constructs (EECs) disclosed herein are designed to have minimal UTRs (minUTs), i.e., the EECs are designed to have minimal 5' and / or 3' UTRs that can achieve high expression levels in the intended application.

[0050] Thus, in some embodiments, the present disclosure provides minimal UTRs that significantly improve protein expression. In some embodiments, the present disclosure provides 5'UTRs having 20 to 23 nucleotides. In some embodiments, the present disclosure provides 3'UTRs having 27 or 67 nucleotides, depending on whether or not they include an optional polyA tail. When combined, some embodiments of the 5' and 3'UTR combinations include 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 present disclosure includes the components necessary for in vitro mRNA production and in vivo protein production. For in vitro mRNA production, the present disclosure employs the use of RNA polymerase from T7 bacteriophage (T7P). T7P encodes a specific DNA double-stranded sequence (5'-TAATACGACTCACTATA G -3' (SEQ ID NO: 45)) and initiates RNA synthesis introducing a 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'), which stabilizes the transcription complex and facilitates T7P clearance and elongation of the RNA polymer.

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

[0053] In certain embodiments, a minimal promoter is selected for use in the 5'UTR. A minimal promoter does not have the activity of promoting gene expression by itself, but can be activated to promote gene expression when linked to a proximal promoting element. Examples of minimal promoters include minBglobin, minCMV, minCMV with the SacI restriction site removed, minRho, minRho with the SacI restriction site removed, Hsp68 minimal promoter (proHSP68), and the like. In certain embodiments, the minimal promoter comprises a minimal T7 promoter (miniT7 promoter).

[0054] An example of a disclosed 5'UTR includes the mini-enhancer sequence (CAUACUCA) of the present application. The mini-enhancer sequence may be placed between a minimal promoter (e.g., T7) and a Kozak consensus sequence to generate a minimal 5'UTR having 20 to 23 nucleotides (depending on whether a start codon is specified as part of the UTR). Generally, eukaryotic translation is initiated by the AUG codon, but other start codons may be included. Additionally, mammalian cells can utilize leucyl-tRNA to decode the CUG codon to initiate translation at the amino acid leucine, and the mitochondrial genome uses AUA and 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 mRNAs are guanylylated by adding an inverted 7-methylguanosine to the 5'triphosphate (i.e., m7GpppN, where N represents the first base of the mRNA). m7GpppN, the 5' cap structure of an mRNA, is involved in nuclear export and binds to the mRNA Cap Binding Protein (CBP) for mRNA translational competence.

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

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

[0058] In certain embodiments, the 5'UTR is operably linked to a coding sequence. As used herein, "operably linked" refers to a functional bond formed between a nucleotide expression control sequence (e.g., a promoter sequence or a UTR) and another nucleotide sequence, thereby allowing and effecting the transcription and / or translation of the other nucleotide sequence by the control sequence.

[0059] The present disclosure also provides 3'UTRs, optionally used in conjunction with the disclosed 5'UTRs. As shown herein, the combination of the disclosed 5'UTRs with the disclosed 3'UTRs can result in EECs with significantly improved protein expression compared to using only the disclosed 5'UTRs or only the disclosed 3'UTRs.

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

[0061] As previously described, the disclosed 3'UTRs include a spacer, a stem-loop structure, and optionally a polyadenine tail (polyA tail), and in some embodiments disclosed herein, the 3'UTR is described as being 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 ]AAA (e.g., UGCAUA, UGCAAA, UGAAA, GCAUA, UAAA, and GAUA), where N is any nucleotide such as A, G, C, T, or U. The subscript numbers indicate the number of nucleotides. For example, [N 1-3 ] contains 1, 2, or 3 nucleotides and stands for N, NN, or NNN.

[0064] Stem loops (SLs), also called hairpins or hairpin loops, are characteristic of highly expressed transcripts in the 3'UTR. SLs are specific secondary structures in which complementary nucleotides are paired as a double helix (i.e., stem), sometimes with other sequences intervening to form a loop. Specific examples of secondary structures represented by SLs include a continuous nucleic acid sequence that includes a stem and a (terminal) loop, also called a hairpin loop, in which the stem is formed by two adjacent fully or partially complementary sequence elements, which are intervened by a short sequence (e.g., 3 to 10 nucleotides) to form the loop of the SL structure. The two adjacent fully or partially complementary sequence elements may be referred to, for example, as SL elements stem 1 and stem 2. An SL is a double-stranded nucleic acid sequence in which two adjacent, wholly or partially reverse-complementary sequence elements (e.g., SL elements stem 1 and stem 2) base-pair with each other, but are not paired at the ends, forming a loop with a short sequence interposed between SL elements stem 1 and stem 2. Thus, an SL contains two stems (stem 1 and stem 2). The two stems base-pair with each other at the secondary structure level of the nucleic acid molecule and are interposed by a short sequence that is not part of stem 1 or stem 2 at the primary structure level of the nucleic acid molecule. Diagrammatically, the secondary depiction of an SL resembles a lollipop structure. The formation of a stem-loop structure requires a sequence that can fold back to form a paired duplex, which is formed by stem 1 and stem 2. The stability of the paired SL elements is typically determined by their length, i.e., the number of nucleotides in stem 1 that can form base pairs (preferably standard base pairs, more preferably Watson-Crick base pairs) with nucleotides in stem 2, relative to the number of nucleotides in stem 1 that cannot form such base pairs with nucleotides in stem 2 (mismatches or bulges).According to the present invention, the optimal loop length is 3 to 10 nucleotides, more preferably 4 to 7 nucleotides, such as 4 nucleotides, 5 nucleotides, 6 nucleotides, or 7 nucleotides. If a nucleic acid sequence forms a SL, the corresponding complementary nucleic acid sequence typically also forms a SL. The SL is typically formed by a single-stranded RNA molecule. 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 long.

[0065] SLs are present within the 3′UTRs of highly expressed transcripts (e.g., those encoding abundant cellular proteins such as histones) where they promote translation independent of the polyadenine tail (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 six base pairs, two of which are GC pairs, three of which are pyrimidine-purine (YR) pairs, and one of which is AU pair, and the loop contains four ribonucleic acids, two of which are uridines (U), one of which is purine (Y), and one of which is 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] SLs associate with stem-loop binding proteins (SLBPs) for replication-dependent mRNA stability / processing / metabolism / translation. Structural evidence indicates that SLBPs make direct contact with SLs at the guanosine nucleotide (G7) at the base of SLs (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, adenosines adjacent to the stem, more specifically, the upstream AAA, influence SLBP binding and function (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). The 3′UTR also plays a role in stabilizing protein-coding transcripts while enhancing translational competence.According to some embodiments, the present disclosure provides the design of synthetic SLs that introduce a SL configuration of three groups of GC pairs interposed by a sequence having adjacent spacer sequences such as multiple adenosines (UAACGGUCUU (sequence number 34)), improving SLBP binding and mRNA translation.

[0067] In particular, it is important to note that the stem loop used in EEC is independent of the orientation of the sequence and may be a) a configuration containing CCUC and GAGG, b) a configuration containing GAGG and CCUC, c) a configuration containing AAACCUC and GAGG, and d) a configuration containing AAAGAGG and CCUC. Furthermore, the distance between the two arms of the stem (where CCUC and GAGG are base-paired) must be long enough to form a loop. In certain embodiments, the stem loop may be a configuration containing 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 some embodiments, 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 examples, the loop segment of SL comprises UAACGGUCUU (SEQ ID NO: 34). According to certain embodiments, the SL sequence comprises 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 RRYRYYYYYYYRRRYRRYRRRYRYY (SEQ ID NO: 74), RRYRYYYYRYRYRRRYRRYRYRRRYRYY (SEQ ID NO: 75), RYYYRYYYRRYYRRRRRRRYYRRYRRRY (SEQ ID NO:76), YYYYRYRRYRRYRYRYRRYYYYYRRRR (SEQ ID NO:77), YRYRRYRYRYRYRYRYRYRYRYRYRYYYRYR (SEQ ID NO:78), YRRYYYYRYRRYRRRRRYYYRYRRRYYR (SEQ ID NO:79), RYRYYYYYYRRRRRYRRRYYRRYRY (SEQ ID NO:80), YRRYYYRRRYRYRYRYRYRYYYYYRRYYR (SEQ ID NO:81), RYRYYYYRRYRYRYRYRYRYYRYYRRRYRY (SEQ ID NO:82) or YYRRRYRRRYYRRRYYRRRYRYRYRYYYYYYYRR (SEQ ID NO:83), where 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] Optional polyA 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, freeing the 3' hydroxyl group. Poly-A polymerase then adds a chain of adenine nucleotides to the RNA. This process is called polyadenylation, which adds a poly-A tail, for example, 100 to 250 residues long. For in vitro RNA synthesis, poly-A tails are encoded on DNA templates and introduced during the in vitro transcription process.

[0070] In some examples, the polyA tail is in the range of 0 to 500 nucleotides in length (e.g., 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides in length). In some examples, a polyA tail 40 nucleotides in length is used. Length can also be expressed in units or as a function of binding of polyA binding protein. In such embodiments, the polyA tail is long enough to bind four monomers of polyA binding protein, three monomers of polyA binding protein, two monomers of polyA binding protein, or one monomer of polyA binding protein. The polyA binding protein monomer binds to a sequence of 38 nucleotides.

