Compositions and methods for expressing proteins with RNA

Abstract

The compositions and methods provided herein include a ribonucleic acid (RNA) encoding a nucleoplasm transporter (NCT) inhibitory protein to improve expression of a target protein, such as expression of a target protein encoded by a DNA vector, messenger RNA, self-amplifying RNA, or RNA comprising an unmodified uridine nucleotide. The compositions and methods provided herein can be used to improve the expression of any target proteins, such as viral protein antigens (e.g., for vaccines).

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 323,332, filed March 24, 2022; U.S. Provisional Patent Application No. 63 / 329,050, filed April 8, 2022; U.S. Provisional Patent Application No. 63 / 375,350, filed September 12, 2022; and U.S. Provisional Patent Application No. 63 / 478,633, filed January 5, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Regarding the merging of referenced sequence lists

[0004] The contents of the electronic sequence list (EXCI_004_04WO_SeqList_ST26.xml; size: 258,708 bytes; creation date: March 23, 2023) are incorporated herein by reference in their entirety. Background Technology

[0005] In recent years, significant progress has been made in the therapeutic application of target protein expression for disease treatment and prevention. For example, exogenous proteins have been engineered to replace abnormal or defective proteins in various diseases and to induce immune responses as vaccines. However, improving target protein expression remains a long-standing challenge: exogenous ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) trigger cellular stress signals that directly inhibit protein production; RNA is rapidly degraded in cells, leading to low protein expression levels; and DNA often results in protein overexpression. Therefore, there is a need for compositions and methods to overcome these challenges and improve target protein expression. Summary of the Invention

[0006] This document provides a composition comprising RNA encoding a nucleocytoplasmic transport (NCT) repressor protein and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a target protein. A method for improving the expression of the target protein using the RNA encoding the NCT repressor protein is also provided.

[0007] In some aspects, the compositions of this disclosure comprise self-amplified ribonucleic acid (saRNA) encoding the nucleocytoplasmic transport (NCT) repressor protein. In some aspects, the RNA encoding the target protein and / or the RNA encoding the NCT repressor protein is messenger RNA (mRNA).

[0008] In some aspects, the saRNA encoding the NCT repressor protein encodes the target protein. In some aspects, the composition comprising the saRNA encoding the NCT repressor protein comprises mRNA encoding the target protein.

[0009] In some respects, the mRNA or saRNA encoding the target protein contains at least one unmodified uridine nucleotide.

[0010] In some embodiments, the saRNA comprises a sequence of any one of SEQ ID NO:79-97, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0011] In some embodiments, the NCT inhibitory protein comprises: the sequence of Table 4, the sequence of SEQ ID NO: 65, 66, 67 or 68, and a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with it.

[0012] In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is selected from Table 3.

[0013] In some embodiments, the composition comprises a delivery medium. In some embodiments, the delivery medium is lipid nanoparticles (LNPs).

[0014] In some respects, this article provides methods for improving the expression of target proteins, the methods comprising expressing ribonucleic acid (RNA) encoding a nucleocytoplasmic transport (NCT) repressor protein, wherein the target protein is encoded by ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0015] In some embodiments of the method, the RNA encoding the target protein is self-amplifying ribonucleic acid (saRNA). In some embodiments of the method, the RNA encoding the target protein is messenger ribonucleic acid (mRNA). In some embodiments of the method, the RNA encoding the NCT repressor protein is messenger RNA (mRNA). In some embodiments of the method, the saRNA encoding the target protein also encodes an NCT repressor protein.

[0016] In some embodiments of the method, the RNA encoding the target protein contains at least one unmodified uridine nucleotide.

[0017] In some embodiments of the method, the expression of the NCT inhibitor protein increases the expression level of the target protein by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200-fold, about 500-fold, or about 1000-fold.

[0018] In some embodiments of the method, the expression of the NCT inhibitor protein reduces the expression level of the target protein by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0019] In some embodiments of the method, the expression of the NCT inhibitor protein increases the activity of the target protein by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold.

[0020] In some embodiments of the method, the expression of the NCT inhibitory protein causes the EC of the target protein to... 50 The reduction is approximately 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, or 300 times.

[0021] In some embodiments of the method, the NCT inhibitory protein and the target protein are expressed in cells or a subject. In some embodiments of the method, the NCT inhibitory protein increases the duration of the target protein in cells or a subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold.

[0022] In some embodiments of the method, co-expression of the NCT inhibitory protein reduces the coefficient of variation (CV%) of the target protein in the subject's tissue by approximately 1.2-fold, approximately 1.3-fold, approximately 1.4-fold, approximately 1.5-fold, approximately 1.6-fold, approximately 1.7-fold, approximately 1.8-fold, approximately 1.9-fold, approximately 2-fold, approximately 2.1-fold, approximately 2.2-fold, approximately 2.3-fold, approximately 2.4-fold, approximately 2.5-fold, approximately 2.7-fold, approximately 2.8-fold, approximately 2.9-fold, or approximately 3-fold.

[0023] In some embodiments of the method, the NCT inhibitory protein comprises: the sequence of Table 4, the sequence of SEQ ID NO: 65, 66, 67 or 68, or the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with it.

[0024] In some embodiments of the method, the target protein is a viral antigen.

[0025] In some respects, this article provides methods for reducing cellular signaling responses to stressors, methods comprising introducing polynucleotides encoding NCT inhibitors into cells.

[0026] In some embodiments of the method, the cellular signaling response to the stressor is the expression of interferon-β, and the introduction of an NCT inhibitor reduces the expression of interferon-β in cells by about 10-fold, about 100-fold, about 1000-fold, about 10000-fold, or about 100,000-fold. Attached Figure Description

[0027] Figure 1A-1E This shows an example DNA template used for RNA preparation.

[0028] Figure 2A and 2B Showing the RNA construct encoding luciferase. Figure 2A This shows an example RNA encoding luciferase. Figure 2B This shows an example RNA encoding an NCT repressor protein, which can be used with... Figure 2A RNA co-expression.

[0029] Figure 3A and 3B The RNA construct encoding the Sars-CoV2 spike protein is shown. Figure 3A Example RNA encoding the Sars-CoV2 spike protein is shown. Figure 3B This shows an example RNA encoding an NCT repressor protein, which can be used with... Figure 3A RNA co-expression.

[0030] Figure 4A and 4B The RNA construct encoding glucosylceramidase (GBA) is shown. Figure 4A This shows an example RNA encoding the GBA protein. Figure 4B This shows an example RNA encoding an NCT repressor protein, which can be used with... Figure 4A RNA co-expression.

[0031] Figure 5Aand 5B The DNA construct configuration is shown for in vitro transcription (IVT) to generate single mRNA constructs encoding L protein (L) and nanoluciferase (nLuc). Figure 5A This shows the DNA construct used for IVT to generate a single mRNA construct (L-NLuc) encoding L and nLuc separated by a p2A separator element. Figure 5B This shows the DNA construct used for IVT to generate a single mRNA construct (NLuc-L) encoding nLuc and L separated by a p2A separator element.

[0032] Figure 6A and 6B This diagram shows the luciferase activity assay in mammalian cells expressing luciferase at 24, 48, and 72 hours post-transfection. Measurements were performed as four replicates in a bar chart, showing the mean. Error bars indicate the standard deviation. Figure 6A and 6B Display using encoding a single L protein (L), a single nanoluciferase (nLuc), or such Figure 5A (L-nLuc) and Figure 5B The (nLuc-L) diagram shows the total luciferase (nLuc) activity, measured in relative light units (RLU), upon transfection with mRNAs encoding two conformations of single mRNAs of L and nLuc. These mRNAs contain approximately 50% native uridine and approximately 50% pseudouridine. Figure 6A The results of transfection of mouse C2C12 cells are shown. The presence of L protein combined with nLuc showed increased activity, because nLuc expression was higher in the presence of L protein, and L-nLuc and nLuc-L increased by up to 3.5-fold and 5.5-fold, respectively, after 72 hours. Figure 6B The results of human BJ cell transfection are shown. The combination of L protein and nLuc showed a more significant increase in activity, because nLuc expression was higher in the presence of L protein. In the presence of mRNA containing about 50% natural uridine, the activities of L-nLuc and nLuc-L increased by up to 6.5-fold and 14-fold, respectively, after 72 hours.

[0033] Figure 7 An assay showing the luciferase activity expressed in mammalian cells in the presence of exogenous interferon (IFN)β. This was performed using proteins encoding only L protein (L), only nLuc, or proteins encoding... Figure 5A (L-nLuc) and Figure 5BC2C12 cells were transfected with mRNAs of two conformations of L and nLuc single mRNAs, as shown in (nLuc-L). These mRNAs contained approximately 50% native uridine and approximately 50% pseudouridine. Six hours post-transfection, a 10-fold serial dilution of IFN-β was added to the cells. Luciferase activity was then measured 48 hours after IFN-β stimulation. The figure shows a decrease in luciferase activity due to reduced luciferase expression in the presence of IFN-β (expressed as a percentage of total nLuc expression in the absence of IFN-β). The EC50 of IFN-β required to reduce luciferase expression for each construct is shown. 50 The approximate value is shown by the dashed line. As with the increase in EC... 50 As demonstrated by the approximate values, the presence of L protein protects cells from the effects of exogenous IFN-β by 10-fold and 12-fold (for L-nLuc and nLuc-L, respectively).

[0034] Figure 8 This demonstrates that IFN-β production in cells serves as an indicator of intracellular stress responses. It is demonstrated using proteins encoding only L protein (L), only nLuc, or proteins encoding... Figure 5A (L-nLuc) and Figure 5B Human BJ cells were transfected with mRNAs of two conformations of L and nLuc single mRNAs, as shown in (nLuc-L). These mRNAs contained approximately 50% native uridine and approximately 50% pseudouridine. The presence of protein L suppressed IFN-β production in cells to baseline levels.

[0035] Figure 9 This study demonstrates the effect of mRNA-based L protein expression on Poly I:C-induced downregulation of target gene expression.

[0036] Figure 10 This study demonstrates the effect of mRNA-based L protein expression on IFN-β-induced downregulation of target gene expression.

[0037] Figure 11A-11C This study demonstrates the effect of mRNA-based L protein expression on the downregulation of target gene expression induced by natural nucleosides. Figure 11A This study demonstrates the effect of using different concentrations of L protein on the expression of the target gene using mRNA containing only natural nucleosides (including natural uridine). Figure 11B This shows the effect of L protein expression on mRNA modified with only non-inflammatory nucleosides (including pseudouridine) using different concentrations of L protein. Figure 11C The effects of L protein on the expression of the target gene from mRNA containing only natural nucleosides and modified mRNA were compared over a 20-day period after transfection.

[0038] Figure 12A and 12BThis study demonstrates the effect of L protein expression on the expression of target genes using self-amplified RNA (saRNA). Figure 12A This study demonstrates the effect of L protein expression on the expression of the target gene using saRNA when different concentrations of L protein are used. Figure 12B The effects of different concentrations of L protein on the expression of the target gene of saRNA were compared over a period of 20 days post-transfection.

[0039] Figure 13 The study showed the effect of L protein expression on the expression of the target gene using self-amplified RNA (saRNA) at day 6 post-transfection, using different concentrations of mutant forms of L protein (SEQ ID NO: 65, 66, 67, 68).

[0040] Figure 14 The study showed the effect of L protein expression on the expression of the target gene using self-amplified RNA (saRNA) at day 9 post-transfection, using different concentrations of mutant forms of L protein (SEQ ID NO: 65, 66, 67, 68).

[0041] Figure 15A and 15B This demonstrates the effects of L protein and autoamplified RNA (saRNA) expressing the target gene on the inflammatory mediator IFN-β. Figure 15A ) and IP-10 ( Figure 15B The influence of related expressions.

[0042] Figure 16 The effect of L protein expression on the expression of target genes based on self-amplified RNA (saRNA) over time was shown. L protein saRNA expression significantly increased the expression of target proteins via saRNA constructs.

[0043] Figure 17 The study showed the effect of L protein expression on tetherin expression in the presence of IFN-β. Tetherin, located downstream of IFN-β, is a negative regulator of VLP generation.

[0044] Figure 18A and 18B The mRNA expression vector is shown, in which the target gene (GOI) is tagged with a NanoLuc reporter protein via an adaptor peptide. G6PC1 ( Figure 18A (SEQ ID NO:99) and SERPINA1 ( Figure 18BThe coding sequence of SEQ ID NO:104 is flanked by hBG 5'UTR (SEQ ID NO:98) and hBG 3'UTR (SEQ ID NO:102). The mRNA expression vector contains a Cap1 mRNA cap structure, N1-methylpseudouridine modification, and a 120-nt polyA tail. The mRNA was generated by in vitro transcription (IVT).

[0045] Figures 19A-19D The expression of G6PC1 and SERPINA1 mRNA in cell lysates was shown. Each experiment was repeated three times. A total of five independent experiments (biological replicates) were performed. Figure 19A G6PC1 expression induced by L protein expression (EG Tech) is shown in relative light units (RLU). Figure 19B This shows the fold change in G6PC1 expression relative to the absence of L protein caused by L protein expression (EG Tech). Figure 19C SERPINA1 expression induced by L protein expression (EG Tech) is shown in relative light units (RLU). Figure 19D This shows the fold change in SERPINA1 expression relative to the absence of L protein caused by L protein expression (EG Tech).

[0046] Figure 20A and 20B The gene encoding lysosomal acid glucosinolate ceramide (GBA) is shown. Figure 20A ) and erythropoietin (EPO) gene ( Figure 20B The plasmid DNA expression vector, GBA and EPO, are each labeled by NanoLuc reporter protein via adaptor peptides and driven by the CMV promoter.