[0071] Based on the above description of the 3'UTR, the 3'UTR constructs disclosed herein may contain one or more spacers (e.g., [N 1-3 ]AUA,[N 1-3]AAA, UGCAUA, or UGCAAA, a hybridization sequence of the stem-loop (e.g., CCUC or GAGG, etc.), a loop segment of the stem-loop (e.g., [N 7-15 ]、 UAACGGUCUU (SEQ ID NO: 34), and / or optionally a polyA tail. If a stop codon is provided as 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 a specific embodiment, other non-UTR sequences may be introduced into the 5'UTR (or 3'UTR). For example, an intron sequence or a part of an intron sequence may be introduced into these regions. Introduction of an intron sequence may further improve protein expression and mRNA amount.

[0074] EEC construct with disclosed 5' and 3' UTRs The disclosed 5'UTR and 3'UTR recombinant sequences, as disclosed herein, when used adjacent to any coding sequence, can be used to generate EECs useful for enhancing protein expression of a variety of proteins, such as green fluorescent protein (GFP), interleukin 2 (IL-2), and POU5F1 (OCT3 / 4), as disclosed herein. Additionally, these inventive 5'UTR and 3'UTR sequences demonstrate similar behavior in a variety of cell types, such as lymphoid cells, adherent and suspension fetal kidney cells, regardless of the mode of transfection.

[0075] An exemplary EEC of the present disclosure is shown in Figure 1. In some embodiments, an "EEC" refers to a polynucleic acid transcript having a 5' and / or 3' UTR as disclosed herein adjacent to a coding sequence that encodes one or more proteins and that retains sufficient structural and / or chemical organization to permit translation of the encoded protein.

[0076] As shown in FIG. 1, the illustrated EEC comprises a coding sequence of linked nucleotides in an open reading frame flanked by a first flanking region and a second flanking region. The coding sequence comprises an RNA sequence encoding a protein. The protein may comprise one or more signal sequences at the 5' end encoded by a signal sequence region. The first flanking region may comprise a region of linked nucleotides including one or more complete or incomplete 5' UTR sequences. The first flanking region may also comprise a 5' end cap. Linking the 5' end of the coding sequence to the first flanking region is a first functional segment. Conventionally, the functional segment comprises a start codon. Alternatively, the functional segment may comprise any translation initiation sequence or signal including a start codon.

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

[0078] The second flanking region may be configured to include a region of linked nucleotides including one or more complete or incomplete 3'UTRs. The flanking region may also be configured to include a 3' tailing sequence (e.g., a polyA tail). The 3'UTR may also be configured to be divided into three segments, such as a stop codon, a spacer, and a stem-loop segment.

[0079] A second functional segment links the 3' end of the coding sequence to the second flanking region. Conventionally, this functional segment comprises a stop codon. Alternatively, the functional segment may comprise any translation initiation sequence or signal that comprises a stop codon. In accordance with the present disclosure, the functional segment may comprise multiple consecutive stop codons.

[0080] In general, the minimum length of a coding sequence for an EEC may be a length of nucleic acid sequence sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In other embodiments, the length may be sufficient to encode a peptide of 2 to 30 amino acids, e.g., a peptide of 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 20 amino acids. The length may be sufficient to encode a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or a peptide of 40 or fewer amino acids, e.g., a peptide 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 the polynucleotide sequences can encode include carnosine and anserine.

[0081] Generally, the length of the coding sequence is greater than 30 nucleotides (e.g., 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, 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 up to 100,000 nucleotides in length).

[0082] In some embodiments, the EEC comprises between 30 and 100,000 nucleotides (e.g., between 30 and 50, between 30 and 100, between 30 and 250, between 30 and 500, between 30 and 1,000, between 30 and 1,500, between 30 and 3,000, between 30 and 5,000, between 30 and 7,000, between 30 and 10,000, between 30 and 25,000, between 30 and 50,000, between 30 and 70,000, between 100 and 250, between 100 and 500, between 100 and 1,000). pcs, 100pcs to 1,500pcs, 100pcs to 3,000pcs, 100pcs to 5,000pcs, 100pcs to 7,000pcs, 100pcs to 10,000pcs, 100pcs to 25,000pcs, 100pcs to 50,000pcs, 100pcs to 70,000pcs, 100pcs to 100,000pcs, 500pcs to 1,000pcs, 500pcs to 1,500pcs, 500pcs to 2,000pcs, 500pcs to 3,000pcs, 500pcs to 5,000pcs, 500pcs to 7,000pcs, 500pcs to 10,000pcs, 500pcs to 25,000pcs pcs, 500pcs to 50,000pcs, 500pcs to 70,000pcs, 500pcs to 100,000pcs, 1,000pcs to 1,500pcs, 1,000pcs to 2,000pcs, 1,000pcs to 3,000pcs, 1,000pcs to 5,000pcs, 1,000pcs to 7,000pcs, 1,000pcs to 10,000pcs, 1,000pcs to 25,000pcs, 1,000pcs to 50,000pcs, 1,000pcs to 70,000pcs, 1,000pcs to 100,000pcs, 1,500pcs to 3,000pcs, 1,500pcs to 5,00 0 pcs, 1,500 pcs to 7,000 pcs, 1,500 pcs to 10,000 pcs, 1,500 pcs to 25,000 pcs, 1,500 pcs to 50,000 pcs, 1,500 pcs to 70,000 pcs, 1,500 pcs to 100,000 pcs, 2,000 pcs to 3,000 pcs, 2,000 pcs to 5,000 pcs, 2,000 pcs to 7,000 pcs, 2,000 pcs to 10,000 pcs, 2,000 pcs to 25,000 pcs, 2,000 pcs to 50,000 pcs, 2,000 pcs to 70,000 pcs, and 2,000 pcs to 100,000 pcs).

[0083] According to the present disclosure, the first and second flanking regions may each independently range from 5 to 100 nucleotides in length (e.g., 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, 47, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 108, 110, 111, 112, 113, 114, 115, 116, 117 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 nucleotides in length).

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

[0085] According to the present disclosure, the first or second functional segment may be in the range of 3 to 40 nucleotides in length, for example, 5 to 30, 10 to 20, 15, or at least 4, or up to 30 nucleotides in length, and may be configured to include one or more signal sequences and / or restriction sequences in addition to start and / or stop codons.

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

[0087] Several research groups have proposed that in order to achieve sufficiently high levels of protein expression from IVT-RNA transfected into cells due to the activation of defense mechanisms, the mRNA transcripts must contain modified nucleotides (see, e.g., issued U.S. Patent No. 9,750,824, filed August 4, 2012, assigned to the University of Pennsylvania) or contain immune evasion factors-containing proteins or other reagents in the form of IVT-RNA (see, e.g., issued U.S. Patent No. 10,207,009, filed May 7, 2015, assigned to BioNTech). These immune evasion factors include viral genes that encode proteins that attenuate cellular immune responses, for example by interfering with IFN receptor binding by extracellular IFN (e.g., B18R from vaccinia virus), inhibiting intracellular IFN signaling (e.g., both E3 and K3 from vaccinia virus), or by performing both functions (e.g., NS1 from influenza) (Liu et al., Sci Rep 9: 11972, 2019). In certain embodiments, the immune evasion protein comprises B18R, E3, K3, NS1, or ORF8 (from SARS-CoV2).

[0088] Aspects of the present disclosure are designed to overcome activated defense mechanisms by introducing secondary and tertiary structures into mRNA transcripts rather than using modified nucleotides, microRNAs, or immune evasion factors, etc. According to other embodiments, certain embodiments do not use modified nucleotides or microRNAs to enhance protein expression. According to still other embodiments, modified nucleotides or microRNAs are not used to extend translation from IVT-RNA transfected into cells or for other purposes.

[0089] In some embodiments, the EEC does not contain a microRNA binding site and / or a modified NTP in the 5'UTR, in the 3'UTR, in the 5'UTR and 3'UTR, or in the entire EEC.

[0090] MicroRNAs (miRNAs) are non-coding RNAs of 19 to 25 nucleotides in length that bind to the 3'UTR of a nucleic acid molecule and suppress gene expression by reducing the stability of the nucleic acid molecule or inhibiting translation. In some embodiments, the EEC is configured to not contain a known microRNA target sequence, a microRNA sequence, or a microRNA seed.

[0091] The microRNA seed is the sequence from position 2 to 8 of the mature microRNA that has perfect Watson-Crick complementarity to the miRNA target sequence.

[0092] In some embodiments, the EECs of the present disclosure are designed to not specifically include modified NTPs. Modified NTPs are those that have additional chemical groups attached to them to modify their chemical structure. Examples of these modified NTPs include pseudouridine, methylpseudouridine, N1-methylpseudouridine, methyluridine (m5U), 5-methoxyuridine (mo5U), and 2-thiouridine (s2U). The 5' cap does not modify the NTP.

[0093] In some embodiments, the EEC comprises messenger RNA (mRNA). As used herein, "messenger RNA" (mRNA) refers to any polynucleotide that encodes a protein and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo.