[0047] Figure 21 Display Figure 20A GBA expression was assessed 48 hours after transfection with a DNA expression vector and mRNA encoding the L protein (EG Tech). Stimulation of cells with the pro-inflammatory molecules IFN-β and Poly I:C reduced GBA expression. However, in cells stimulated with IFN-β and Poly I:C, L protein mRNA expression increased GBA expression compared to the absence of L protein.

[0048] Figure 22 CCL2 induction was observed in cells transfected with GBA expression plasmid DNA. CCL2 induction decreased when L protein mRNA was also added to the cells.

[0049] Figures 23A-23E It shows that it has been transfected. Figure 20AThe cells containing GBA-encoding plasmid DNA were also stimulated with Poly I:C to induce high levels of the inflammation-associated molecule CCL2. Figure 23A ), CCL5 ( Figure 23B ), CXCL10 induces ( Figure 23C ), IFN-α ( Figure 23D ) and IFN-β Figure 23E The secretion of inflammatory molecules was observed. However, in the presence of L protein mRNA, the levels of each inflammatory molecule decreased to baseline levels in untransfected and unstimulated cells.

[0050] Figures 24A-24E It shows that it has been transfected. Figure 20B The cells contain EPO-encoded plasmid DNA, and they are also stimulated with Poly I:C to induce high levels of the inflammation-related molecule CCL2 (CCL2). Figure 24A ), CCL5 ( Figure 24B ), CXCL10 Figure 24C ), IFN-α ( Figure 24D ) and IFN-β Figure 24E The secretion of inflammatory molecules was observed. However, in the presence of L protein mRNA, the levels of each inflammatory molecule decreased to the baseline of untransfected and unstimulated cells.

[0051] Figures 25A-25C Showing the coding sequence for the L protein ( Figure 25A ), L protein mutant coding sequence ( Figure 25B ) or disordered negative control sequence ( Figure 25C ) and the self-amplifying RNA vector construct of the nano-luciferase reporter gene.

[0052] Figure 26 Shown on days 2 and 5 post-transfection, such as Figures 25A-25C The expression level of L protein mediated by the self-amplified RNA construct is shown. L protein significantly increases the expression of saRNA-mediated nanoluciferase.

[0053] Figures 27A-27C The single construct encoding the L protein and luciferase gene was shown to be effective in human fibroblasts 48 hours after transfection. Figure 27A ), A549 cells ( Figure 27B ) and C2C12 mouse muscle cells ( Figure 27C The effect of this single construct is that the mRNA contains a pro-inflammatory ARCA cap and a mixture of 50%-50% natural uridine and pseudouridine.

[0054] Figures 28A-28C Shown in human fibroblasts ( Figure 28A ), A549 cells ( Figure 28B ) and C2C12 mouse muscle cells ( Figure 28CIn the study, the expression of L protein mRNA almost eliminated the induction of IFN-β expression by ARCA-capped mRNA containing 50% natural uridine, as measured by IFN-β ELISA.

[0055] Figures 29A-29D Displayed Figures 27A-27C and Figure 28A In the experiment described in -28D, the induction of other pro-inflammatory regulatory factors: IFNβ ( Figure 29A ), IFNλ1( Figure 29B ), IFNλ2 / 3 ( Figure 29C ) and IP-10 ( Figure 29D It was also eliminated by the expression of L protein mRNA.

[0056] Figures 30A-30H Example conformations of a self-amplifying RNA construct are shown, encoding non-structural proteins (NSP1, NSP2, NSP3, and NSP4), a subgenomic promoter (SGP), a target gene (GOI), an internal ribosome entry site (IRES), a P2A self-cleavage site, an L protein (EG tech), and 5' and 3' UTRs, as well as an RNA cap and a polyadenylated tail. The saRNA constructs provided herein can be designed as cis-replicons, where the target gene (GOI), L protein, and saRNA replicase cassette are part of the same sequence as the saRNA. Figures 30A-30C ), can be designed as trans replicons, wherein at least one RNA molecule containing GOI and / or L proteins does not contain a saRNA replicase cassette, and the saRNA replicase cassette is provided on different saRNA molecules ( Figure 30D-30F Alternatively, it can be designed as a partial saRNA replicon assembly, wherein at least one saRNA component, such as a component of the replicase cassette GOI and / or L protein, is provided as non-replicating RNA, for example, as messenger RNA. Figure 30G-30H ). Detailed Implementation

[0057] This document discloses various compositions, methods, and kits for improving target protein expression. In one embodiment, the compositions, methods, and kits disclosed herein provide ribonucleic acid (RNA) encoding a nucleocytoplasmic transport (NCT) repressor protein to improve said target protein expression. As provided herein, RNA encoding an NCT repressor protein can improve the duration, quality, and / or quantity of expression of a target protein encoded by RNA or DNA introduced into cells. In some embodiments, the NCT repressor protein improves the expression of self-amplified RNA (saRNA) of the target protein introduced into cells. In some embodiments, the RNA encoding an NCT repressor protein improves the expression of target protein RNA containing at least one unmodified uridine nucleotide introduced into cells.

[0058] In some embodiments of this disclosure, the target protein is encoded by DNA, and the NCT repressor protein is encoded by RNA, such as messenger RNA. In some embodiments, the target protein is encoded by RNA (e.g., messenger RNA), and the NCT repressor protein is encoded by the same or different messenger RNAs. In some embodiments, the target protein is encoded by self-amplifying RNA (saRNA), and the NCT repressor protein is encoded by the same or separate saRNAs, or by mRNA. As provided herein, RNA encoding an NCT repressor protein can improve the duration, quality, and / or quantity of expression of the target protein encoded by DNA or RNA (e.g., saRNA or mRNA) introduced into the cell.

[0059] In some embodiments, the mRNA or saRNA encoding the target protein disclosed herein contains at least one unmodified uridine nucleotide. As provided herein, the introduction of RNA encoding an NCT repressor protein can improve the duration, quality, and / or quantity of expression of the target protein encoded by RNA containing at least one unmodified uridine nucleotide introduced into cells.

[0060] This article also provides a combination of RNA-encoded target proteins and DNA-encoded NCT inhibitory proteins.

[0061] In some embodiments, the nucleocytoplasmic transport (NCT) inhibitor may be selected from the group consisting of piconexvirus leader (L) protein, piconexvirus 2A protease, rhinovirus 3C protease, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0062] As described throughout this document, the inhibition of nucleocytoplasmic transport by RNA-mediated NCT inhibitory proteins can suppress cellular signaling in response to cellular stress, such as interferon-β, and can improve cellular responses to innate immune signaling, pro-inflammatory signaling, delivery-mediated inflammation, native nucleoside mRNA transfection, self-amplified RNA transfection, and the expression of the target protein. Without being bound by theory, it is believed that the compositions and methods of this disclosure prevent the activation of cellular regulatory mechanisms in response to the expression of the target protein, and that this improves the yield and / or functionality of the target protein.

[0063] In some embodiments, the methods and systems of this disclosure, for example, introduce RNA encoding an NCT inhibitor, showing improved regulation of cellular stress responses and prevention of adverse effects on the expression of the target protein caused by in vivo inflammation and interferon induction associated with the introduction of the DNA and RNA into the cells.

[0064] I. Definition

[0065] Unless otherwise defined herein, the scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the terms and techniques used herein in relation to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry are well-known and commonly used in the art.

[0066] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a pharmaceutical candidate” means a single pharmaceutical candidate or a mixture of such candidates, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so on.

[0067] As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value that is quantitatively similar to the reference value and / or within a similar range. In some embodiments, unless otherwise stated or apparent from the context, the term "approximately" or "about" means a value falling within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (unless that number exceeds 100% of the possible value).

[0068] When providing numerical ranges, it should be understood that, unless the context clearly indicates otherwise, every intermediate value between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other specified or intermediate values ​​within the specified range, are included in this application. The upper and lower limits of these smaller ranges may be independently included within the smaller range (which is also included in this application) any specifically excluded limit value belonging to the range. When the range includes one or two limit values, the range excluding one or both of those included limit values ​​is also included in this application.

[0069] As used herein, the terms “polypeptide,” “peptide,” and “protein” refer to polymers of amino acids of any length. The term also includes modified amino acid polymers, for example, those modified to include disulfide bond formation, glycosylation, esterification, phosphorylation, or conjugation with labeled components.

[0070] As used herein, the terms “natural” or “wild-type” refer to nucleotide sequences, such as genes, or gene products, such as RNA or polypeptides, that are present in wild-type cells, tissues, organs, or organisms. The term “variant” as used herein refers to a mutant of a reference polynucleotide or polypeptide sequence (e.g., a natural polynucleotide or polypeptide sequence), i.e., one that has less than 100% sequence identity with the reference polynucleotide or polypeptide sequence.

[0071] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to any subject requiring treatment or therapy. The subject may be a mammalian subject. Mammal subjects include, for example, humans, non-human primates, rodents (e.g., rats, mice), rabbits (e.g., rabbits), ungulates (e.g., cattle, sheep, pigs, horses, goats, etc.), etc. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus monkey. In some embodiments, the subject is a companion or service animal (e.g., a cat or dog).

[0072] As used herein, the terms “treatment” or “treatment” generally refer to the use of a therapeutic agent to achieve a desired pharmacological and / or physiological effect. The term treatment or treatment may include treatment of a single cell, multiple cells, tissue, or subject. This effect may be preventative, i.e., complete or partial prevention of a disease or its symptoms, such as reducing the likelihood of the disease or its symptoms occurring in a subject, and / or therapeutic, i.e., complete or partial relief of symptoms, or partial or complete cure of the disease and / or side effects caused by the disease. As used herein, “treatment” includes any treatment of mammalian diseases, including: (a) preventing the occurrence of said disease in subjects who may be susceptible to said disease but have not yet been diagnosed with said disease; (b) inhibiting or slowing the onset or development of the disease; or (c) alleviating the disease, e.g., causing the disappearance of the disease or disease-related symptoms. The therapeutic agent may be administered before, during, or after the onset of the disease. For the treatment of an ongoing disease, particular attention may be warranted when such treatment stabilizes or reduces adverse clinical symptoms in the patient. In some embodiments, treatment is performed before complete loss of function in the affected tissue. In some implementations, personalized therapy is administered before the symptom stage of the disease; and in some implementations, personalized therapy is administered during the symptom stage of the disease; and in some implementations, personalized therapy is administered after the symptom stage of the disease.

[0073] It should be understood that this application is not limited to specific methods, products, apparatuses, and factors, as these methods, apparatuses, and formulations can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application, which is limited only by the appended claims.

[0074] II. Target Protein

[0075] This article provides compositions comprising RNA encoding a nucleocytoplasmic transport (NCT) repressor protein and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a target protein. This article also provides methods for improving target protein expression using RNA encoding an NCT repressor protein. Example target protein sequences that can be introduced into cells along with RNA encoding an NCT repressor protein include, but are not limited to, those in Tables 1 and 2.

[0076] In some embodiments, the target protein is an antibody; an antibody-like molecule; a receptor; a monoclonal antibody; an antibody fraction or fragment; a nanobody; a bispecific or multispecific antibody; or a bispecific or multispecific antibody-like molecule. In some embodiments, the antibody is adalimumab. In some embodiments, the antibody is abciximab, alemtuzumab, alirocumab, amivantamab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, or brentuximab. Vedotin, Brodalumab, Brolucizumab, Burosumab, Canakinumab, Caplacizumab, Capromab, Catuxomab, Cemiplimab, Certolizumab pegol, cetuximab, crizanlizumab, daclizumab, daratumumab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emapalumab, emicizumab, enfortumab vedotin, eptinezumab, errenumab, ertumaxomab, etaracizumab, evolocumab, Fremanezumab, Galcanezumab, gemtuzumab / oczomidinozogamicin, golimumab, guselkumab, ibazumab, tiuxetan, edarucizuma, imciromab, infliximab, Inotuzumab ozogamicin, ipilimumab, isatuximab, itolizumab, ixekizumab, lanadelumab, lokivetmab, mepolizumab, mogamulizumab, moxetumomab Pasudotox, Natalizumab, Necitumumab, Nimotuzumab, Nivolumab, Obiltoxaximab, Obinutuzumab, Ocrelizumab, Ofatumab, Olaratumab, Omalizumab, Palivizumab, Panitumab, Pembrolizumab, Pertuzumab, Polatuzumab vedotin, Racotumomab, Ramucirumab, Ranibizumab, Raxibacumab, Ravulizumab, Reslizumab, Risankizumab, Rituximab, Rmab, Romosozumab, Rovelizumab, Ruplizumab, SacituzumabThe following are listed: govitecan, Sarilumab, Secukinumab, Siltuximab, Talquetamab, Teclistamab, Teprotumumab, Tildrakizumab, Tocilizumab, Tositumomab, Trastuzumab, Trastuzumab duocarmazine, Trastuzumab emtansine, Ustekinumab, and Vedolizumab. The peptide sequences of such antibodies are publicly available—for example, the Thera-SAbDab database (opig.stats.ox.ac.uk), as described in Raybould et al. (2020) Thera-SAbDab: the Therapeutic Structural Antibody Database. Nucleic Acids Res. 48(D1): gkz827.

[0077] In some embodiments, the target protein is a bispecific or multispecific antibody; or a bispecific or multispecific antibody-like molecule. In some embodiments, the bispecific antibody is Blinatumomab and Emicizumab. In some embodiments, the target protein is a bispecific T-cell adaptor (BiTE), such as Blinatumomab (MT103) and Solitomab. In some embodiments, the target protein is a protein framework-based binding ligand or conjugate (e.g., adnectin, antiicalin, avimer, fynomer, Kunitz domain, Knottin, affibody, or DARPin).