[0094] The EEC encodes a protein or a fragment thereof. By "protein" is meant a polymer of amino acid residues (natural or non-natural) linked together, often by peptide bonds. The term encompasses polypeptides and peptides of any size, structure, or function. Although the encoded protein may be smaller than 50 amino acids, such proteins are referred to as peptides. If the protein is a peptide, it has at least two linked amino acids. Proteins include naturally occurring proteins, synthetic proteins, homologs, orthologs, paralogs, fragments, recombinant proteins, fusion proteins, and other equivalents, variants, and analogs thereof. Proteins may be single proteins or multi-molecular complexes such as dimers, trimers, and tetramers. They may also include single chain proteins or multi-chain proteins such as antibodies and insulin, which may be associated or linked. Disulfide bonds are typically found in multi-chain proteins. The term protein also applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

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

[0096] The EEC may encode a protein selected from any of several target categories including biologics, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane associated proteins, nuclear proteins, human disease associated proteins, target sites, or proteins encoded by the human genome that have yet to be identified as therapeutic utilities but are useful in the fields of research and discovery. A particular protein may fall into one or more of these categories.

[0097] In some embodiments, specific sequences are used that code for specific proteins. Examples of these specific proteins include green fluorescent protein (GFP), interleukin 2 (IL-2), and POU5F1 or OCT3 / 4 (see FIG. 15). GFP is a protein that fluoresces bright green when exposed to light. Human POU5F1 or OCT3 / 4 (hOCT4 here) is a key nuclear transcription factor that is important for stem cell reprogramming and maintenance. IL-2 is one of the interleukins, a class of cytokine signaling molecules in the immune system. It is a 15.5 to 16 kDa protein that controls the activity of white blood cells. IL-2 is part of the body's natural response to bacterial infections, distinguishing between foreign ("non-self") and "self". IL-2 exerts its effects by binding to the IL-2 receptor expressed on lymphocytes. The primary source of IL-2 is activated CD4+ and activated CD8+ T cells.

[0098] The EECs disclosed herein may encode one or more biologics. A "biologic" includes a protein used in the treatment, cure, mitigation, prevention, or diagnosis of a disease or condition. Examples of biologics include allergen extracts (e.g., for allergy injections and testing), blood components, gene therapy products, human tissue or cell products for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytic agents, 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), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single chain molecules), and antibody fragments. The term "immunoglobulin (Ig)" is used synonymously with "antibody" herein. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogenous antibody population, i.e., an individual antibody comprising the population that is identical except for what may be minor naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), etc. Monoclonal antibodies are highly specific and directed against a single antigenic site.

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

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

[0102] Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, may be encoded by coding sequences including heavy chains designated α, δ, ε, γ, and μ, respectively. Also included are polynucleotide sequences encoding subclasses such as γ and μ. Thus, any of the subclasses of antibodies may be partially or entirely encoded, including subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

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

[0104] Particular embodiments encode an anti-SARS-Cov2 antibody, an anti-SARS antibody, an anti-RSV antibody, an anti-HIV antibody, an anti-Dengue virus antibody, an anti-Bordetella pertussis antibody, an anti-Hepatitis C antibody, an anti-Influenza virus antibody, an anti-Parafenza virus antibody, an anti-Metapneumovirus (MPV) antibody, an anti-Cytomegalovirus antibody, an anti-Epstein-Barr virus antibody, an anti-Herpes simplex virus antibody, an anti-Clostridium difficile toxin antibody, or an anti-Tumor necrosis factor (TNF) antibody.

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

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

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

[0108] An example of an anti-B. pertussis antibody is that described in US Pat. No. 9,512,204.

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

[0110] Anti-influenza virus antibodies are described in US Pat. No. 9,469,685, and examples include C102 (manufactured by Thermo Fisher).

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

[0112] Examples of anti-cytomegalovirus (CMV) antibodies include MCMV5322A, MCMV3068A, LJP538, and LJP539. Also see, 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, e.g., Wilcox et al., N Engl J Med 376(4) 305-317 (2017).

[0115] Those of skill in the art will recognize that numerous other antibody sequences are available and may be used within the teachings of the present 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. As used herein, "vaccine" refers to a composition that stimulates an immune response to generate adaptive immunity against a particular pathogen or infectious agent that causes and / or is necessary for the development of a disease or infection, thereby improving immunity against the pathogen or infectious agent. For example, a vaccine is a preparation that pre-exposes the immune system to a particular antigen, thereby producing an immune system response against the antigen. The pathogen antigen may be an intact but non-infectious form of the pathogen (e.g., heat-killed). The antigen may also 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 or cancer cell). Recognition of an antigen by the immune system after prior exposure leads to long-term immunological memory, which allows the immune system to mount a rapid and effective response if re-exposed to the antigen.

[0117] For example, viral vaccine antigens may be derived from adenoviruses, arenaviruses, bunyaviruses, coronaviruses, flaviviruses, hantaviruses, hepadnaviruses, herpesviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, sponge viruses, or togaviruses. In certain embodiments, vaccine antigens include peptides expressed by viruses such as CMV, EBV, influenza virus, hepatitis A, B, or C, herpes simplex, HIV, influenza, Japanese encephalitis, measles, polio, rabies, RS, rubella, smallpox, varicella-zoster, West Nile fever, and / or Zika.

[0118] Examples of vaccine antigens derived from whole pathogens include the attenuated poliovirus used in the live oral polio vaccine (OPV) and the killed poliovirus used in the inactivated polio vaccine.

[0119] Other specific examples include: exemplary SARS-CoV-02 vaccine antigens, such as spike protein or fragments thereof (e.g., receptor binding domain (RBD)); exemplary CMV vaccine antigens, such as envelope glycoprotein B and CMV pp65; exemplary EBV vaccine antigens, such as EBV EBNAI, EBV P18, and EBV P23; exemplary hepatitis vaccine antigens, such as hepatitis B virus S, M, L proteins, hepatitis B virus pre-S antigen, HBCAG DELTA, HBV HBE, hepatitis C virus RNA, HCV NS3, and HCV NS4; exemplary herpes simplex vaccine antigens, such as immediate early proteins and glycoproteins; exemplary 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, HIV Examples of antigens include gag, pol, and env genes such as GP36, Nef protein, and reverse transcriptase; examples of viral antigens of human papillomavirus (HPV) include L1 protein; examples of influenza virus antigens include hemagglutinin and neuraminidase; examples of Japanese encephalitis vaccine antigens include proteins E, ME, ME-NS1, NS1, NS1-NS2A, and 80%E; examples of malaria vaccine antigens include Plasmodium protein circumsporozoite (CSP), glutamate dehydrogenase, lactate dehydrogenase, and fructose bisphosphate aldolase; examples of measles vaccine antigens include measles virus fusion protein; examples of rabies virus antigens include rabies glycoprotein and rabies nucleoprotein; RS vaccine antigens include RSV fusion protein and M2 protein; examples of rotavirus vaccine antigens include VP7sc; examples of rubella vaccine antigens include proteins E1 and E2;Examples of varicella zoster virus antigens include gpl and gpll; examples of Zika vaccine antigens include premembrane, envelope (E), and domain (III) of E protein and nonstructural proteins 1-5;

[0120] Specific examples of other 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: (AFTFTKIPAETLHTVTEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITEGTENSKMMLELDPPFGDSYIVIGVGE (SEQ ID NO: 54)). For other examples of viral antigens, see Fundamental Virology, 2nd ed., 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, such as Bacillus anthracis, Gram-negative bacillus, Chlamydia, Corynebacterium diphtheriae, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis, Pertussis toxin, Streptococcus pneumoniae, Rickettsia, Staphylococcus aureus, Streptococcus pyogenes, and Clostridium tetani.

[0122] Specific examples of bacterial vaccine antigens include anthrax vaccine antigens such as protective antigen of Bacillus anthracis; gram-negative bacillus vaccine antigens such as lipopolysaccharide; Haemophilus influenzae vaccine antigens such as capsular polysaccharide; diphtheria vaccine antigens such as diphtheria toxin; tuberculosis vaccine antigens such as mycolic acid, heat shock protein 65 (HSP65), 30 kDa secretory protein, and antigen 85A; pertussis toxin vaccine antigens such as hemagglutinin, pertactin, FIM2, FIM3, and adenylate cyclase; pneumococcal vaccine antigens such as pneumolysin and pneumococcal capsular polysaccharide, and an example of a rickettsial vaccine antibody such as rompA; streptococcal vaccine antigens such as M protein; and tetanus vaccine antigens such as tetanus toxin.

[0123] In certain embodiments, the vaccine antigens are derived from multi-drug resistant "superbugs," such as Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., etc.).

[0124] Vaccine antigens may also be configured to include proteins specifically and preferentially expressed by cancer cells to activate the immune system to fight 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; mesothelin; MUC; These 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. As with DNA, RNA can be transferred into cells to elicit both cellular and humoral immune responses in vivo. In particular, different strategies for immunotherapy using in vitro transcribed RNA (IVT-RNA) have been attempted, and both have shown good experimental results in various animal models. RNA can be directly injected into patients by various immunological routes, or IVT-RNA can be transfected into cells in vitro using known transfection methods, and the transfected cells can be administered to patients. For example, RNA can be translated into proteins expressed on MHC molecules on the cell surface to elicit an immune response.

[0126] Therapeutic protein refers to a protein that, when expressed by a cell, treats an existing condition or disease. By "treatment" is meant that expression of the protein alleviates the cause of the existing condition or disease and / or alleviates the side effects of the condition or disease (e.g., pain, inflammation, congestion, fatigue, fever, chills, etc.).

[0127] Cell-permeable proteins The EECs disclosed herein may encode one or more cell penetrating proteins (CCPs, also referred to as cell penetrating peptides). A CCP refers to a protein that can facilitate the uptake or absorption of a molecule by a cell. In general, cell penetrating peptides are (short) peptides that can carry various cargo molecules through the cell membrane, thereby facilitating the cellular uptake of various molecular cargoes (ranging from nano-sized particles to small chemical molecules to large fragments of DNA, etc.). Typically, the association of the cargo with the peptide is via a covalent chemical linkage or non-covalent interactions. Although cell penetrating peptides vary in size, amino acid sequence, and charge, all CCPs share in common the ability to penetrate the plasma membrane and facilitate the delivery of various molecular cargoes into the cytoplasm and organelles of a cell. Currently, the theory of CPP internalization suggests that there are three main mechanisms of internalization: direct penetration of the cell membrane, endocytosis-mediated entry, and internalization via the formation of transient structures (Jafari S, Solmaz MD, Khosro A, 201 5, Bioimpacts 5(2): 103-1 1 1 ; Madani F, Lindberg S, Langel LI, Futaki S, Graslund A, 201 1 , 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 such as 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)); venom-derived proteins such as melittin (Dempsey, C. E., Biochim Biophys Acta, 1 990. 1031 (2): p. 143-61), mastoparan (Konno, K. et al., Toxicon, 2000. 38(11): p. 1 505-1 5), maurocalcine (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 buforin (Kobayashi, S. et al., Biochemistry, 2004. 43(49): p. 1 561 0-6)); or synthetic CPPs, such as polyarginines (R8, R9, R10 and R12) (Futaki, S. et al., J Biol Chem, 2001. 276(8): p. 5836-40) or 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, partially or not at all encode a detectable label. The cell-penetrating peptide may also contain a signal sequence. As used herein, the term "signal sequence" refers to a sequence of amino acid residues that is attached to the amino terminus of a nascent protein during protein translation. The signal sequence may be used to signal the secretion of the cell-penetrating polypeptide.

[0130] The EEC-encoded CPP may post-translationally form a complex, which may comprise a charged protein linked to a cell-penetrating polypeptide.

[0131] In certain embodiments, the CPP may have a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. Here, the "protein-binding partner" may be an antibody or a functional fragment thereof, a scaffold protein, or a peptide. The CPP may further comprise an intracellular binding partner for the protein-binding partner. The CPP may be secretable from a cell into which the EEC has been introduced. The CPP may also be capable of entering a cell into which the EEC has been introduced.

[0132] In another embodiment, the CPP is capable of entering a second cell. The second cell may be from the same site as the first cell or from a different site, such as a tissue or an organ. The second cell may be proximal or distal to the first cell.

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

[0134] Secreted Proteins Human and other eukaryotic cells are compartmentalized by membranes into numerous compartments with different functions. Each membrane-enclosed compartment, or organelle, contains a variety of proteins essential for the function of that organelle. Cells target proteins to specific organelles using "sorting signals," amino acid motifs located within proteins.

[0135] A type of sorting signal, also called a signal sequence, signal peptide, or leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). Proteins that are targeted to the ER by a signal sequence can be released into the extracellular space as secreted proteins. Similarly, proteins that reside in the plasma membrane can be secreted into the extracellular space by proteolytic cleavage of a "linker" that holds the protein in the plasma membrane.

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

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

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

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

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

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

[0142] In some embodiments, EECs can be used to express proteins with currently unknown therapeutic functions.

[0143] In some instances, the EECs encode one or more proteins currently available or under development. Polynucleic acids encoding proteins currently available or under development can be introduced into the EECs to enhance expression of the proteins as disclosed herein.

[0144] An EEC can encode one or more proteins by including a coding sequence for a self-cleaving peptide or by including a ribosomal skipping element in its coding sequence.

[0145] EEC encoded proteins may be used to treat conditions and diseases in many therapeutic areas, including hematology, cardiology, CNS, poisoning (e.g., antitoxins), dermatology, endocrinology, gastroenterology, medical imaging, musculoskeletal system, oncology, immunology, respiratory, sensory, and anti-infective.

[0146] When used to treat a subject, the EECs may be formulated for administration.

[0147] Preparation of the EEC formulations can be performed by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include combining the EEC with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping, and / or packaging the formulation into the desired single or multiple dosage units.

[0148] The relative amounts of EEC, pharma- ceutically acceptable excipients, and / or any other auxiliary materials in a formulation according to the present disclosure will vary depending on the subject's identity, body size, and / or condition, and also on the route by which the formulation is administered. For example, the formulation may contain from 0.1% to 100% active ingredient, e.g., 0.5% to 50%, 1% to 30%, 5% to 80%, at least 80% (w / w).

[0149] EEC formulations can include one or more excipients to: (1) improve stability; (2) improve cell transfection; (3) sustain or delay release (e.g., from a depot formulation); (4) modify biodistribution (e.g., to target specific tissues or specific cell types); and / or (5) modify the release profile of the encoded protein in vivo. In addition to traditional excipients, such as any and all solvents, dispersion media, diluents, or other liquid carriers, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, examples of excipients include lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with EEC (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0150] In vitro synthesis of EEC

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

[0152] In in vitro transcription, cDNA from the desired construct is produced by methods known in the art. Any such cDNA may be transcribed using an in vitro transcription (IVT) system. Such IVT can provide in vitro synthesis of the mRNA of the disclosed EEC. The system typically includes a transcription buffer, nucleotide triphosphates (NTPs), ribonuclease (RNAse) inhibitors, and a polymerase. The NTPs may be made 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 polymerase may be selected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and mutant polymerases such as polymerases capable of introducing modified nucleic acids.

[0153] Transfection of EEC into mammalian cells The EECs designed and synthesized as described herein can be transfected into various cell types and can translate the proteins encoded in their open reading frames into the desired proteins. Transfection can be performed using any method known in the art, such as, for example, electroporation or lipofection. Exemplary cell types include any mammalian cell known in the art or that will become known in the future. Exemplary mammalian cells that can be used include Jurkat cells, Raji cells, HEK293, primary fibroblasts, primary blood cells (such as various white blood cells), primary kidney cells, primary hepatic cells, primary pancreatic cells, and primary neural cells.

[0154] The present disclosure provides an EEC comprising an in vitro synthesized RNA comprising a coding sequence in an open reading frame for translation in a mammalian cell. The 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 can be configured with a 5'UTR comprising any 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 comprising SEQ ID NO:4, 5, 6, or 7. The 5'UTR can also comprise a T7 polymerase promoter, a mini-enhancer sequence (CAUACUCA), or a Kozak sequence. Additionally, the bacteriophage T7 promoter can be selected from a T7 class III promoter (SEQ ID NO:2) in a recombinant sequence.

[0155] The present disclosure also provides an EEC for an in vitro synthesized RNA comprising a coding sequence in an open reading frame for translation in a mammalian cell, the EEC may further comprise a 3'UTR comprising SEQ ID NO: 4, 5, 6, or 7 with any stop codon (UAA / UAG / UGA). The in vitro synthesized mRNA may further comprise a 3'UTR comprising a) CCUC and GAGG, or b) GAGG and CCUC. Each of the 3'UTR sequence sets of a) CCUC and GAGG, or b) GAGG and CCUC may be separated by 7 or more nucleotides, or may be more than 7 nucleotides. The total number of nucleotides in the 3'UTR sequence is preferably 50 nucleotides or less.

[0156] The present disclosure provides an EEC and method for recombinant in vitro synthesized mRNA, which may comprise a 5'UTR comprising any one of the mini-enhancer sequences (CAUACUCA), SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:38, and SEQ ID NO:39, and a 3'UTR comprising any one of SEQ ID NO:4, 5, 6, or 7. The recombinant in vitro synthesized mRNA may comprise a 5'UTR comprising any one of the mini-enhancer sequences (CAUACUCA), SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:38, or ... 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 comprising any one of SEQ ID NO:4, 5, 6, or 7. Additionally, the EECs of the present disclosure may comprise a recombinant mRNA having a coding sequence encoding any one of green fluorescent protein (GFP), interleukin 2 (IL-2), and human POU5F1 (i.e., OCT3 / 4). The present disclosure also provides EECs in which in vitro synthesized RNA enhances expression of the encoded protein. Additionally, in some embodiments, the EECs of the present disclosure are free of modified nucleotides and / or free of microRNA binding sites. In other embodiments, the EECs of the present disclosure are free of modified nucleotides, free of microRNA binding sites, and free of immune evasion factors.