[0078] In some implementations, the target protein is a blood protein. Non-limiting examples of the blood proteins mentioned include transferrin, t-PA, hirudin, C1 esterase inhibitors, antithrombin, plasma-type kallikrein inhibitors, plasmin, prothrombin complex, complement components, prealbumin (transthyretin), α1-antitrypsin, α-1-acid glycoprotein, α-1-fetoprotein, α2-macroglobulin, gamma globulin, β-2-microglobulin, haptoglobulin, ceruloplasmin, complement component 3, complement component 4, C-reactive protein (CRP), lipoproteins (chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL)), transferrin, prothrombin, mannose-binding lectin (MBL), albumin, globulin, fibrinogen, regulatory factors, and coagulation factors, such as coagulation factors I, II, III, IV, V, VI, VII, IX, X, XI, XII, XIII, and von Willebrand factor. Willeband factor, prokallikrein, Fitzgerald factor, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-associated protease inhibitor, plasminogen, α2-antiplasmin, tissue plasminogen activator, urokinase, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and cancer-procoagulant substances. In some embodiments, the target protein is a thrombolytic agent. Non-limiting examples of the thrombolytic agent include eminase, retavase, streptase (streptokinase, kabikinase), alteplase, t-PA (including activase-based drugs), TNKase, abokinase, and kinlytic (prokinase).

[0079] In some embodiments, the target protein is a growth factor. Non-limiting examples of the growth factors include: erythropoietin (EPO), insulin-like growth factor-1 (IGF-1), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-GCF), bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), adrenomedullin (AM), angiopoietin (Ang), autocrine motor factors, bone morphogenetic protein (BMP), and the ciliary neurotrophic factor family. Ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), colony-stimulating factor, macrophage colony-stimulating factor (M-CSF), epidermal growth factor (EGF), hepatocyte glycoproteins—hepatocyte glycoprotein A1, hepatocyte glycoprotein A2, hepatocyte glycoprotein A3, hepatocyte glycoprotein A4, hepatocyte glycoprotein A5, hepatocyte glycoprotein B1, hepatocyte glycoprotein B2, hepatocyte glycoprotein B3, fibroblast growth factor (FGF) 1, FGF 2, FGF 3, FGF 4, FGF 5, FGF 6, FGF 7, FGF 8, FGF 9, FGF 10, FGF 11. Each of FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23; fetal bovine growth hormone (FBS); GDNF family ligands; glial cell-derived neurotrophic factor (GDNF); neuroturin; persephin; artemin; growth differentiation factor-9 (GDF9); hepatocyte growth factor (HGF); hepatocellular carcinoma-derived growth factor (HDGF); insulin; insulin-like growth factors: insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2); interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7; keratinocyte growth factor (KGF); migration-stimulating factor (MSF); macrophage-stimulating protein (MSP), also known as hepatocyte growth factor-like protein (HGFLP).The target proteins include myostatin (GDF-8), neurotrophic factor 1 (NRG1), neurotrophic factor 2 (NRG2), neurotrophic factor 3 (NRG3), neurotrophic factor 4 (NRG4), neurotrophic factor, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophic factor-3 (NT-3), neurotrophic factor-4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), reninase (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and the Wnt signaling pathway. In some embodiments, the target proteins are hormones. Non-restricted examples of hormones include glucagon-like peptide-1, insulin, human growth hormone, follicle-stimulating hormone, calcitonin, luteinizing hormone, glucagon-like peptide-2, leptin, parathyroid hormone, human chorionic gonadotropin, thyroid-stimulating hormone, erythropoietin, and glucagon.

[0080] In some embodiments, the target protein is an enzyme. Non-limiting examples of the enzyme include α-glucosidase, glucocerebrosidase, iduronate-2-sulfatase, α-galactosidase, uricase, N-acetylgalactosidase, carboxypeptidase, hyaluronidase, DNase, asparaginase, uricase, adenosine deaminase and other enterokinases, cyclases, caspases, cathepsins, oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Target proteins expressed using the compositions and methods described herein may include proteins associated with enzyme substitutes, such as agalsidase beta, agalsidase alfa, imiglucerase, taligulcerase alfa, velaglucerase alfa, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, C3 inhibitors, glucose-6-phosphatase catalytic subunit 1, phenylalanine hydroxylase, Hurler, and Hunter corrector factors. In some embodiments, the compositions and methods described herein are used for enzyme production. The enzymes may be used to produce clinical diagnostic kits or other diagnostic assays.

[0081] In some embodiments, the target protein is a membrane protein. Exemplary membrane proteins include ion channels, gap junctions, ion channel receptors, transport proteins, integrated membrane proteins (e.g., cell surface receptors), and proteins that shuttle between the membrane and cytoplasm in response to signal transduction. In some embodiments, the cell surface receptor is a G protein-coupled receptor (GPCR), a tyrosine kinase receptor, an integrin, etc. In some embodiments, the cell surface receptor is a G protein-coupled receptor. In some embodiments, the target protein is a seven-transmembrane domain receptor (i.e., a 7-transmembrane (7-TM) receptor), a heptaspiral receptor, a serpentine receptor, or a G protein-linked receptor (GPLR). In some embodiments, the target protein is a class A, class B, class C, class D, class E, or class F GPCR. In some embodiments, the target protein is a class 1, class 2, class 3, class 4, class 5, or class 6 GPCR. In some embodiments, the target protein is a rhodopsin-like GPCR, a secretin receptor family GPCR, a metabolite glutamate / pheromone GPCR, a fungal mating pheromone receptor, a cyclic AMP receptor, or a coiled / smooth GPCR. In some embodiments, the cell surface receptor is an IL-1 receptor, IL-1Ra, a tumor necrosis factor receptor (TNFR), or a vascular endothelial growth factor receptor (VEGFR). In some embodiments, the target protein is a receptor mimic. In some embodiments, the target protein is a protein that shuttles between the membrane and cytoplasm in response to signal transduction, such as Ras protein, Rac protein, Raf protein, Gα subunit, repressor protein, Src protein, and other effector proteins.

[0082] In some embodiments, the target protein is a nucleoside enzyme, NAD+ nucleoside enzyme, hydrolase, glycosylation enzyme, glycosylation enzyme that hydrolyzes N-glycosyl compounds, NAD+ glycosyl hydrolase, NAD enzyme, DPN enzyme, DPN hydrolase, NAD hydrolase, pyridine diphosphate nucleoside enzyme, nicotinamide adenine dinucleotide nucleoside enzyme, NAD glycosyl hydrolase, NAD nucleoside enzyme, or nicotinamide adenine dinucleotide glycosyl hydrolase. In some embodiments, the target protein is an enzyme involved in nicotinic acid and nicotinamide metabolism and the calcium signaling pathway.

[0083] In some embodiments, the target protein is selected from the group consisting of: abatacept, aflibercept, agalsidase beta, albiglutide, aldesleukin, alefacept, alglucerase, alglucosidase alfa, aliskiren, and alpha-1 proteinase inhibitors. Inhibitors, Alteplase, Anakinra, Ancestim, Anistreplase, Human anthrax immunoglobulin, Antihemophilic factor, Antithrombin Alpha, Human antithrombin III, Antithymocyte globulin, Antithymocyte globulin (horse), Antithymocyte globulin (rabbit), Aprotinin, Arcitumomab, Asfotase Alfa, Asparaginase, Erwinia chrysogena asparaginase Chrysanthemi), Becaplermin, Belatacept, Beractant, Bivalirudin, Botulinum toxin type A, Botulinum toxin type B, Buserelin, C1 esterase inhibitor (human), C1 esterase inhibitor, Human chorionic gonadotropin alpha, Human chorionic gonadotropin, Human chorionic gonadotropin, Coagulation factor IX, Coagulation factor VIIa, Human coagulation factor X, Coagulation factor XIII A subunit, Collagenase, Conestalfa, Corticotropin, Cosyntropin, Daptomycin, Darbepoetin alfa, Defibrotide, Denileukin diftitox, Desirudin, Dornase alfa, Drotrecogin alfa, Dulaglutide, Efalizumab, Efmoroctocog alfa, Elosulfase alfaalfa, Enfuvirtide, Epoetin alfa, Epoetin zeta, Eptifibatide, Etanercept, Exenatide, Factor IX complex (human), Fibrinogen concentrate (human), Fibrinolysin (aka plasmin), Filgrastim, Filgrastim-sndz, Follicle-stimulating hormone α, Follicle-stimulating hormone β, Galsulfase, Gastric intrinsic factor, Glatiramer acetate, Glucagon recombinant, Glucarpidase, Gramicidin D D), Hepatitis A vaccine, Hepatitis B immunoglobulin, Human calcitonin, Human tetanus toxoid immunoglobulin, Human rabies virus immunoglobulin, Human Rho(D) immunoglobulin, Human serum albumin, Human varicella-zoster immunoglobulin, Hyaluronidase, Hyaluronidase, Ibritumomab, Idursulfase, Imiglucerase, Human immunoglobulin, Infliximab, Insulin aspart, Insulin Beef, Insulin Degludec, Insulin detemir, Insulin glargine, Insulin glulisine, Insulin Lispro, Insulin Pork, Regular insulin, Regular insulin Regular insulin, porcine-derived insulin, isopranin insulin, recombinant interferon alpha-2a, interferon alpha-2b, and interferon alfacon-1.Alfacon-1, Interferon α-n1, Interferon α-n9, Interferon β-1a, Interferon β-1b, Interferon γ-1b, Intravenous Immunoglobulin, Ipilimumab, Ixekizumab, Laronidase, Lenograstim, Lepirudin, Leuprolide, Liraglutide, Lucinactant, Lutropinalfa, Lutropin alfa, Mecasermin, Menotropins, Epoetin beta, Metreleptin, Muromonab, Alpha Interferon interferon, Nesiritide, Ocriplasmin, Omalizumab, Oprelvekin, OspA lipoprotein, Oxytocin, Palifermin, Pancrelipase, Poractantalfa, Pramlintide, Preotact, Protein S human, Rasburicase, Reteplase, Rilonacept, Rituximab, Romiplostim, Sacrosidase, Salmon Calcitonin, Sargramostim, Satumomab Pendetide, Sebelipase Alfa, Secretin, Secukinumab, Sermorelin, Serum albumin, Serum albumin iodonated, Simococog Alfa, Sipuleucel-T, Somatotropin Recombinant, Somatotropinrecombinant, streptokinase, sulodexide, susoctocog alfa, taliglucerase alfa, teduglutide, teicoplanin, tenecteplase, teriparatide, tesamorelin, thrombomodulin alfa, thymalfasin, thyroglobulin, thyrotropin alfa, thyrotropin alfa, tocilizumab, tositumomab, tuberculin purified protein derivative, turoctocog alfa, urofollitropin, urokinase, vasopressin, and velaglucerase alfa.

[0084] In some embodiments, the target protein is a biosimilar. In some embodiments, the target protein is a therapeutic polypeptide, such as a biopharmaceutical drug, also known as a biologic; a biomarker-enabling polypeptide, such as a diagnostic, prognostic, or predictive biomarker; a prophylactic polypeptide, such as an adjuvant, a soluble antigen, a subviral particle, or a virus-like particle; a helper polypeptide, such as a polypeptide that supports the activity of another molecule or binds to or inhibits another protein-protein interaction; or a polypeptide for research, such as an antigen, a reporter protein, or a tool polypeptide for studying physiological or pathological processes in animal models and the effects of drugs on these processes. In some embodiments, the target protein is a protein that can be used for microscopy and imaging, such as a fluorescent protein. In some embodiments, the target protein is not a reporter protein, such as luciferase. In some embodiments, the target protein is a human protein.

[0085] In some embodiments, the target protein is an immunomodulator. Non-limiting examples of immunomodulators include cytokines, chemokines, interleukins, and interferons. In some embodiments, the target protein is an antigen used as a vaccine or for research. In some embodiments, the target protein is a structural protein, such as a structural protein that plays a role in the assembly of protein complexes. In some embodiments, the target protein is an antimicrobial peptide; or an antiviral peptide. In some embodiments, the target protein is a tumor suppressor. In some embodiments, the target protein is a transcription factor or translation factor. In some embodiments, the target protein is a pharmacokinetic regulator, a small molecule binding protein, an RNA-binding protein, or a protein that binds to other proteins.

[0086] In some embodiments, the target protein is dopamine receptor 1 (DRD1), cystic fibrosis transmembrane transport regulator (CFTR), C1 esterase inhibitor (C1-Inh), IL2-induced T-cell kinase (ITK), or NADase. In some embodiments, the target protein is firefly luciferase.

[0087] In some embodiments, the target protein is an antigen of a pathogenic organism (e.g., a virus or bacteria). In some embodiments, the target protein is a bacterial antigen—a protein or glycoprotein antigen. In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is derived from a virus selected from the group consisting of coronaviruses, influenza viruses, hepatitis B viruses, human papillomavirus (HPV), West Nile virus, and human immunodeficiency virus (HIV). In some embodiments, the viral protein is derived from a coronavirus. In some embodiments, the coronavirus is a beta-coronavirus. In some embodiments, the beta-coronavirus is a severe acute respiratory syndrome (SARS) virus. In some embodiments, the SARS virus is a SARS-CoV-2 virus. In some embodiments, the beta-coronavirus is a Middle East respiratory syndrome (MERS) virus.