[0157] As described herein, the EEC enhances expression of a protein, which may be relative to the natural expression level of the protein, relative to a coding sequence that does not include the subject mini-enhancer sequence in the 5'UTR, relative to a coding sequence that does not include the subject stem-loop sequence in the 3'UTR, relative to a coding sequence that does not include the subject mini-enhancer sequence in the 5'UTR and does not include the subject stem-loop sequence in the 3'UTR, relative to a coding sequence that includes modified nucleotides but does not include the EEC disclosed herein, and / or relative to the extent to which the protein has been expressed in the past or previously. In some examples, improved protein expression may be at least 10% improved protein expression, at least 20% improved protein expression, at least 30% improved protein expression, at least 40% improved protein expression, at least 50% improved protein expression, at least 60% improved protein expression, at least 70% improved protein expression, at least 80% improved protein expression, at least 90% improved protein expression, at least 100% improved protein expression, at least 200% improved protein expression, at least 300% improved protein expression compared to a relevant control system or condition.

[0158] Exemplary embodiments and examples are provided to illustrate certain embodiments of the present disclosure. Those skilled in the art will appreciate with reference to this disclosure that many variations can be made to the examples disclosed herein and still obtain similar or similar results without departing from the spirit and scope of the present disclosure.

[0159] Exemplary embodiments 1. A recombinant expression construct (EEC) having a 5' untranslated region (UTR) operably linked to a coding sequence, the 5'UTR having a sequence designated as CAUACUCA between a minimal promoter and a Kozak sequence. 2. The EEC of embodiment 1, wherein the minimal promoter is a T7 promoter. 3. The EEC of embodiment 2, wherein the T7 promoter has the sequence shown as GGGAGA. 4. An EEC according to any of the preceding claims, wherein the Kozak sequence has the sequence shown as GCCRCCAUG, where R is A or G. 5. The 5'UTR comprises: (i) the sequence set forth in SEQ ID NO:2 operably linked to a start codon, or (ii) An EEC according to any one of embodiments 1 to 4, having the sequence set forth in SEQ ID NO:3 operably linked to a start codon. 6. The EEC of embodiment 5, wherein the sequence set forth in SEQ ID NO:2 operably linked to the start codon has the sequence set forth in SEQ ID NO:38. 7. The EEC of embodiment 5, wherein the sequence set forth in SEQ ID NO:3 operably linked to the start codon has the sequence set forth in SEQ ID NO:39. 8. The EEC of any of the preceding embodiments, wherein the 5'UTR has fewer than 30 nucleotides. 9. The EEC of any one of embodiments 1 to 8, further comprising a 3'UTR. 10. The EEC of embodiment 9, wherein the 3'UTR comprises a spacer and a stem-loop structure operably linked to a stop codon. 11. The EEC of embodiment 10, wherein the stop codon has the sequence UAA, UGA, or UAG. 12. The spacer has the sequence [N 1-3 ]AUA or [N 1-3 12. The EEC of embodiment 10 or 11, having AAA. 13. The EEC of embodiment 10 or 11, wherein the spacer has the sequence UGCAUA or UGCAAA. 14. An EEC according to any of embodiments 10 to 13, wherein the stem-loop structure has hybridization sequences shown as CCUC and GAGG. 15. An EEC according to any of embodiments 10 to 13, wherein the stem-loop structure has hybridization sequences represented as AAACCUC and GAGG, or as AAAGAGG and CCUC. 16. The EEC of any of embodiments 10 to 15, wherein the stem-loop structure has a loop segment having at least 7 nucleotides. 17. An EEC according to any of embodiments 10 to 16, wherein the stem-loop structure has a loop segment having from 7 to 15 nucleotides. 18. An EEC according to any of embodiments 10 to 17, wherein the stem-loop structure has a loop segment having the sequence shown as UAACGGUCUU (sequence number 34). 19. The EEC of any of embodiments 9 to 18, wherein the 3'UTR has fewer than 30 nucleotides. 20. The EEC of any of embodiments 9 to 19, wherein the 3'UTR further comprises a polyadenine (polyA) tail. 21. The EEC of embodiment 20, wherein the polyA tail has 60 or fewer residues. 22. The EEC of embodiment 20 or 21, wherein the polyA tail has 40 residues. 23. An EEC according to any of embodiments 9 to 22, wherein the 3'UTR has a sequence as set forth in SEQ ID NO: 4, 5, 6, 7, 8, or 9. 24. An EEC according to any of embodiments 9 to 22, wherein the 3'UTR has a sequence as set forth in SEQ ID NO: 10, 11, or 12. 25. An EEC according to any of embodiments 9 to 24, wherein the 3'UTR has a sequence as set forth in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, or 21. 26. An EEC according to any one of embodiments 1 to 25, comprising an in vitro synthesized messenger RNA (mRNA). 27. An EEC according to any of the preceding embodiments, wherein the coding sequence encodes green fluorescent protein (GFP), human interleukin 2 (IL-2), or human POU5F1 (i.e., OCT3 / 4). 28. An EEC according to any of the preceding embodiments, having a sequence as set forth in SEQ ID NO: 56, 58, or 60. 29. An EEC according to any of the preceding embodiments, wherein the coding sequence encodes a therapeutic protein. 30. The EEC of embodiment 29, wherein the therapeutic protein comprises an antibody or a binding fragment thereof. 31. The EEC of embodiment 30, wherein the antibody or binding fragment thereof comprises an anti-SARS-Cov2 antibody or binding fragment thereof, an anti-SARS antibody or binding fragment thereof, an anti-RSV antibody or binding fragment thereof, an anti-HIV antibody or binding fragment thereof, an anti-Dengue virus antibody or binding fragment thereof, an anti-Bordetella pertussis antibody or binding fragment thereof, an anti-Hepatitis C antibody or binding fragment thereof, an anti-Influenza virus antibody or binding fragment thereof, an anti-Parafenza virus antibody or binding fragment thereof, an anti-Metapneumovirus (MPV) antibody or binding fragment thereof, an anti-Cytomegalovirus antibody or binding fragment thereof, an anti-Epstein-Barr virus antibody, an anti-Herpes simplex virus antibody or binding fragment thereof, an anti-Clostridium difficile toxin antibody or binding fragment thereof, or a tumor necrosis factor (TNF) antibody or binding fragment thereof. 32. The EEC of any of the preceding embodiments, wherein the coding sequence encodes a vaccine antigen. 33. The EEC of embodiment 32, wherein the vaccine antigen comprises a SARS-CoV-02 vaccine antigen, a CMV vaccine antigen, an EBV vaccine antigen, a hepatitis vaccine antigen, a herpes simplex virus vaccine antigen, a human immunodeficiency virus (HIV) vaccine antigen, a human papillomavirus (HPV) virus antigen, an influenza vaccine antigen, a Japanese encephalitis vaccine antigen, a malaria vaccine antigen, a measles vaccine antigen, a rabies vaccine antigen, a respiratory syncytial vaccine antigen, a rotavirus vaccine antigen, a varicella zoster vaccine antigen, or a Zika vaccine antigen. 34. An EEC according to any of the preceding embodiments, wherein the coding sequence encodes a cytokine. 35. An EEC according to any one of embodiments 1 to 26, wherein the coding sequence encodes a cell-penetrating protein. 36. The EEC of embodiment 35, wherein the cell-permeable protein comprises penetratin, a minimal domain of TAT, VP22, ZEBRA, melittin, mastoparan, maurocalcine, crotamine, buforin, polyarginine, or transportan. 37. The EEC of any of the preceding embodiments, which does not contain modified nucleotides. 38. An EEC according to any one of the preceding embodiments, which does not contain a microRNA binding site. 39. An EEC according to any of the preceding embodiments, wherein the coding sequence encodes an immune evasion factor. 40. The EEC of embodiment 40, wherein the immune evasion factor comprises B18R, E3, K3, NS1, or ORF8. 41. An EEC according to any of embodiments 1 to 38, which does not contain an immune evasion factor. 42. The EEC of any of embodiments 1 to 41, formulated for administration to a subject. 43. A recombinant expression construct (EEC) having a coding sequence operably linked to a 5' untranslated region (UTR) comprising the sequence set forth in SEQ ID NO:38, and a 3' UTR comprising the sequence set forth in SEQ ID NO:13, 14, or 15. 44. An enhancer sequence comprising the sequence shown as CAUACUCA. 45. Recombinant expression (EEC) constructs carrying 1, 2, 3, 4, or 5 copies of the sequence designated as CAUACUCA. 46. ​​The EEC of embodiment 44, wherein the enhancer sequence is operably linked to a promoter. 47. The EEC of embodiment 46, wherein the promoter is a minimal promoter. 48. A recombinant expression construct comprising an in vitro synthesized RNA comprising a coding sequence in an open reading frame encoding a protein to be translated in a mammalian cell, wherein the in vitro synthesized RNA further comprises a 5' untranslated region comprising CAUACUCA and one of the 3' untranslated regions comprising any of SEQ ID NOs: 4, 5, 6, or 7. 49. A recombinant expression construct comprising an in vitro synthesized RNA comprising a coding sequence in an open reading frame encoding a protein to be translated in a mammalian cell, wherein the in vitro synthesized RNA further comprises a 5' untranslated region comprising SEQ ID NO:2 or SEQ ID NO:3, and a 3' untranslated region comprising any of SEQ ID NOs:4, 5, 6, or 7. 50. A recombinant expression construct comprising an in vitro synthesized RNA containing an open reading frame encoding a protein to be translated in a mammalian cell, wherein the in vitro synthesized RNA further comprises a T7 polymerase promoter, a sequence designated CAUACUCA, and a 5' untranslated region comprising a Kozak sequence. 51. The recombinant expression construct of any 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 synthesized RNA containing an open reading frame encoding a protein to be translated in a mammalian cell, wherein the in vitro synthesized RNA comprises a 3' untranslated region comprising SEQ ID NO: 4, 5, 6, or 7 and a stop codon. 53. The recombinant expression construct of embodiment 52, wherein the stop codon is UAA, UAG, or UGA. 54. A recombinant expression construct comprising an in vitro synthesized RNA comprising an open reading frame encoding a protein to be translated in a mammalian cell, said in vitro synthesized RNA comprising a 3' untranslated region comprising either a) CCUC and GAGG, or b) GAGG and CCUC, said 3' untranslated region comprising a set of sequences, each of which is separated by 7 or more nucleotides. 55. A recombinant expression construct (EEC) comprising an in vitro synthesized RNA comprising an open reading frame encoding a protein to be translated in a mammalian cell, said in vitro synthesized RNA comprising a 3' untranslated region comprising either a) AAACCUC and GAGG, or b) AAAGAGG and CCUC, and wherein each sequence in the set of sequences of said 3' untranslated region is separated by 7 or more nucleotides. 56. A 5' untranslated region (UTR) containing a sequence designated as CAUACUCA, located between the minimal promoter and the Kozak sequence. 57. The 5'UTR of embodiment 56, wherein the minimal promoter is a T7 promoter. 58. The 5'UTR of embodiment 57, wherein the T7 promoter has the sequence shown as GGGAGA. 59. The 5'UTR of any of embodiments 56 to 58, wherein the Kozak sequence has the sequence shown as GCCRCCAUG, where R is adenosine or guanine. 60. The 5'UTR of any of embodiments 56 to 59, comprising the sequence set forth in SEQ ID NO:2 operably linked to a start codon. 61. The 5'UTR according to any of embodiments 56 to 60, comprising the sequence set forth in SEQ ID NO:3 operably linked to the start codon. 62. The 5'UTR of embodiment 60, wherein the sequence set forth in SEQ ID NO: 2 operably linked to the start codon has the sequence set forth in SEQ ID NO: 38. 63. The 5'UTR of embodiment 61, wherein the sequence set forth in SEQ ID NO: 3 operably linked to the start codon has the sequence set forth in SEQ ID NO: 39. 64. The 5'UTR of any of embodiments 56 to 63, wherein the 5'UTR has fewer than 30 nucleotides. 65. A 3'UTR comprising a spacer and a stem-loop structure operably linked to a stop codon, wherein the stop codon has the sequence UAA, UGA, or UAG, and the spacer has the sequence [N 1-3 ]AUA or [N 1-3 ] 3'UTR with AAA. 66. The 3'UTR of embodiment 65, wherein the spacer has the sequence UGCAUA or UGCAAA. 67. The 3'UTR of embodiment 65 or 66, wherein the stem-loop structure comprises hybridization sequences shown as CCUC and GAGG. 68. The 3'UTR of any of embodiments 65 to 67, wherein the stem-loop structure comprises hybridization sequences represented as AAACCUC and GAGG, or as AAAGAGG and CCUC. 69. The 3'UTR of any of embodiments 65 to 68, wherein the stem-loop structure has a loop segment having at least 7 nucleotides. 70. The 3'UTR of any of embodiments 65 to 69, wherein the stem-loop structure has a loop segment having 7 to 15 nucleotides. 71. The 3'UTR of any of embodiments 65 to 70, wherein the stem-loop structure has a loop segment having the sequence shown as UAACGGUCUU (sequence number 34). 72. The 3'UTR of any of embodiments 65 to 71, wherein the number of nucleotides is less than 30. 73. The 3'UTR of any of embodiments 65 to 72, further comprising a polyadenine (polyA) tail. 74. The 3'UTR of embodiment 73, wherein the polyA tail has 60 or fewer residues. 75. The 3'UTR of embodiment 73 or 74, wherein the polyA tail has 40 residues. 76. The 3'UTR of any of embodiments 65 to 75, having a sequence as set forth in SEQ ID NO: 4, 5, 6, 7, 8, or 9. 77. The 3'UTR of any of embodiments 65 to 75, having a sequence as set forth in SEQ ID NO: 10, 11, or 12. 78. The 3'UTR of any of embodiments 65 to 77, having a sequence as set forth in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, or 21. 79. The 3'UTR of any of embodiments 65 to 78, operably linked to a coding sequence. 80. The 3'UTR of embodiment 79, wherein the coding sequence encodes a therapeutic protein, a vaccine antigen, a cytokine, or a fluorescent protein. 81. The 3'UTR of embodiment 79 or 80, wherein the coding sequence encodes an immune evasion factor. 82. The 3'UTR of embodiment 81, wherein the immune evasion factor comprises B18R, E3, K3, NS1, or ORF8. 83. The 3'UTR of any of embodiments 79-80, wherein the coding sequence does not contain an immune evasion factor. 84. The 3'UTR of any of embodiments 79 to 83, wherein the coding sequence encodes a cell-penetrating protein. 85. The 3'UTR of embodiment 84, wherein the cell permeability protein comprises penetratin, a minimal domain of TAT, VP22, ZEBRA, melittin, mastoparan, maurocalcine, crotamine, buforin, polyarginine, or transportan. 86. The 5'UTR and / or 3'UTR sequences disclosed herein. 87. A 5'UTR and / or 3'UTR sequence disclosed herein operably linked to a coding sequence.