[0088] Example target protein sequence

[0089] In some embodiments, RNA encoding an NCT inhibitory protein (e.g., L protein) improves the expression of a target protein, wherein the target protein is an antibody selected from the group consisting of the following antibodies (see also Table 1): adalimumab, pembrolizumab, nivolumab, trastuzumab, bevacizumab, ustekinumab, and ozogluconate. The RNAs listed in Table 1 of this disclosure are: zumab, secukinumab, vedolizumab, ibalizumab, nirsevimab, atoltivimab, maftivimab, odesivimab, casivimab, imdevimab, and brolucizumab. In some embodiments, one or more sequences in Table 1 of this disclosure, or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with them.

[0090] Table 1: Example Antibody Target Proteins

[0091]

[0092]

[0093] In some embodiments, introducing RNA encoding an NCT inhibitory protein (e.g., L protein) can improve the expression of target proteins, wherein the target proteins are selected from blood proteins or immuno-oncology proteins including the following (see also Table 2): rFIX-Fc coagulation factor IX, taliglucerase, agalsidase beta, alglucosidase alfa, laronidase, idursulfase, HLA class I α chain (mouse K2-D1) and B2m (mouse), Nlrc5 (mouse), NLRC5 (human), scIL-12 (mouse), scIL-12 (human), and HLA class I α chain (human) and B2M (human). In some embodiments, one or more sequences in the RNA coding table 2 of this disclosure, or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0094] Table 2: Exemplary target proteins of hematologic and immuno-oncology

[0095]

[0096]

[0097] In some embodiments, RNA encoding an NCT repressor protein (e.g., L protein) improves the expression of a target protein, wherein the target protein is an antigen. In some embodiments, the target protein is a bacterial antigen—a protein or glycoprotein antigen. In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen protein is a viral antigen protein derived from a virus selected from the group consisting of coronaviruses, influenza viruses, hepatitis B viruses, human papillomavirus (HPV), West Nile virus, and human immunodeficiency virus (HIV). In some embodiments, the viral protein is derived from a coronavirus. In some embodiments, the coronavirus is a beta-coronavirus. In some embodiments, the beta-coronavirus is a severe acute respiratory syndrome (SARS) virus. In some embodiments, the SARS virus is a SARS-CoV-2 virus. In some embodiments, the beta-coronavirus is a Middle East respiratory syndrome (MERS) virus.

[0098] In some embodiments, the viral protein is derived from West Nile virus. In some embodiments, the viral protein is a precursor membrane protein (preM), an envelope glycoprotein (E), or a combination thereof.

[0099] In some embodiments, the viral antigen comprises one or more proteins derived from viruses listed in Table 3 below. In some embodiments, the RNA encoding one or more sequences in Table 3 of this disclosure, or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with them.

[0100] Table 3: Exemplary viral antigen proteins

[0101]

[0102]

[0103]

[0104] III. Nucleocytoplasmic Transport (NCT) Inhibitor Protein

[0105] This document provides compositions comprising RNA encoding a nucleocytoplasmic transport (NCT) repressor protein and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a target protein. This document also provides methods for improving the expression of said target protein using RNA encoding an NCT repressor protein. Example NCT repressor proteins (e.g., L proteins) include, but are not limited to, those listed in Table 4, or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with them.

[0106] In some embodiments, the NCT inhibitory protein is a nuclear pore-blocking viral protein. In some embodiments, the NCT inhibitory protein is a native or synthetic peptide capable of blocking nuclear pores to inhibit nucleocytoplasmic transport (“NCT”). In some embodiments, the NCT inhibitory protein is a viral protein. In some embodiments, the NCT inhibitory protein is selected from the group consisting of piconevirus leader (L) protein, piconevirus 2A protease, rhinovirus 3C protease, coronavirus ORF6 protein, Ebola virus VP24 protein, Venezuelan equine encephalitis virus (VEEV) capsid protein, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0107] The NCT inhibitory protein may be a functional variant of any protein disclosed herein. As used herein, the term "functional variant" refers to a protein that is homologous to and / or substantially sequence similar to the original protein (e.g., having an amino acid sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) and shares one or more functional features of the original protein. For example, a functional variant of the NCT inhibitory protein retains the ability to inhibit NCT.

[0108] In some embodiments, the NCT inhibitory protein is a leader (L) protein derived from a piconese virus or a functional variant thereof. In some embodiments, the NCT inhibitory protein is a leader protein derived from a cardiovirus, hepatovirus, or aphthovirus. For example, the NCT inhibitory protein may be derived from Bovine rhinitis A virus, Bovine rhinitis B virus, Equine rhinitis A virus, Foot-and-mouth disease virus, Hepatovirus A, Hepatovirus B, Marmota himalayana hepatovirus, Phopivirus, Cardiovirus A, Cardiovirus B, Theiler's Murineencephalomyelitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), Theiler-like rat virus (TRV), or Saffold virus (SAF-V).

[0109] In some embodiments, the NCT inhibitory protein is the L protein of Theerevirus or a functional variant thereof. In some embodiments, the L protein comprises SEQ ID NO:1. In some embodiments, the NCT inhibitory protein may comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1.

[0110] In some embodiments, the L protein is the L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof. In some embodiments, the L protein comprises SEQ ID NO:2, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0111] In some embodiments, the L protein is selected from the L protein of poliovirus, the L protein of HRV16, the L protein of mengo virus, and the L protein of Saffold virus 2, or functional variants thereof.

[0112] In some embodiments, the NCT inhibitory protein is a piconetic viral 2A protease or a functional variant thereof. In some embodiments, the NCT inhibitory protein is a 2A protease derived from enteroviruses, rhinoviruses, foot-and-mouth disease viruses (Aphtoviruses), or cardiviruses.

[0113] In some embodiments, the NCT inhibitory protein is a rhinovirus 3C protease or a functional variant thereof. In some embodiments, the NCT inhibitory protein is a picornavirus 3C protease. In some embodiments, the NCT inhibitory protein is a 3C protease derived from enteroviruses, rhinoviruses, foot-and-mouth disease viruses, or cardiviruses. For example, in some non-limiting embodiments, the NCT inhibitory protein is a 3C protease derived from poliovirus, Coxsackievirus, rhinovirus, foot-and-mouth disease virus, or hepatitis A virus.

[0114] In some embodiments, the NCT inhibitory protein is a coronavirus ORF6 protein or a functional variant thereof. In some embodiments, the NCT inhibitory protein is a viral protein that disrupts the formation of the nuclear import complex and / or disrupts the transport of STAT1 to the nucleus.

[0115] In some embodiments, the NCT inhibitory protein is the Ebola virus VP24 protein or a functional variant thereof. In some embodiments, the NCT inhibitory protein is the Ebola virus VP40 protein or VP35 protein. In some embodiments, the NCT inhibitory protein is a viral protein that binds to the importer nucleotransfer protein-α (KPNA). In some embodiments, the NCT inhibitory protein is a viral protein that inhibits the binding of STAT1 to KPNA.

[0116] In some embodiments, the NCT inhibitory protein is a Venezuelan equine encephalitis virus (VEEV) capsid protein or a functional variant thereof. In some embodiments, the NCT inhibitory protein is a viral capsid protein that interacts with the nuclear pore complex.

[0117] In some embodiments, the NCT inhibitory protein is the herpes simplex virus (HSV) ICP27 protein or a functional variant thereof. In some embodiments, the NCT inhibitory protein is the HSV ORF57 protein.

[0118] In some embodiments, the NCT inhibitory protein is a rhabdovirus matrix (M) protein or a functional variant thereof. In some embodiments, the NCT inhibitory protein is an M protein derived from the genera Cytorhabdovirus, Dichorhavirus, Ephemerovirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Sprivivirus, Tibrovirus, Tupavirus, Varicosavirus, or Vesiculovirus.

[0119] In some embodiments, the NCT inhibitory protein may have an amino acid sequence comprising one or more of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with it.

[0120] Table 4: Exemplary NCT ​​Inhibitor Proteins

[0121]

[0122]

[0123]

[0124] In some embodiments, the NCT inhibitor, such as the L protein, comprises: the sequence of SEQ ID NO:161, a mutated sequence of any one of SEQ ID NO:163, 164, 166 or 168, or a sequence having about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with it.

[0125] In some implementations, the expression of the NCT inhibitor protein increases the expression level of the target protein by approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, or 1000-fold.

[0126] In some implementations, the expression of the NCT inhibitor protein reduces the expression level of the target protein by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90%.

[0127] In some implementations, expression of the NCT inhibitor protein increases the activity of the target protein by approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, or 300-fold.

[0128] In some implementations, the expression of the NCT inhibitor protein causes the target protein's EC... 50 The reduction is approximately 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, or 300 times.

[0129] In some implementations, the NCT inhibitory protein and target protein are expressed in cells or subjects.

[0130] In some implementations, expression of the NCT inhibitor protein increases the duration of the target protein in cells or subjects by approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.

[0131] In some implementations, expression of the NCT inhibitor protein reduces the coefficient of variation (CV%) of the target protein in the subject's tissue by approximately 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.7-fold, 2.8-fold, 2.9-fold, or 3-fold.

[0132] In some embodiments, the expression of the NCT inhibitor protein reduces cellular signaling in response to stressors. In some embodiments, the cellular signaling in response to stressors is the expression of interferon-β. In some embodiments, the introduction of the NCT inhibitor reduces interferon-β expression in cells by approximately 10-fold, approximately 100-fold, approximately 1000-fold, approximately 10000-fold, or approximately 100000-fold.

[0133] In some embodiments, the NCT repressor protein improves target protein expression characteristics, including but not limited to: yield, quality, folding, post-translational modifications, activity, localization, and downstream activity. In some embodiments, the NCT repressor protein can reduce one or more of misfolding, altered activity, incorrect post-translational modifications, and / or toxicity. In some embodiments, expression of the NCT repressor protein increases the duration, quality, and / or quantity of the target protein.

[0134] In some implementations, RNA encoding an NCT inhibitor protein reduces interferon sensitivity and inflammation associated with the expression of the target protein.

[0135] This application also considers that antibodies against NCT proteins and short interfering RNAs (siRNAs) for NCT proteins can improve the expression of target proteins. For example, mRNAs encoding anti-Xpo7 nanobodies, anti-NFT2 antibodies, RanQ69L mutant proteins, MxB K131A mutant proteins, myristylated ERK-derived phosphomimicrystic peptides, myristylated SMAD-derived phosphomimicrystic peptides, siRNAs targeting Ran mRNA, NTF2 mRNA, MxB mRNA, Xpo7 mRNA, or Nup93 mRNA can be used. For example, NCT inhibitory siRNAs are commercially available as chemically synthesized sequences (IDT Technologies).

[0136] IV. RNA Composition

[0137] This document provides compositions comprising RNA encoding a nucleocytoplasmic transport (NCT) repressor protein and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a target protein. In some embodiments, the RNA encoding the NCT repressor protein and the RNA encoding the target protein are messenger RNAs. In some embodiments, the RNA encoding the NCT repressor protein and the RNA encoding the target protein are self-amplifying RNAs. In some embodiments, the RNA encoding the NCT repressor protein is a messenger RNA, and the RNA encoding the target protein is a self-amplifying RNA. In some embodiments, the RNA encoding the NCT repressor protein is a self-amplifying RNA, and the RNA encoding the target protein is a messenger RNA.

[0138] In some embodiments, the compositions of this disclosure comprise messenger RNA encoding an NCT repressor protein and a target protein. In some embodiments, the compositions comprise a first messenger RNA encoding an NCT repressor protein and a second messenger RNA encoding a target protein.

[0139] In some embodiments, the RNA disclosed herein, for example, saRNA or mRNA encoding an NCT inhibitor, is delivered to cells simultaneously or on the same day as saRNA, mRNA, or DNA encoding a target protein. In some embodiments, the RNA disclosed herein, for example, saRNA or mRNA encoding an NCT inhibitor, is delivered to cells separately from saRNA, mRNA, or DNA encoding a target protein or on a different day.

[0140] In some embodiments, RNA, such as saRNA or mRNA encoding an NCT inhibitor, is delivered to the cell before saRNA, mRNA, or DNA encoding the target protein. In some embodiments, RNA (e.g., saRNA or mRNA encoding an NCT inhibitor) is delivered to the cell approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 10 hours, approximately 14 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 1 week, approximately 2 weeks, or approximately 3 weeks before the saRNA, mRNA, or DNA encoding the target protein is delivered to the cell.

[0141] In some embodiments, RNA, such as saRNA or mRNA encoding an NCT inhibitor, is delivered to the cell after saRNA, mRNA, or DNA encoding the target protein. In some embodiments, RNA (e.g., saRNA or mRNA encoding an NCT inhibitor) is delivered to the cell approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 10 hours, approximately 14 hours, 1 day, approximately 2 days, approximately 3 days, approximately 4 days, or approximately 5 days, approximately 1 week, approximately 2 weeks, or approximately 3 weeks after the saRNA, mRNA, or DNA encoding the target protein is delivered to the cell.

[0142] In some embodiments, one or more saRNA vectors are used to express the L protein and / or the target gene (GOI). Any saRNA vector known in the art can be used, including alpha virus- and flavivirus-based saRNA vectors, or any novel saRNA vector. Non-exhaustive examples include saRNA vectors derived from Venezuelan equine encephalitis virus (VEEV), Semliki forest virus (SFV), Sindbis virus (SINV), VEE-SINV chimera, tick-borne encephalitis virus (TBEV), classical swinefever virus (CSFV), and the like. saRNA expression vectors can be designed as cis replicons (where GOI, L protein, and saRNA replicase cassette are part of the same RNA sequence as the saRNA), trans replicons (where at least one RNA molecule containing GOI and / or L protein does not contain the saRNA replicase cassette, and the saRNA replicase cassette is provided on a different saRNA molecule), or combinations of partial saRNA replicons (where at least one saRNA component, such as a replicase cassette, a component of GOI and / or L protein, is provided as a replication-defective RNA, such as as regular mRNA). Figures 30A-30H Illustrations of selected cis, trans, and partial saRNA replicons are provided.