[0160] Experimental Examples Example 1: Materials and Methods UTR design and structure prediction Minimal transcriptional and translational elements, each 4 to 10 nucleotides long (e.g., the present 5'UTR enhancer, T7 hexamer, and Kozak sequence, each disclosed herein), are assembled to construct the UTR of the present disclosure. Based on the above stem-loop characteristics, synthetic 3' sequences for testing were assembled. The secondary structure prediction web server (rna.urmc.rochester.edu / RNAstructureWeb / ) was used with default parameters to examine 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 adenosine) sequences using the oligonucleotides listed in Table 1. Using these templates, transcription reaction was performed using T7 RNA polymerase and 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 appropriately stored.

[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 incubated at 37°C, 5% CO 2The HEK293 cells were maintained in Eagle's minimum essential medium (EMEM) (ATCC 30-2003) supplemented with 10% fetal bovine serum (FBS). TM Jurkat and Raji cells were cultured in RPMI-1640 medium (ATCC 30-2001) supplemented with 10% FBS. TM ) was maintained. 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 increasing amounts of EEC containing the full-length UTR. As a control experiment, 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-encoding EEC supplemented with jetMessenger (PolyPlus). For electroporation, the Neon Transfection System (Thermo Fisher Scientific) was used according to the manufacturer's manual. Optimal electroporation parameters (voltage, time, and number of pulses) were determined by consulting a database.

[0165] Flow cytometry Cells were fixed with 4% paraformaldehyde for 30-60 min and stored in phosphate-buffered saline (PBS). For hOCT3 / 4 staining, cells were permeabilized, incubated with antibodies (BioLegend), washed twice with PBS, and stored in PBS until use. Cells were analyzed on a FACSCalibur flow cytometer (BD) and a CytoFlex flow cytometer (Beckman Coulter) and analyzed with FlowJo software.

[0166] ELISA 24 hours after transfection, cell culture media were harvested, centrifuged, and diluted as appropriate. Human IL2 ELISA (BioLegend) was used to quantify expression levels according to the manufacturer's protocol. Briefly, plates (Costar) were coated with capture antibody and then incubated with the diluted cell culture media, detection antibody, and avidin-HRP. Absorbance (450 nm) was measured and analyzed.

[0167] Example 2: Enhanced protein expression by EECs containing the 5'UTR sequence of the present application disclosed herein compared to those without the 5'UTR sequence To test the ability of EEC sequences to enhance protein expression, EECs containing the above modified 5'UTR and 3'UTR sequences and GFP as a reporter protein were transfected into EXPI293 suspension cells (Thermo Fisher Scientific). EXPI293 suspension cells derived from the HEK293 cell line were used first because they are designed for high protein expression. To determine the appropriate amount of mRNA to employ in the experiment, EXPI293 cells were transfected with increasing amounts of GFP-encoding EEC ranging from 0 to 2 pmol (0 to 500 ng) using EXPIfectamine transfection reagent. After 24 hours, the cells were subjected to flow cytometry as described above. In this experiment, the GFP fluorescent signal is proportional to the amount of protein in the cells. As shown in Figure 2, the flow cytometry data showed that the GFP median intensity was 0.01–0.25% at 6.0 × 10 cells. 5 Saturation occurs when the amount of mRNA per cell is 0.4 pmol (100 ng).

[0168] Cells were then transfected with equimolar amounts of mRNA with various 5'UTRs and analyzed for GFP expression at 3 and 24 hours post-transfection. The various 5'UTR sequences included (M1) (T7 promoter) with a Kozak sequence (M3); (M1, M2 ((CAUACUCA), and M3) or no other 5'UTR beyond the 5' methyl cap and T7 hexamer. Each construct contains a 5' methyl cap and a T7 hexamer. The results of this experiment are shown in Figures 3A and 3B, which show flow cytometry graphs of GFP intensity (FL1-H) on the x-axis and cell number on the y-axis at 3 hours post-transfection (Figure 3A) and 24 hours post-transfection (Figure 3B) for GFP-encoding EEC 5'UTR mutants. Figure 3 shows the results of the transfection of GFP in cells transfected with the 5'UTR transcript (construct containing only T7 hexamer GGGAGA) and a bar graph depicting the data. As expected, the median intensity of the GFP signal in cells transfected with the UTR-less transcript (construct containing only T7 hexamer GGGAGA) was the lowest at all time points after transfection (Figures 3A and 3B). Addition of the Kozak consensus sequence with an A at the R position increased protein expression by 200%, while further addition of our translational enhancer (CAUACUCA) increased GFP expression by 635% and 240% at 3 and 24 h, respectively, compared to the GFP signal detected in cells not treated with the 5'UTR transcript.

[0169] Thus, this experiment demonstrated that the genetically engineered 5'UTR of the present application, in particular the 5'UTR having the translation enhancer of the present application, is important in significantly improving protein expression.

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

[0171] Thus, this experiment demonstrates that including the 3' stem loop of the present invention in the mRNA is important to further improve protein expression levels, if desired.

[0172] Example 4: Enhanced protein expression of various protein types by EECs having the 5'UTR and 3'UTR sequences of the present application To confirm the utility of the engineered UTRs in improving expression of various proteins, we demonstrated their advantage in coding multiple proteins of different nature. Figure 5 illustrates the targeted expression of three different types of proteins tested in the EECs disclosed herein, namely, proteins in the cytoplasm (GFP), organelles (nuclear compartment, human POU5F1 or OCT3 / 4 in this example), and extracellular compartment (secreted protein, IL2 in this example). See Examples 2 and 3 and Figures 3A-4B for experiments on the effect of the 5'UTR and 3'UTR sequences of the present application on the expression of cytoplasmic proteins (GFP).

[0173] We verified the effect of the UTRs of the present invention on the expression of organelle-bound proteins using human POU5F1 or OCT3 / 4 (hOCT4 in this example), which are key 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 increasing amounts (0-4.8 pmol) of hOCT4-encoding mRNA resulted in increased protein abundance in the cells after 24 hours (Figure 11A and Figure 11C). The percentage of hOCT4-positive cells was 2.0×10 5 A maximum (75%) was reached at 1.2 pmol of mRNA in 10 cells. When HEK293 cells were transfected with equal amounts of hOCT4 transcripts with 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, the median (n=2) hOCT4 intensity was 4-fold higher in cells treated with hOCT4 mRNA with full-length 5'UTR (SEQ ID NO:2) and 3'UTR (SEQ ID NO:10) (Figures 11A and 11B).

[0174] Finally, we examined the effect of the UTRs of the present invention on the expression of the secreted protein human interleukin-2 (hIL2). hIL2 activates T lymphocytes and is currently a target for 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). As in the previous experiment, HEK293 were transfected with increasing amounts of hIL2-encoding mRNA, including the full-length 5'UTR (SEQ ID NO:2) and 3'UTR (SEQ ID NO:10). A proportional increase in hIL2 protein was observed by ELISA 24 hours after transfection (Figure 11A). Furthermore, when hIL2 mRNA was transfected with the UTR mutants, the highest expression of hIL2 protein was observed when both the full-length 5'UTR (sequence number 2) and 3'UTR (sequence number 10) were present (Figure 11B).

[0175] Thus, this experiment demonstrated that mRNAs containing both recombinant UTRs of the present invention are useful in enhancing protein expression using a variety of protein types.

[0176] Example 5: Enhanced protein expression of various protein types by EECs with the 5'UTR and 3'UTR sequences of the present invention

[0177] To test whether the improved protein expression could be reproduced in other cell types, the above experiment was repeated in the following cell lines: HEK293 (ATCC CRL1573™), Jurkat (clone E6-1; ATCC TIB-152), and Raji (ATCC CCL-86) lymphocytes. Adherent HEK293 cells were derived from human embryonic kidney cells transformed by 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 increasing amounts of a new lot of GFP-encoding EEC containing the 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 cells showed GFP signals that reached signal saturation at 1 pmol (250 ng) of GFP-encoding EEC. Increasing the amount of mRNA treated cells only slightly improved the percentage of GFP-positive cells. The median GFP intensity, which indicates protein expression, reached saturation at 2 pmol (500 ng).

[0178] Then, HEK293 cells were treated with various EECs containing the 5'UTR (SEQ ID NO:2) and 3'UTR (SEQ ID NO:10) disclosed herein at 0.4 to 1 pmol amounts (Figures 7A and 7B). All mRNA variants resulted in GFP expression in 60% to 80% of cells, but only cells treated with full-length UTR showed 5-fold higher GFP median intensity than the others (Figure 7C). Median intensity was calculated from duplicate experiments.

[0179] We then performed the above experiments with lymphocyte lines Jurkat and Raji cells, which were 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's lymphoma (Osunkoya, The preservation of burkitt tumour cells at moderately low temperature. Br. J. Cancer (1965), doi:10.1038 / bjc.1965.87; Pulvertaft, A Study of Malignant Tumours 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 jetMessenger reagent (Polyplus-transfection®). Unlike HEK293 cells, which had a transfection efficiency of 90% (i.e., GFP-positive cells), only 10% of Jurkat cells showed GFP signal at the maximum amount of mRNA (16 pmol) (Figure 7A). Furthermore, while cells treated with equimolar amounts of the various constructs showed some GFP signal, those transfected with transcripts containing the 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 are observed in Raji cells (data not shown).

[0180] Thus, this experiment demonstrated that the recombinant UTRs of the present invention are useful in enhancing protein expression in a variety of cell types.

[0181] Example 6: The transfection method is less important for the effect of enhancing protein expression in EECs disclosed herein To examine whether the method of transfection is important in the expression enhancing effects exhibited by the EECs disclosed herein, Jurkat cells were electroporated with increasing amounts of EECs carrying a coding sequence encoding GFP.

[0182] Electroporation has been shown to improve the transfer of nucleic acids into 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 be used in this experiment, Jurkat cells were electroporated with increasing amounts of GFP-encoding EEC using a Neon electroporation system (Thermo Fisher Scientific) as shown in Example 1. This resulted in a proportional increase in GFP signal. Next, Jurkat cells were electroporated with 4 pmol and 8 pmol of GFP-encoding EEC with UTR mutants. 6 to 8 × 10 5At 4 pmol (1 μg) of GFP-encoding EEC per cell, up to 10% of cells were GFP positive, with cells treated with mRNA with 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 with 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 when the highest amount of mRNA was used (Figures 10A, 10B, and 10C).

[0183] In Example 7, in which the recombinant EEC of the present invention was used, protein amounts similar to those observed in Examples 2, 3, and 4 were observed, indicating that the transfection method does not affect the effect of the EEC.

[0184] Example 7: Reversing the stem-loop sequence of the 3'UTR has no effect on improving protein expression To examine 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 to replace CCUC with GAGG (Figure 13A, 3'UTR-B). GFP-encoding EECs were constructed containing either 3'UTR-A or 3'UTR-B and tested for GFP expression in HEK293 cells (transfected with MessengerMax lipofectamine). Using 1-2 pmol, 60-70% of cells showed GFP expression (number of experiments = 2-3), with no difference between constructs with either 3'UTR (Figure 13B). Also, GFP median intensity (number of experiments = 2-3) was similar between EECs with either 3'UTR (Figure 13C). We also tested GFP expression using EECs with another 3'UTR in which a single nucleotide substitution (U to A) was made at the -2 position preceding GGAG in 3'UTR-B (Figure 13A, 3'UTR-C). This sequence resembles histone stem-loops, where the stem region is preceded by a truncate of adenosines that are important for stem-loop binding proteins (SLBPs) and mRNA coupling to translation (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).Addition of 3'UTR-C did not increase the percentage of GFP positive cells as much as the 3'UTR, but did increase the median GFP intensity in transfected cells by 60% (FIGS. 13B and 13C).

[0185] Thus, as observed in the above examples, a recombinant 3'UTR containing a stem-loop with flanking sequences of the present application enhances GFP expression in human cells.

[0186] Example 8: When transfected into fibroblasts, recombinant mRNAs containing the 5'UTR sequence of the present application enhance protein expression compared to mRNAs using modified nucleotides

[0187] To compare the amount of protein produced from the disclosed recombinant mRNA with that produced by mRNA using modified nucleotides (N1-methylpseudouridine), several Oct4-expressing mRNA constructs, such as 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 FIG. 14, OCT4 expression was highest when unmodified mRNA (UO) was used. The percentage of OCT4-positive cells was highest at 50.7% for unmodified recombinant transcripts using 800 ng of mRNA. Also, as shown in FIG. 14, the percentage of OCT4-positive cells was significantly lower when modified nucleotide pseudouridine (PUO and PUMD) was used compared to the recombinant mRNA disclosed herein (36.9% vs. 50.7%). These results demonstrated that the 5'UTR and 3'UTR disclosed herein resulted in higher protein expression compared to transcripts obtained with modified nucleotides such as N1-methylpseudouridine.