[0143] Exemplary NCT ​​inhibitor mRNA sequence

[0144] In some embodiments, the messenger RNA (mRNA) encoding a wild-type NCT inhibitor (e.g., L protein) is encoded by SEQ ID NO:160 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0145] In some embodiments, the messenger RNA (mRNA) encoding a mutant NCT inhibitor (e.g., an L protein with an E39A mutation) is encoded by SEQ ID NO:162, or by a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0146] In some embodiments, the messenger RNA (mRNA) encoding a mutant NCT inhibitor (e.g., an L protein with a K35Q mutation) is encoded by SEQ ID NO:164, or by a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0147] In some embodiments, the messenger RNA (mRNA) encoding a mutant NCT inhibitor (e.g., an L protein with a Y36F mutation) is encoded by SEQ ID NO:166, or by a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0148] In some embodiments, the messenger RNA (mRNA) encoding a mutant NCT inhibitor (e.g., an L protein with a D37A mutation) is encoded by SEQ ID NO:168, or by a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0149] In some implementations, the messenger RNA (mRNA) encoding an NCT inhibitor (e.g., L protein) comprises a UTR sequence encoded by the sequence of SEQ ID NO:174 and / or SEQ ID NO:175.

[0150] In some implementations, the messenger RNA (mRNA) encoding an NCT inhibitor (e.g., L protein) is encoded by the sequence SEQ ID NO:177.

[0151] Exemplary target protein mRNA sequence

[0152] In some embodiments, the messenger RNA (mRNA) of this disclosure is expressed by the following expression vectors... Figure 18A and Figure 18B The sequence sequence represented in the diagram encodes the mRNA, or is encoded by a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the mRNA of this disclosure is encoded by a sequence of an expression vector having a different coding sequence, such as SERPINA1 (SEQ ID NO: 104) or other coding sequences, or having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it:

[0153] hBG 5'UTR sequence: (SEQ ID NO:98)

[0154] G6PC1 encoded sequence: (SEQ ID NO:99),

[0155] Connector sequence: (SEQ ID NO:100)

[0156] NanoLuc reporter gene coding sequence: (SEQ ID NO:101), and

[0157] hBg 3'UTR sequence: (SEQ ID NO:102).

[0158] Exemplary saRNA sequence

[0159] In some embodiments, the self-amplified RNA (saRNA) of this disclosure is encoded by an expression vector having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the target gene (GOI) sequence. In some embodiments, the saRNA of this disclosure is encoded by an expression vector having a different target gene (GOI) sequence, according to... Figure 30A The arrangement shown encodes a Venezuelan equine encephalitis virus (VEEV) replicon, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the saRNA of this disclosure is encoded by an expression vector having a different target gene (GOI) sequence, according to... Figure 30A The arrangement shown is a replicon based on Venezuelan equine encephalitis virus (VEEV), or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it:

[0160] 5'CSE: (SEQ ID NO:79),

[0161] NSP1-4 and SGP box: (SEQ ID NO:80)

[0162] GOI / antigen kit (e.g., firefly luciferase): (SEQ ID NO:81)

[0163] Internal ribosome entry site (IRES): (SEQ ID NO:82)

[0164] EG Tech L protein coding sequence: (SEQ ID NO:83), and

[0165] 3'CSE and PolyA: (SEQ ID NO:84).

[0166] In some embodiments, the saRNA of this disclosure is encoded by an expression vector having a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the target gene (GOI) sequence. In some embodiments, the saRNA of this disclosure is encoded by an expression vector having a different target gene (GOI) sequence, according to... Figure 30A The arrangement shown is a replicon of Semliki Forest Virus (SFV), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it:

[0167] 5'CSE: (SEQ ID NO:85),

[0168] NSP and SGP boxes: (SEQ ID NO:86)

[0169] GOI / antigen kit (e.g., mKate2): (SEQ ID NO:87)

[0170] Internal ribosome entry site (IRES): (SEQ ID NO:88)

[0171] EG Tech L protein coding sequence: (SEQ ID NO:89)

[0172] Part A of SFV 26S RNA: (SEQ ID NO:90), and

[0173] SFV 26S RNA, part B, 3'CSE and polyA: (SEQ ID NO:91).

[0174] In some embodiments, the saRNA of this disclosure is encoded by an expression vector having a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the target gene (GOI) sequence. In some embodiments, the saRNA of this disclosure is encoded by an expression vector having a different target gene (GOI) sequence, according to... Figure 30A The arrangement shown represents a replicon of Venezuelan equine encephalitis virus (VEEV), or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it:

[0175] 5'CSE: (SEQ ID NO:92),

[0176] NSP1-4 and SGP box: (SEQ ID NO:93)

[0177] GOI / antigen kit (e.g., full-length influenza HA as antigen): (SEQ ID NO:94)

[0178] Internal ribosome entry site (IRES): (SEQ ID NO:95)

[0179] EG Tech L protein coding sequence: (SEQ ID NO:96), and

[0180] 3'CSE and PolyA: (SEQ ID NO:97).

[0181] RNA modification

[0182] The RNAs provided herein, such as messenger RNAs or self-amplifying RNAs for the expression of target proteins and NCT repressor proteins, can be modified as described herein. For example, RNA can be modified to reduce immunogenicity in cells or tissues. The RNAs provided herein may contain one or more untranslated regions (UTRs), a 5' cap, and a polyadenylated tail.

[0183] In some embodiments, RNA can be synthesized as unmodified or modified RNA. RNA can be modified to enhance stability and / or evade immunodetection and degradation. Modified RNA may include, for example, one or more nucleotide modifications, nucleoside modifications, backbone modifications, sugar modifications, and / or base modifications. In some embodiments, the modified nucleoside is pseudouridine or a pseudouridine analogue. In some embodiments, the pseudouridine analogue is N-1-methylpseudouridine.

[0184] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, 95%, 99%, or 100% of the uracil nucleoside in the RNA is depleted and replaced by a uracil nucleoside analog (e.g., pseudouridine, 5-methoxyuridine, or N-1-methyl-pseudouridine).

[0185] In some implementations, the RNA may contain RNA backbone modifications. Typically, backbone modifications are modifications in which the phosphate groups of the nucleotide backbone contained in the RNA are chemically modified. Example backbone modifications may include, but are not limited to, modifications in which the phosphodiester bond is replaced by a member selected from the group consisting of peptides, methylphosphonates, methylphosphamides, phosphoramides, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphate)), boron phosphates, and / or positively charged guanidinium groups, or other ways of replacing the phosphodiester bond.

[0186] hat structure

[0187] In some embodiments, the RNA may include a 5' cap structure. The 5' cap may contain, for example, a triphosphate bond and a 7-nitromethylated guanine nucleotide. In some embodiments, the 5' cap is 5'-7-methylguanosine (m7G). Various m7G cap analogs are known in the art, many of which are commercially available. These include the aforementioned m7GpppG, as well as ARCA 3'-OCH3 and 2'-OCH3 cap analogs (Jemielity, J. et al., RNA, 9:1108-1122 (2003)). Other cap analogs used in embodiments of the present invention include N7-benzylated dinucleotide tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10:1479-1487 (2004)), phosphate thiophosphate cap analogs (described in Grudzien-Nogalska, E. et al., RNA, 13:1745-1755 (2007)) and cap analogs (including biotinylated cap analogs), described in U.S. Patent Nos. 8,093,367 and 8,304,529, which are incorporated herein by reference.

[0188] In some embodiments, the 5' cap comprises or is composed of an internal ribosome entry site (IRES). In some embodiments, the IRES is located within the 5' UTR. In some embodiments, the 5' cap comprises or is composed of a 2A self-cleaving peptide (e.g., one or more of P2A, T2A, E2A, and F2A).

[0189] Tail structure

[0190] The presence of a "tail" can protect RNA from exonuclease degradation. The poly-A tail is considered to stabilize both natural messengers and synthetic sense RNA. Therefore, in some embodiments, a long poly-A tail can be added to the RNA molecule to make the RNA more stable. A variety of techniques recognized by those skilled in the art can be used to add the poly-A tail. For example, a long poly-A tail can be added to synthetic or in vitro transcribed RNA using a poly-A polymerase (Yokoe et al., Nature Biotechnology. 1996; 14:1252-1256). The transcription template can also encode the long poly-A tail. Alternatively, the poly-A tail can be added directly from the PCR product via transcription. The poly-A tail can also be ligated to the 3' end of sense RNA using an RNA ligase (see, for example, Molecular Cloning A Laboratory Manual, 2nd edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

[0191] In some embodiments, the RNA may include a 3' poly(A) tail structure. The length of the poly(A) tail may be at least about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 100, about 200, about 300, about 400, or about 500 nucleotides.

[0192] 5' and 3' Untranslated Regions (UTR)

[0193] In some embodiments, the RNA may include a 5' untranslated region (UTR) and / or a 3' UTR. In some embodiments, the 5' UTR may include one or more elements that affect RNA stability or translation. In some embodiments, the length of the 5' UTR may be about 50 to about 100 nucleotides, or about 50 to about 500 nucleotides. In some embodiments, the 3' UTR includes one or more polyadenylated tail signals, binding sites for proteins that may affect RNA stability or localization, or one or more binding sites for miRNAs. In some embodiments, the length of the 3' UTR may be about 0 to about 50 nucleotides, or about 50 to about 100 nucleotides.

[0194] Examples of 3' and 5' UTR sequences may be derived from RNAs with relatively long half-lives (e.g., globin, actin, GAPDH, tubulin, histones, or citrate cycling enzymes) to increase the stability of the positive-sense RNA molecule. For example, the 5' UTR sequence may include a portion of the cytomegalovirus (CMV) early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve the half-life of the polynucleotide. Typically, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to its unmodified counterpart, and include modifications, for example, to improve resistance to in vivo nuclease digestion of such polynucleotides. In some embodiments, the UTR may improve tissue-specific expression. Example UTRs include the hBG 5' UTR (SEQ ID NO: 98) and the hBG 3' UTR (SEQ ID NO: 102).

[0195] DNA template for in vitro RNA production

[0196] The RNA disclosed herein, such as RNA encoding an NCT repressor protein or a target protein, can be produced by in vitro transcription (IVT) of one or more DNA templates having a polynucleotide sequence encoding the desired RNA. The DNA template may contain one or more promoters capable of transcription. In some embodiments, for example, the template may contain a T7 promoter configured to transcribe the nucleotide sequence encoding the target protein and / or the nucleotide sequence encoding the NCT repressor protein. Alternatively, the RNA can be prepared synthetically.

[0197] The template may contain a separatory element for the individual expression of a protein. In various embodiments, the template is a bicistronic or polycistronic template. The separatory element may be an internal ribosome entry site (IRES) or a 2A element. In some embodiments, the template may contain a nucleic acid encoding a 2A self-cleaving peptide. Exemplary 2A self-cleaving peptides include P2A, E2A, F2A, and T2A.

[0198] In some implementations, the first polynucleotide or the second polynucleotide or both are operatively linked to the internal ribosome entry site (IRES).

[0199] In some implementations, the first polynucleotide or the second polynucleotide or both are operatively linked to the 2A element.

[0200] In some implementations, a first polynucleotide or a second polynucleotide, or both, are operatively linked to a stem-loop structure that induces translation stop codon readout. Exemplary stem-loop structures are described in Napthine et al. RNA 18:241-52 (2012) and Houck-Loomis et al. Nature 480:561-64 (2011).

[0201] In some embodiments, the DNA template encoding the RNA disclosed herein may contain or encode according to Figure 1A-1E The sequence is any of the following sequences in the sequence shown, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it. In some embodiments, the DNA template encoding the RNA of this disclosure may contain or encode according to Figure 1A-1E The following sequences that are in any of the sequence sequences shown, or sequences that have at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequences shown, but encode a different target protein sequence:

[0202] T7 promoter SEQ ID NO:70,

[0203] 5'UTR SEQ ID NO:71,

[0204] 3'UTR SEQ ID NO:72,

[0205] Polyadenylate segment SEQ ID NO:73,

[0206] Nanoluciferase – Nucleotide target protein sequence SEQ ID NO:74,

[0207] Nanoluciferase – amino acid sequence SEQ ID NO:75,

[0208] SARS-CoV-2 spike protein – nucleotide target protein sequence SEQ ID NO:76,

[0209] SARS-CoV-2 spike protein – amino acid sequence SEQ ID NO:15,

[0210] GBA signal peptide – nucleotide target protein sequence SEQ ID NO:77, and

[0211] GBA mature protein – nucleotide target protein sequence SEQ ID NO:78.

[0212] V. DNA composition

[0213] Any DNA sequence encoding the target protein can be used with the methods and compositions described herein (i.e., the RNA encoding the NCT repressor protein).

[0214] In some implementations, the plasmid expression vector may contain, according to Figure 20A and Figure 20B The sequence shown is a sequence of the following, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it. In some embodiments, the plasmid expression vector may contain, according to... Figure 20A and Figure 20B The following sequences, or sequences having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown, but encoding different NCT repressor or target protein sequences:

[0215] GBA-NanoLuc / EPO Nano-Luc expression plasmid sequence, expression cassette:

[0216] CMV enhancer and promoter sequences: (SEQ ID NO:108)

[0217] Multiple cloning site (MCS) sequence: (SEQ ID NO:109),

[0218] GBA encoded sequence: (SEQ ID NO:110), or EPO encoded sequence: (SEQ ID NO:116),

[0219] Connector sequence: (SEQ ID NO:111),

[0220] Nano-luciferase reporter sequence: (SEQ ID NO:112), and

[0221] 3'UTR and SV40 poly(A) signal sequences: (SEQ ID NO:113).

[0222] VI. Delivery medium

[0223] In some embodiments, one or more RNA molecules and / or a delivery medium may be delivered to cells or tissues. In some embodiments, the delivery medium is a viral delivery medium. In some embodiments, the delivery medium is a non-viral delivery medium.

[0224] Non-viral delivery medium

[0225] In some embodiments, the delivery medium may be nanoparticles. In some embodiments, the delivery medium is lipid nanoparticles (LNPs), including but not limited to nanoparticles comprising lipids and / or polymers, liposomes, liposomal nanoparticles, cationic lipids, or exosomes. In other embodiments, the delivery medium may include calcium phosphate nucleotides, aptamers, cell-penetrating peptides, or other carrier tags.

[0226] In some embodiments, the nanoparticles are polymer nanoparticles. In some embodiments, the nanoparticles are metal nanoparticles.

[0227] In some embodiments, RNA can be delivered via lipid nanoparticles (LNPs). Example LNPs may comprise one or more different lipids and / or polymers. In some embodiments, LNPs comprise one or more ionizable or cationic lipids, neutral lipids, cholesterol, and / or stable lipids (e.g., PEGylated lipids).

[0228] In some embodiments, the LNP may comprise ionizable lipids. Ionizable lipids can refer to any of a variety of lipid species that have a net positive charge at a selected pH (e.g., physiological pH). The ionizable lipids may also refer to, for example, lipids in an ionized state, such as cationic lipids, such as one or more of the following lipids: 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyarginylglycine bis(octadecylamide) (DOGS), 2,3-dioleoyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propanium (D... OSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-distearate-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleoyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleoyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinoleoyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleoyl-N,N-dimethylammonium chloride (DODAC), or variants thereof.

[0229] In some implementations, LNPs may comprise neutral or zwitterionic lipids. In some embodiments, the neutral lipid is selected from one or more of the following: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or variants thereof.

[0230] In some embodiments, the LNP comprises PEGylated lipids, cholesterol, and one or more ionizable lipids. An example LNP formulation comprises approximately 1.5% DMG-PEG(2000), 38.5% cholesterol, 10% DSPC, and 50% DLin-MC3-DMA.

[0231] Lipid nanoparticles (LNPs) or liposomes carrying RNA can be produced, for example, by mixing lipids or polymers in an organic solvent (e.g., ethanol) with one or more RNAs in an aqueous buffer, followed by purification of the resulting nanoparticles by filtration. In some embodiments, the LNPs or liposomes can be produced using a microfluidic device to rapidly mix reagents and form size-controlled monodisperse particles. In some embodiments, the LNPs or liposomes can encapsulate RNA and / or bind to one or more RNAs via electrostatic interactions.

[0232] Virus delivery medium

[0233] In other embodiments, the delivery medium comprises a recombinant virus or virus-like particles, such as adenovirus, adeno-associated virus (AAV), herpesvirus, or retrovirus (e.g., lentivirus). In some embodiments, the delivery medium comprises a modified viral vector, such as an adenovirus dodecahedron or a recombinant adenovirus aggregate.

[0234] VII. Treatment Methods

[0235] The treatment methods described herein refer to treating a disease or condition in a subject of need by administering one or more RNAs to express a target protein and an NCT repressor protein (e.g., L protein). In some embodiments, the RNA encoding the NCT repressor protein is a messenger RNA. In some embodiments, the RNA encoding the NCT repressor protein is a self-amplifying RNA.

[0236] In some embodiments, the RNA or DNA provided herein for expressing the target protein, having RNA encoding an NCT inhibitory protein (e.g., L protein), encodes adalimumab protein, a CDR sequence, or a variable light or heavy chain sequence. In some embodiments, the RNA or DNA encoding adalimumab protein or its sequence and the RNA encoding the NCT inhibitory protein can treat one or more of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, hidradenitis suppurativa, uveitis, and juvenile idiopathic arthritis.

[0237] In some embodiments, the RNA of this disclosure is associated with an immune response and can be used as a vaccine. In some embodiments, the vaccine is used to treat or prevent disease or infection associated with a pathogen from which the antigen protein originates or with a related pathogen. In some embodiments, the antigen protein is a viral antigen protein. As described herein, the RNA or DNA of this disclosure encoding the target protein can encode any of the viral antigen proteins in Table 3 for administration together with RNA encoding an NCT inhibitory protein as a vaccine. In some embodiments, the RNA or DNA encoding the target protein of this disclosure provides a vaccine against coronaviruses, such as those against Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), or West Nile virus. In some embodiments, the RNA or DNA of this disclosure encoding the target protein provides a vaccine against influenza virus, hepatitis B virus, human papillomavirus (HPV), West Nile virus, or human immunodeficiency virus (HIV).

[0238] In some embodiments, RNA encoding the L protein (e.g., messenger RNA or self-amplifying RNA) is applied to the cells at a concentration of about 0.01 ng, about 0.05 ng, about 0.1 ng, about 0.15 ng, about 0.2 ng, about 0.3 ng, about 0.4 ng, about 0.5 ng, about 0.6 ng, about 0.7 ng, about 0.8 ng, about 0.9 ng, about 1.0 ng, about 1.1 ng, about 1.2 ng, about 1.3 ng, about 1.4 ng, about 1.5 ng, about 1.6 ng, about 1.7 ng, about 1.8 ng, or about 2.0 ng per well of a 96-well plate or its equivalent.

[0239] In some embodiments, the expression of the NCT inhibitory protein and the target protein increases the expression level of the target protein in cells or a subject. In some embodiments, the expression of the NCT inhibitory protein and the target protein increases the expression level of the target protein in cells or a subject by approximately 2-fold, approximately 3-fold, approximately 4-fold, approximately 5-fold, approximately 6-fold, approximately 7-fold, approximately 8-fold, approximately 9-fold, approximately 10-fold, approximately 20-fold, approximately 30-fold, approximately 40-fold, 50-fold, approximately 60-fold, approximately 70-fold, approximately 80-fold, approximately 90-fold, approximately 100-fold, approximately 200-fold, approximately 500-fold, or approximately 1000-fold.

[0240] In some embodiments, the RNA encoding the NCT inhibitor protein reduces the expression level of the target protein in cells or a subject. In some embodiments, the expression of the NCT inhibitor protein and the target protein reduces the expression level of the target protein in cells or a subject by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90%.

[0241] In some implementations, the RNA encoding the NCT inhibitor protein increases the expression level of the target protein in cells or subjects by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 200%, approximately 300%, approximately 400%, approximately 500%, or approximately 1000%.

[0242] In some embodiments of this disclosure, RNA encoding an NCT inhibitory protein increases the activity of the target protein in the subject's cells or in the subject. In some embodiments, the expression of the NCT inhibitory protein and the target protein increases the activity of the target protein in the subject's cells or in the subject by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold.

[0243] In some embodiments of this disclosure, the RNA encoding the NCT inhibitor protein increases the duration of the target protein's presence in the subject's cells or in the subject. In some embodiments, the RNA encoding the NCT inhibitor protein increases the duration of the target protein's presence in the subject's cells or in the subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold.

[0244] In some embodiments of this disclosure, RNA encoding an NCT inhibitor protein reduces the coefficient of variation (CV%) of the target protein in the tissues or in the subject. In some embodiments, RNA encoding an NCT inhibitor protein reduces the coefficient of variation (CV%) of the target protein in the tissues or in the subject by approximately 1.2-fold, approximately 1.3-fold, approximately 1.4-fold, approximately 1.5-fold, approximately 1.6-fold, approximately 1.7-fold, approximately 1.8-fold, approximately 1.9-fold, approximately 2-fold, approximately 2.1-fold, approximately 2.2-fold, approximately 2.3-fold, approximately 2.4-fold, approximately 2.5-fold, approximately 2.7-fold, approximately 2.8-fold, approximately 2.9-fold, or approximately 3-fold.

[0245] In some embodiments of this disclosure, the RNA encoding the NCT repressor protein reduces the degradation of the target protein. In some embodiments, the RNA encoding the NCT repressor protein reduces the degradation of the target protein by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold.

[0246] Route of administration

[0247] In some embodiments, one or more RNAs encoding an NCT inhibitor protein (e.g., L protein) can be delivered orally, subcutaneously, intravenously, intranasally, intradermally, percutaneously, intraperitoneally, intramuscularly, intrapulmonaryly, vaginally, rectally, or intraocularly. In exemplary embodiments, the RNA can be administered intramuscularly or subcutaneously.

[0248] VIII. Reagent Kit

[0249] This document also provides a kit containing RNA encoding the NCT repressor protein disclosed herein for improving the expression of the target protein. The kit may include, for example, suitable containers, such as vials or tubes, and instructions for use thereof.

[0250] In some embodiments, the kits of this disclosure include RNA encoding an NCT inhibitor, such as the L protein, comprising: the sequence of Table 4, the sequence of SEQ ID NO: 161, a mutated sequence of any one of SEQ ID NO: 163, 164, 166 or 168, or a sequence having about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with it.

[0251] In some embodiments, the kits of this disclosure comprise RNA encoding an NCT inhibitor or a delivery medium, said RNA encoding an NCT inhibitor being packaged in or together with a delivery medium (e.g., LNP). In some embodiments of this disclosure, the kit comprises RNA encoding an L protein (e.g., messenger RNA or self-amplifying RNA) present in vials or tubes in amounts of about 0.01 ng, about 0.05 ng, about 0.1 ng, about 0.15 ng, about 0.2 ng, about 0.3 ng, about 0.4 ng, about 0.5 ng, about 0.6 ng, about 0.7 ng, about 0.8 ng, about 0.9 ng, about 1.0 ng, about 1.1 ng, about 1.2 ng, about 1.3 ng, about 1.4 ng, about 1.5 ng, about 1.6 ng, about 1.7 ng, about 1.8 ng, or about 2.0 ng for use in each well of a 96-well plate or its equivalent.

[0252] List of implementation plans

[0253] Implementation Scheme I-1. A method for improving the expression of target proteins in cells or subjects, said method comprising administering one or more ribonucleic acid (RNA) encoding the target protein and an NCT inhibitor protein.

[0254] Implementation Scheme I-2. The method of Implementation Scheme I-1, wherein the NCT inhibitor protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the NCT inhibitor protein amino acid sequence in Table 4.

[0255] Implementation Scheme I-3. The method of Implementation Scheme I-1, wherein the NCT inhibitory protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the L protein sequence of SEQ ID NO:2.

[0256] Implementation Scheme I-4. The method of any one of Implementation Schemes I-1 to I-3, wherein the target protein is selected from any sequence of Tables 1, 2 and 3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with it.

[0257] Implementation Scheme I-5. The method of any one of Implementation Schemes I-1 to I-4, wherein the target protein is an antibody.

[0258] Implementation Scheme I-6. The method of Implementation Scheme I-5, wherein the target protein is adalimumab, wherein optionally, the target protein comprises: the adalimumab heavy chain amino acid sequence of SEQ ID NO:13 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith; and / or the adalimumab light chain amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith.

[0259] Implementation Scheme I-7. The method of any one of Implementation Schemes I-1 to I-4, wherein the target protein is an antigen of a pathogen, optionally, is the SARS-CoV-2 spike (S) protein, wherein optionally, the target protein comprises the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0260] Implementation Scheme I-8. The method of any one of Implementation Schemes I-1 to I-7, wherein the method comprises delivering one or more RNAs with lipid nanoparticles (LNPs).

[0261] Implementation Scheme I-9. The method described in Implementation Scheme I-8, wherein the LNP comprises PEGylated lipids, cholesterol and neutral lipids, and ionizable lipids.

[0262] Implementation Scheme I-10. The method of Implementation Scheme I-8, wherein the LNP comprises DMG-PEG (2000), cholesterol, DOPC and DLin-KC2-DMA in a ratio of about 1% to about 5% DMG-PEG (2000), about 30% to about 50% cholesterol, about 5% to about 15% DOPC and about 40% to about 60% DLin-KC2-DMA.

[0263] Implementation Scheme I-11. A method for treating a disease or condition in a subject in need, said method comprising administering to the subject one or more messenger ribonucleic acid (RNAs) encoding a target protein and an NCT inhibitor protein.

[0264] Implementation Scheme I-12. The method of Implementation Scheme I-11, wherein the NCT inhibitor protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the NCT inhibitor protein amino acid sequence in Table 4.

[0265] Implementation Scheme I-13. The method of Implementation Scheme I-11, wherein the NCT inhibitory protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the L protein sequence of SEQ ID NO:2.

[0266] Implementation Scheme I-14. The method of any one of Implementation Schemes I-11 to I-13, wherein the target protein is selected from any sequence of Tables 1, 2 and 3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with it.

[0267] Implementation Scheme I-15. A method for vaccinating a subject in need, the method comprising administering to the subject one or more messenger ribonucleic acid (RNAs) encoding a target protein and an NCT inhibitory protein, wherein the target protein is an antigen protein.

[0268] Implementation Scheme I-16. The method of Implementation Scheme I-15, wherein the NCT inhibitor protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the NCT inhibitor protein amino acid sequence in Table 4.

[0269] Implementation Scheme I-17. The method of Implementation Scheme I-15 or I-16, wherein the antigen protein is selected from any sequence in Table 3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0270] Implementation Scheme I-18. The method described in any of the preceding implementation schemes, wherein the co-expression of the NCT inhibitor protein and the target protein increases the expression level of the target protein in cells or subjects by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200-fold, about 500-fold, or about 1000-fold.