[0188] See FIG. 15 for the sequences used in each example experiment.

[0189] Conclusion Each embodiment disclosed herein may comprise, consist essentially of, or consist of certain elements, steps, materials, or ingredients described herein, as will be apparent to one of ordinary skill in the art. Thus, the term "include" or "including" should be interpreted to mean "comprising, consist essentially of, or consist of." In this application, the transitional term "comprise" or "comprises" in a claim means that the claim may include, and may or may not include, elements, steps, materials, or ingredients not specified in the claim, possibly in large amounts. The transitional term "consisting of" means that the claim does not include elements, steps, materials, or ingredients not specified in the claim. The transitional term "consisting essentially of" means that the scope of the embodiment is limited to the elements, steps, materials, or ingredients specified in the claim and those that do not have a material effect on the embodiment. As used herein, a significant effect means an effect that may result in a statistically significant reduction in the improvement in protein expression observed by using an EEC comprising SEQ ID NO:2 in its 5'UTR and SEQ ID NO:10 in its 3'UTR.

[0190] Unless otherwise indicated, all numerical values ​​used in the specification and claims should be construed in all instances as being modified by the term "about". Thus, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be achieved by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed, at the very least, in light of the number of significant digits set forth and by applying ordinary rounding techniques. In cases where further clarity is required, the term "about" when used in conjunction with a set forth numerical value or numerical range shall have the meaning reasonably given to it by one of ordinary skill in the art. That is, the term "about" indicates something somewhat greater or less than a stated value or range, and is intended to indicate within the range of ±20% of the stated value, ±19% of the stated value, ±18% of the stated value, ±17% of the stated value, ±16% of the stated value, ±15% of the stated value, ±14% of the stated value, ±13% of the stated value, ±12% of the stated value, ±11% of the stated value, ±10% of the stated value, ±9% of the stated value, ±8% of the stated value, ±7% of the stated value, ±6% of the stated value, ±5% of the stated value, ±4% of the stated value, ±3% of the stated value, ±2% of the stated value, or ±1% of the stated value.

[0191] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical values ​​necessarily contain certain errors resulting from the standard deviation in their experimental measurements.

[0192] Additionally, the proteins and variants of EEC (including the 5'UTR and 3'UTR) disclosed herein have sequences that have at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the reference sequence.

[0193] "Percent sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" refers to the degree of sequence relatedness between protein, nucleic acid, or gene sequences, as determined by the degree to which the sequence matches. "Identity" (often also referred to as "similarity") can be readily calculated by known methods, such as those described in 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). Methods for determining identity are preferably designed to best indicate the match between the sequences being tested. Methods for determining identity and similarity are codified in commercially available computer programs. Sequence alignment and percent identity calculation may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin).Alternatively, multiple alignment of sequences may be performed using the cluster 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 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). In the context of this disclosure, when sequence analysis software is used for the analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program. As used herein, "default values" refers to any original values ​​or parameters that the software loads when first initialized.

[0194] In the text describing the present invention (particularly in the text of the claims below), the terms "a," "an," "the," and similar references are to be construed as including the singular and plural alike, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely used as a shorthand method of referring to each value within the range individually. Unless otherwise indicated, each individual value is included herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Any examples or illustrative language (e.g., "etc.") used herein are merely used to better illustrate the present invention and are not intended to limit the scope of the invention as otherwise claimed in the claims. No language used herein should be construed as indicating any element essential to the practice of the invention that is not recited in the claims.

[0195] Combinations of alternative elements or embodiments of the invention in the disclosure herein are not to be construed as limitations. Each element in a combination may be referenced or claimed individually or in any combination with other elements in the combination or other elements described herein. It is understood that one or more elements in a combination may be included or omitted in another combination for ease of description and / or patentability. If practiced with any inclusion or omission, the specification shall be deemed to include such modified combinations, and thus satisfy the recitation requirement for all Markush groups used in the appended claims.

[0196] Some embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, modifications to these described embodiments will become apparent to those skilled in the art upon reading the above description. The inventors anticipate that such modifications will occur to those skilled in the art, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Thus, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Additionally, all possible variations of the combinations of the elements described above are intended to be within the scope of the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0197] Additionally, this specification references a number of publications, patents and / or patent applications (collectively referred to as "References"), each of which is individually incorporated herein by reference for the teachings thereof specifically cited therein.

[0198] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other possible variations are within the scope of the invention. Thus, by way of non-limiting example, alternative configurations of the invention may be utilized in accordance with the teachings herein. That is, the invention is not limited to that precisely as shown and described herein.

[0199] The specific configurations shown herein are for purposes of example and are presented only to illustrate preferred embodiments of the invention and to provide what is believed to be the most useful and easily understood method for explaining the principles and conceptual aspects of various embodiments of the invention. In this regard, the details of the configurations of the invention have not been described in more detail than is necessary for a fundamental understanding of the invention, since it will be apparent to one of ordinary skill in the art upon reading the above description with reference to the figures and / or examples how several aspects of the invention may be practiced. Definitions and explanations used in this disclosure are meant and intended to provide for future interpretations, unless clearly and unambiguously changed in a particular instance, or unless application of the meaning would render the interpretation meaningless or substantially meaningless. In cases where interpretation of a term would render it meaningless or substantially meaningless, the definition as set forth in Webster's Dictionary, Third Edition, or in a dictionary 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. An in vitro synthesized RNA, (i) a 5' untranslated region (UTR) having the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:38 or SEQ ID NO:39; (ii) a 3'UTR having a sequence as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, 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) contains an open reading frame encoding a protein; The present invention is characterized in that it does not contain modified nucleosides and does not contain a microRNA binding site; An in vitro synthesized RNA, wherein the open reading frame is located between the 5'UTR and the 3'UTR.

2. The in vitro synthesized RNA according to claim 1, characterized in any one of the following (a) to (h): (a) the 5' UTR has the sequence set forth in SEQ ID NO:2 and the 3' UTR has the sequence set forth in SEQ ID NO:10; (b) the 5' UTR has the sequence set forth in SEQ ID NO:3 and the 3' UTR has the sequence set forth in SEQ ID NO:10; (c) the 5' UTR has the sequence set forth in SEQ ID NO: 38 and the 3' UTR has the sequence set forth in SEQ ID NO: 10; (d) the 5' UTR has the sequence set forth in SEQ ID NO: 39 and the 3' UTR has the sequence set forth in SEQ ID NO: 10; (e) the 5' UTR has the sequence set forth in SEQ ID NO:2 and the 3' UTR has the sequence set forth in SEQ ID NO:13; (f) the 5' UTR has the sequence set forth in SEQ ID NO:3 and the 3' UTR has the sequence set forth in SEQ ID NO:13; (g) the 5' UTR has the sequence set forth in SEQ ID NO: 38 and the 3' UTR has the sequence set forth in SEQ ID NO: 13; (h) the 5' UTR has the sequence set forth in SEQ ID NO: 39, and the 3' UTR has the sequence set forth in SEQ ID NO:

13.

3. The in vitro synthesized RNA of claim 1 , formulated for administration to a subject.

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

5. A recombinant expression construct (EEC) containing an enhancer consisting of the sequence designated as CAUACUCA.

6. The EEC of claim 5 , further comprising a promoter.

7. The EEC of claim 6 , wherein the promoter is a minimal promoter.

8. The EEC of claim 7, wherein the promoter has a sequence represented as GGGAGA.

9. The EEC of claim 7 , wherein the enhancer and the minimal promoter are operably linked to an open reading frame encoding a protein.

10. The EEC of claim 5 which does not contain any modified nucleosides.

11. The EEC of claim 5, which does not contain a microRNA binding site.

12. The EEC of claim 5, which does not contain modified nucleosides and does not contain a microRNA binding site.

13. The EEC of claim 5, comprising a 5'UTR having the sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:38 or SEQ ID NO:

39.

14. The EEC of claim 5, comprising a 3'UTR having the sequence shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, 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.

15. The EEC of claim 5, comprising any one of the following (a) to (h): (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 set forth in SEQ ID NO:3 and a 3'UTR having the sequence set forth in SEQ ID NO:10; (c) a 5'UTR having the sequence set forth in SEQ ID NO:38 and a 3'UTR having the sequence set forth in SEQ ID NO:10; (d) a 5'UTR having the sequence set forth in SEQ ID NO:39 and a 3'UTR having the sequence set forth in SEQ ID NO:10; (e) a 5'UTR having the sequence set forth in SEQ ID NO:2 and a 3'UTR having the sequence set forth in SEQ ID NO:13; (f) a 5'UTR having the sequence set forth in SEQ ID NO:3 and a 3'UTR having the sequence set forth in SEQ ID NO:13; (g) a 5'UTR having the sequence set forth in SEQ ID NO:38 and a 3'UTR having the sequence set forth in SEQ ID NO:13; (h) a 5'UTR having the sequence set forth in SEQ ID NO:39 and a 3'UTR having the sequence set forth in SEQ ID NO:

13.

16. The EEC of claim 5 , further comprising a Kozak sequence.

17. The EEC of claim 5 , further comprising a promoter and a Kozak sequence, wherein the enhancer is located between the promoter and the Kozak sequence.

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