[0271] Implementation Scheme I-19. The method of any one of the preceding implementation schemes, wherein the co-expression of the NCT inhibitory protein and the target protein reduces the expression level of the target protein in cells or subjects by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0272] Implementation Scheme I-20. The method described in any of the preceding implementation schemes, wherein the co-expression of the NCT inhibitor protein and the target protein increases the activity of the target protein in the subject's cells or in the subject by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold.

[0273] Implementation Scheme I-21. The method of any of the preceding implementation schemes, wherein the co-expression of the NCT inhibitor protein and the target protein increases the duration of presence of the target protein in the subject's cells or in the subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold.

[0274] Implementation Scheme I-22. The method described in any of the preceding implementation schemes, wherein the co-expression of the NCT inhibitory protein and the target protein reduces the coefficient of variation (CV%) of the target protein in the subject's tissues or in the subject by approximately 1.2-fold, approximately 1.3-fold, approximately 1.4-fold, approximately 1.5-fold, approximately 1.6-fold, approximately 1.7-fold, approximately 1.8-fold, approximately 1.9-fold, approximately 2-fold, approximately 2.1-fold, approximately 2.2-fold, approximately 2.3-fold, approximately 2.4-fold, approximately 2.5-fold, approximately 2.7-fold, approximately 2.8-fold, approximately 2.9-fold, or approximately 3-fold.

[0275] Implementation Scheme I-23. The method described in any of the preceding implementation schemes, wherein the co-expression of the NCT inhibitor protein and the target protein reduces the degradation of the target protein by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold.

[0276] Implementation Scheme I-24. The method described in any of the preceding embodiments, wherein the co-expression of the NCT inhibitory protein and the target protein causes the ECG of the target protein to be reduced. 50 The reduction is approximately 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, or 300 times.

[0277] Implementation Scheme I-25. A composition comprising one or more ribonucleic acid (RNA) encoding a target protein and an NCT inhibitor protein.

[0278] Implementation Scheme I-26. The composition of Implementation Scheme I-24, wherein the NCT inhibitor protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the NCT inhibitor protein amino acid sequence in Table 4.

[0279] Implementation Scheme I-27. The composition of Implementation Scheme I-24, wherein the NCT inhibitory protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the L protein sequence of SEQ ID NO:2.

[0280] Implementation Scheme I-28. The composition of any one of Implementation Schemes I-24 to I-27, wherein the target protein is selected from any sequence of Tables 1, 2 and 3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with it.

[0281] Implementation Scheme I-29. The composition of any one of Implementation Schemes I-24 to I-27, wherein the target protein is an antibody.

[0282] Implementation Scheme I-30. The composition of Implementation Scheme I-28, wherein the target protein is adalimumab, wherein optionally, the target protein comprises: the adalimumab heavy chain amino acid sequence of SEQ ID NO:13 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith; and / or the adalimumab light chain amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith.

[0283] Implementation Scheme I-31. The composition of any one of Implementation Schemes I-24 to I-27, wherein the target protein is an antigen of a pathogen, optionally, is the SARS-CoV-2 spike (S) protein, wherein optionally, the target protein comprises the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0284] Implementation Scheme I-32. The composition of any one of Implementation Schemes I-24 to I-30, wherein the target protein is selected from any sequence of Tables 1, 2 and 3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with it.

[0285] Implementation Scheme I-33. The composition of any one of Implementation Schemes I-24 to I-31, wherein the method comprises delivering one or more RNAs with lipid nanoparticles (LNPs).

[0286] Implementation Scheme I-34. The composition of Implementation Scheme I-32, wherein the LNP comprises PEGylated lipids, cholesterol and neutral lipids and ionizable lipids.

[0287] Implementation Scheme I-35. The composition of Implementation Scheme I-32, wherein the LNP comprises DMG-PEG (2000), cholesterol, DOPC and DLin-KC2-DMA in a ratio of about 1% to about 5% DMG-PEG (2000), about 30% to about 50% cholesterol, about 5% to about 15% DOPC and about 40% to about 60% DLin-KC2-DMA.

[0288] Implementation Scheme I-36. A system comprising:

[0289] (i) one or more polynucleotides encoding an NCT inhibitor; and

[0290] (ii) Delivery medium.

[0291] Implementation scheme I-37. The system described in implementation scheme I-36, wherein the polynucleotide encoding the NCT inhibitor is RNA.

[0292] Implementation Scheme I-38. The system described in Implementation Scheme I-36, wherein the delivery medium is a non-viral delivery medium.

[0293] Implementation scheme I-39. The system described in implementation scheme I-38, wherein the non-virus delivery medium is LNP.

[0294] Implementation scheme I-40. The system described in implementation scheme I-36, wherein the delivery medium is a virus delivery medium.

[0295] Implementation Scheme I-41. The system described in Implementation Scheme I-36, comprising the RNA encoding the target protein.

[0296] Implementation Scheme I-42. The system described in Implementation Scheme I-41, wherein the RNA comprises at least one unmodified uridine nucleotide.

[0297] Implementation scheme I-43. The system described in implementation scheme I-41, wherein the RNA is self-amplifying RNA (saRNA).

[0298] Implementation Scheme I-44. A method for reducing cellular signaling in response to stressors, the method comprising administering a polynucleotide encoding an NCT inhibitor to cells.

[0299] Implementation Scheme I-45. The method described in Implementation Scheme I-44, wherein the stressor is selected from: innate immune signaling; pro-inflammatory signaling; delivery vector-induced inflammation; natural nucleoside mRNA transfection; self-amplified RNA transfection; and expression of target proteins.

[0300] Implementation Scheme I-46. The method described in Implementation Scheme I-44, wherein the polynucleotide encoding the NCT inhibitor is RNA.

[0301] Implementation Scheme I-47. The system described in Implementation Scheme I-36, wherein the polynucleotide encoding the NCT inhibitor comprises the sequence of SEQ ID NO: 65, 66, 67 or 68.

[0302] Implementation scheme I-48. The method of implementation scheme I-11, wherein the target protein is an antigen for use in a vaccine.

[0303] Implementation scheme I-49. The system of any one of implementation schemes I-41 to I-43, wherein the target protein is an antigen for use in a vaccine.

[0304] Implementation scheme I-50. The method of implementation scheme I-44, the method comprising co-expressing a target protein, wherein the target protein is an antigen for use in a vaccine.

[0305] Example

[0306] Example 1: RNA Production Method

[0307] The compositions and methods disclosed herein provide RNA encoding target proteins (e.g., therapeutic proteins, viral proteins, antibodies, and reporter proteins) and nucleocytoplasmic transport (NCT) inhibitory proteins (e.g., NCT inhibitor L protein). RNA containing NCT inhibitory proteins can provide one or more of the following: a) prolonged expression time of the target protein; b) reduced cellular inflammation in vitro; c) reduced inflammation in vivo; d) higher quality and / or higher quantity of the target protein expressed in vivo and in vitro; and / or e) improved cytokine secretion.

[0308] Construction of RNA molecules

[0309] All assemblies were constructed into a plasmid backbone capable of propagating in *E. coli*, containing a promoter controlling the high copy number origin of replication (ColE1), followed by terminators (rrnB T1 and T2 terminators). This is followed by a promoter controlling an antibiotic resistance gene, which is separated from the rest of the plasmid template by a second terminator (a transcription terminator from λ phage). Genes containing the backbone elements were synthesized via phosphoramide chemosynthesis. Figure 1A-1E The target gene (GOI) can be displayed in RNA separated from NCT inhibitors (such as NCT inhibitor L protein). Figure 1A Delivery on the same RNA and co-transfection, or delivery on the same RNA. Figure 1B-1E In the 5' to 3' direction, NCT inhibitors (e.g., L protein) on the same RNA can be present before or after the target gene. Protease cleavage sites, such as furin sites, may be present. Figure 1B and Figure 1C Internal ribosome entry sites (IRES) may also exist. Figure 1D and Figure 1E ).

[0310] The structural genes used to construct the plasmids were chemically synthesized using phosphoramide, amplified, and cloned into the vectors described above using isothermal assembly reactions (such as NEB HI-FI or Gibson assembly).

[0311] Figure 5A and 5B This shows the DNA template construct configuration for in vitro transcription (IVT) to generate single mRNA constructs encoding L protein (L) and nanoluciferase (nLuc). Figure 5A This shows the DNA template construct used for IVT to generate a single mRNA construct (L-NLuc) encoding L and nLuc separated by a p2A separator element. Figure 5B This shows the DNA template construct used for IVT to generate a single mRNA construct (NLuc-L) encoding nLuc and L separated by a p2A separator element.

[0312] DNA template preparation

[0313] The fully cloned DNA template contained the T7 promoter, 5'UTR, ORF, 3'UTR, Poly A region, and BSAI site. The DNA template was linearized by digestion with BSAI (NEB r3733), and the linearized plasmid was separated from the circularized plasmid by gel electrophoresis (1% agarose). The linear fragment was extracted by phenol / chloroform extraction with an equal volume of 1:1 phenol / chloroform. The DNA was precipitated by adding 1 / 10 volume of 3M sodium acetate (pH 5.2) and two volumes of ethanol. The DNA was incubated at -20°C for 30 min, followed by microcentrifugation for 15 min. The DNA precipitate was washed with 500 μl of 70% ethanol and centrifuged at maximum speed for 15 min. The ethanol was carefully removed, and the precipitate was air-dried before resuspending in nuclease-free water. In some cases, the DNA template used for RNA synthesis was generated using PCR, instead of linearizing the fully cloned DNA with BSAI restriction enzymes as described above. For these PCR reactions, the forward primers were designed to hybridize with the 3'UTR region of the T7 promoter and the fully cloned plasmid DNA template, ensuring that the 5' end of the PCR amplicon was compatible with the CleanCap RNA capping reagent used downstream. The reverse primers were designed to hybridize with the 3'UTR region of the fully cloned plasmid DNA template, ensuring that the intact 3'UTR of the DNA template was preserved within the amplicon. The DNA templates generated by PCR were purified by gel electrophoresis using the Zymoclean Gel DNA Recovery Kit (Zymo Research) and the Monarch PCR & DNA Purification Kit (NEB), following the manufacturer's instructions. The DNA templates generated by either method were then used as templates for RNA synthesis, as described below.

[0314] RNA synthesis

[0315] To generate RNA from in vitro transcription, HiScribee was used. TM The T7 ARCA RNA kit generates a 5',7-methylguanosine (m7G) cap during co-transcription. In some embodiments, RNA in vitro transcription was performed using the Hiscri T7 mRNA kit with the CleanCap Reagent AG kit (NEB) and 100% N1-pseudouridine (Trilink), with the corresponding gel-purified PCR products as templates for RNA synthesis, following the manufacturer's instructions. All components were thawed on ice prior to reaction assembly. The reaction was assembled on ice according to the following specifications:

[0316] Table 5A: RNA synthesis reactions using the HiScribe T7 ARCA RNA kit

[0317]

[0318] Table 5B: RNA synthesis reactions performed using the HiScribe T7 mRNA kit and the CleanCap Reagent AG kit

[0319] Reagents quantity Final concentration 10x reaction buffer 2μl ATP 2 μl 6mM GTP 2 μl 5mM CTP 2 μl 5mM N1-Methylpseudouridine 2 μl 4mM CleanCap AG 2 μl 4mM Nuclease-free water 1 μl DNA template (+water) 6μl 0.1-0.5μg T7 RNA polymerase mixture 2μl Total reaction volume 20μl

[0320] Mix the reaction mixture, briefly centrifuge, and incubate at 37°C for 1–2 hours. To remove the added DNA template, add 2 μL of DNase I, mix thoroughly, and incubate at 37°C for 15 minutes. Purify the synthesized RNA using the Monarch RNA Purification Kit (NEB). Then, assemble the Poly(A) tailing reaction on ice according to the following specifications:

[0321] Table 6: In vitro transcribed (IVT) RNA poly(A) tailing reaction

[0322] Reagents quantity Nuclease-free water 20uL IVT reactants 20μl Poly(A) Polymerase Reaction Buffer (10x) 5μl Poly(A) polymerase 5μl Total reaction volume 50μl

[0323] Mix the reaction mixture, briefly centrifuge, and incubate at 37°C for 30 minutes. Purify the poly(A) tailed RNA using the Monarch RNA Purification Kit (NEB).

[0324] RNA purification

[0325] As described above, RNA was purified using the Monarch RNA Purification Kit (NEB) following the in vitro transcription and Poly(A) tailing steps, according to the manufacturer's instructions. In some cases, RNA was purified using phenol-chloroform extraction and ethanol precipitation. The reaction mixture was adjusted to 180 μl by adding nuclease water before adding 20 μl of 3M sodium acetate (pH 5.2). RNA was then extracted with an equal volume of a 1:1 phenol / chloroform mixture, followed by two extractions with chloroform. The aqueous phase was collected and transferred to a new tube. RNA was precipitated by adding 2 volumes of ethanol. The reaction mixture was incubated at -20°C for at least 30 minutes, and the precipitate was collected by centrifugation. The supernatant was removed, the precipitate was washed with 500 μl of cold 70% ethanol, and then resuspended in 50 μl of 0.1 mM EDTA and stored at -20°C.

[0326] Cell lines - culture and transfection

[0327] HEK293 cells were used to illustrate the application of the systems, methods, and compositions of the present invention in human eukaryotic cells. HEK293 adherent cells (CLS) were cultured in Dulbecco modified Eagle high-glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 Anti-Anti (Gibco). HEK293 cells were grown to 80% confluence at 37°C and 5% CO2, and then transiently transfected using lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. After 48 hours, the cells expressing proteins were analyzed as described in the examples.

[0328] Vero cells were used to illustrate the application of the systems, methods, and compositions of the present invention in eukaryotic cells. VeroE6 adherent cells (ATCC) were cultured in Dulbecco modified Eagle high-glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 U Anti-Anti (Gibco). Vero cells were grown to 80% confluence at 37°C and 5% CO2, and then transiently transfected using lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. After 48 hours, the cells expressing proteins were analyzed as described in the examples.

[0329] C2C12 cells were used to illustrate the improved cytokine sensitivity of the systems, methods, and compositions of the present invention in animal cells. C2C12 adherent cells (ATCC) were cultured in Dulbecco modified Eagle high-glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 Anti-Anti (Gibco). C2C12 cells were grown to 80% confluence at 37°C and 5% CO2, and then transiently transfected using lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. After 48 hours, the cells expressing the proteins were analyzed as described in the examples.

[0330] BJ fibroblasts were used to illustrate the improved cytokine sensitivity of the systems, methods, and compositions of the present invention in human cells. BJ adherent fibroblast cells (ATCC) were cultured in Dulbecco modified Eagle high-glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 Anti-Anti (Gibco). C2C12 cells were grown to 80% confluence at 37°C and 5% CO2, and then transiently transfected using lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. After 48 hours, the expression of proteins in the cells was analyzed as described in the examples.

[0331] Unless otherwise specified, in vitro RNA transfection is generally performed using the Mirus TransIT mRNA Transfection Kit (Mirus) according to the manufacturer's instructions. For co-transfection experiments (i.e., when multiple RNA molecules are used in a dose-titration study), all indicated RNA molecules are first mixed in Opti-MEM. In these experiments, inert filler RNA is used to ensure the same total RNA concentration for each transfection. The amount of RNA used is indicated in the respective examples. Add the Mirus TransITBoost reagent to the pre-diluted RNA sample. Then, add the Mirus TransIT transfection reagent, gently tapping and inverting the tube to mix thoroughly. Incubate the tube at room temperature for up to 5 minutes, then add pre-warmed complete DMEM medium (Gibco) to “quench” the reaction. The transfection complex is then added to the cells. In experiments stimulating cells with Poly I:C or IFN-β, the stimulant is added to the cells at the indicated final concentration within 5 minutes after treatment with the transfection complex.

[0332] LNP-RNA production

[0333] The example LNP formulation provided in this article is: 1.5% DMG-PEG(2000), 38.5% cholesterol, 10% DSPC, and 50% DLin-MC3-DMA. To produce the LNP-mRNA formulation used in the experiments, the lipids were dissolved in ethanol. The RNA was dissolved in an aqueous acidification buffer, for example, 50 mM sodium citrate (pH 4). The lipids were combined with the RNA solution by rapid mixing, for example, at a volume ratio of 1:3 (lipid:RNA) and a nitrogen-to-phosphorus ratio of 6:1 (nitrogen of the ionizable lipids relative to the phosphate of the mRNA). After rapid mixing, the mixture was further diluted 1:1 in a buffer at pH 6 and then percolated against 15 mL of PBS using an Amicon Ultra rotary filter with a molecular weight cutoff of 10 kDa.

[0334] Nano luciferase assay

[0335] For the NanoLuc assay, cell culture supernatant was collected into microcentrifuge tubes, and cell debris was removed by centrifugation. The remaining cells attached to a 24-well plate were lysed at room temperature with 250 μl of PBS containing 0.2% Triton X-100. 50 μl of clear cell culture supernatant or 50 μl of cell lysate was loaded into opaque white 96-well microplates. NanoLuc expression in the collected supernatant and cell lysate samples was measured using the Nano-Glo luciferase assay system (Promega) according to the manufacturer's instructions. In short, 50 μl of fresh 1X Nano-Glo assay reagent in the appropriate assay buffer (Promega) was added to each well, incubated for 2–5 minutes, and then luminescence was measured using a Biotex Synergy LX plate reader. Protein concentration in the cell lysate was further analyzed using the A660 reagent (Thermo Scientific) according to the manufacturer's instructions. In short, 40 μl of sample or standard is mixed with 150 μl of A660 assay reagent, and the absorbance at 660 nm is measured using a Biotek Synergy LX plate reader. Protein concentration is then quantified according to a standard curve. The luminescence value of cell lysates is standardized to per μg of cell lysate protein (i.e., luminescence value is expressed as RLU / μg protein), while the luminescence value of cell culture supernatant is standardized using the volume of supernatant used in the assay (i.e., luminescence value is expressed as RLU / ml supernatant).

[0336] animal

[0337] For animal studies, C57BL / 6 (Charles River Laboratories) or a corresponding disease model was used. Age was indicated in the examples. Animals of the same sex were housed in groups in polycarbonate cages with appropriate bedding. Mice were identified by a visible tattoo on their tail or by implantation of an electronic identification chip. Mice were allowed at least a 5-day acclimatization period before treatment to allow them to adapt to the laboratory environment. Hulling settings were as described in the Guide for the Care and Use of Laboratory Animals, with socialized feeding and chewing aids to enrich the animals' environment. Target environmental conditions were a temperature of 19–25°C, humidity of 30–70%, and a 12-hour light-to-dark cycle. Food (Laboratory Diet Certified Rodent Diet 5CR4) was provided freely in pellet form. Water was provided freely in the form of municipal tap water (treated by reverse osmosis and UV irradiation).

[0338] Example 2: In vitro expression of a reporter gene (luciferase) from RNA in the presence of L protein.

[0339] To investigate the role of the L protein in the mRNA construct, a single mRNA construct encoding luciferase and the L protein (an NCT inhibitor) separated by p2A elements was used. Figure 5A and 5B Interferon-producing mouse C2C12 and human BJ fibroblasts were transfected. These mRNA constructs contained an ARCA cap and a mixture of 50%–50% natural uridine and pseudouridine. Due to the use of natural uridine in the mRNA, these mRNA constructs are inherently pro-inflammatory, as they can activate innate immune signaling pathways in transfected cells, as is known to those skilled in the art. The NCT inhibitor was examined at two locations relative to the target gene (at the 5' and 3' of the gene), demonstrating the flexibility of the NCT inhibitor location. Interestingly, the location of the L protein also modulates the effect of the L protein on the expression of the target gene.

[0340] On day 0, C2C12 and BJ fibroblasts were seeded in 96-well microplates containing 200 μl DMEM (C2C12) or 200 μl MEM (BJ fibroblasts) with 10% FBS. On day 1, Lipofectamine was used according to the manufacturer's instructions. TM MessengerMAX TM Transfection reagent (Invitrogen), used Figure 5A and 5BThe encoded RNA construct was transfected into cells. NanoLuc luciferase activity was measured in transfected cells and cell culture supernatant at 24, 48, and 72 hours post-transfection over the following days. Figure 6A and 6B The study showed luciferase activity, a direct measurement of the enzyme expressed in mouse C2C12 and human BJ cells. Surprisingly, co-expression of the L protein (an NCT inhibitor) led to a more than 10-fold increase in luciferase expression. This suggests that the L protein possesses the ability to protect the expression of the target gene (GOI) against internal stress and inflammatory signals induced by mRNA transfection.

[0341] To further investigate the role of L protein in protecting protein expression against stress and inflammatory signals, exogenous IFN-β (a stress pathway stimulant) was added to cells. Once a threshold concentration of exogenous IFN-β was reached, L protein expression was inhibited. To measure cellular sensitivity to exogenous IFN-β in the presence or absence of L protein, cells encoding L protein alone (L), nLuc alone, or similar proteins were analyzed. Figure 5A (L-nLuc) and Figure 5B C2C12 cells were transfected with mRNAs encoding both L and nLuc single mRNAs, as shown in (nLuc-L). Six hours post-transfection, a 10-fold dilution of recombinant mouse IFN-β was added to the C2C12 cells. Luciferase activity was then measured 48 hours after IFN-β stimulation. The EC50 of IFN-β required to reduce luciferase protein expression was determined. 50 The approximate value was measured as the concentration at which luciferase expression (e.g., an activity indicator, and normalized for the absence of IFN-β) decreased by 50% (dashed line).

[0342] Figure 7 The results show a decrease in luciferase expression in the presence of IFN-β. The EC50 of IFN-β required to decrease luciferase protein expression in each construct is shown. 50 Approximate value. For example, EC value of increased IFN-β. 50 As demonstrated by the approximate values, the presence of the L protein protects cells from exogenous IFN-β stimulation by 10-fold and 12-fold (for L-nLuc and nLuc-L, respectively). Therefore, the L protein protects protein expression from exogenous stress induced by IFN-β.

[0343] EC with IFN sensitivity 50 The values ​​are calculated in Prism (GraphPad) using a logarithmic (inhibitor) relative response-variable slope four-parameter nonlinear regression.

[0344] IFN-β secretion serves as a marker of inflammation. For this purpose, human BJ cells producing IFN-β were transfected with... Figure 5A and5B The construct shown encodes the mRNA. The supernatant was collected 24–72 hours post-transfection and clarified by centrifugation at 500g for 5 minutes. The presence of IFN-β in the clarified supernatant was then analyzed using an ELISA (R&D Systems) according to the manufacturer's instructions.

[0345] Figure 8 This shows the IFN-β levels produced by BJ cells after transfection with the mRNA construct. Notably, the presence of the L protein prevents BJ cells from producing IFN-β in response to mRNA transfection. In the presence of L protein mRNA, intracellular RNA stress induced by pro-inflammatory mRNA containing 50% natural uridine is not propagated by cells, thus eliminating the ability of cells to upregulate IFN-β expression and secretion as part of a stress response.

[0346] Example 3: In vitro expression of viral protein (Covid19 spike protein) from RNA

[0347] To determine whether the Sars-CoV 2 spike protein, a viral protein, is expressed in HEK293T, Vero E6, C2C12, and / or BJ fibroblasts, the following experiments were performed. HEK293T, Vero E6, C2C12, and / or BJ fibroblasts were transfected with RNA expressing only the Sars-CoV 2 spike protein as a control. Figure 3A The virus was co-transfected with two RNA constructs: one expressing the Sars-CoV2 spike protein and the other expressing an NCT inhibitory protein derived from the IVT response. Figure 3A and 3B (as described below).

[0348] On day 0, HEK293T, Vero E6, C2C12, and / or BJ fibroblasts were seeded into 500 μl of DMEM (HEK293T, Vero E6, C2C12) or 500 μl of MEM (BJ fibroblasts) containing 10% FBS in 24-well cell culture microplates. On day 1, Lipofectamine was administered according to the manufacturer's instructions. TM MessengerMAX TM Transfection reagents (Invitrogen) were used to transfect the cells with the corresponding RNA-Spike alone or in combination with RNA-L from the IVT reaction. Over the next few days, NanoLuc luciferase activity was measured in the transfected cells and cell culture supernatant.

[0349] In addition, to measure interferon sensitivity in the presence and absence of NCT inhibitory proteins, cells were treated with IFN-β 6–24 hours post-transfection. For this purpose, recombinant IFN-β (Fisher Scientific) from mice (for C2C12) or humans (for HEK293T, Vero E6, and BJ fibroblasts) was serially diluted 5-fold and added to cells 6 or 24 hours post-transfection with RNA encoding either the Sars-CoV2 spike protein alone or in combination with an NCT inhibitory protein. The proportion of Sars-CoV2 spike protein expression was normalized relative to untreated samples. IFN-sensitivity EC 50 The values ​​are calculated in Prism (GraphPad) using a logarithmic (inhibitor) relative response-variable slope four-parameter nonlinear regression.

[0350] For interferon-producing cell lines (C2C12, BJ fibroblasts), interferon secretion, a marker of inflammation, was measured. To this end, cell supernatants were collected 24–72 hours post-transfection and clarified by centrifugation at 500g for 5 minutes. The presence of IFN-β in the clarified supernatant was then analyzed using ELISA (R&D Systems) according to the manufacturer's instructions.

[0351] In addition, ELISA was used to confirm the secretion of the expressed spike protein. High-binding 96-well plates were coated with cell culture supernatant isolated on day 3 (post-transfection), using 75 μl of cell culture supernatant per well, and incubated overnight at +4°C. On the second day, the coated wells were washed with PBST buffer and blocked with 200 μl of EZ Block reagent per well for 2 hours at +37°C. The wells were washed three times with PBST and incubated for 1 hour at room temperature with primary antibody (rabbit anti-RBD, diluted 1:500 in EZ Block, 75 μl per well). The wells were then washed three times with PBST, and 75 μl of goat anti-rabbit HRP secondary antibody diluted 1:1000 in EZ Block reagent was added per well, and incubated for 1 hour at room temperature. The wells were then washed five times with PBST, and 75 μl of TMB substrate was added per well, incubated for 30 minutes at room temperature, and then 75 μl of stop solution was added. The absorbance at 450 nm was measured using a plate reader.

[0352] Example 4: In vitro expression of GBA

[0353] To determine whether GBA, a secreted protein, is expressed in HEK293T, Vero E6, C2C12, and / or BJ fibroblasts, the following experiments were performed. HEK293T, Vero E6, C2C12, and / or BJ fibroblasts were transfected with RNA expressing only GBA as a control. Figure 4AFurthermore, two RNA constructs were co-transfected simultaneously: one RNA construct expressed the GBA protein, and the other RNA construct expressed the NCT repressor protein derived from the IVT response. Figure 4A and 4B As described below. On day 0, HEK293T, Vero E6, C2C12, and / or BJ fibroblasts were seeded into 500 μl of DMEM (HEK293T, Vero E6, C2C12) or 500 μl of MEM (BJ fibroblasts) containing 10% FBS in 24-well cell culture microplates. On day 1, Lipofectamine was used according to the manufacturer's instructions. TM MessengerMAX TM Transfection reagents (Invitrogen) were used to transfect the cells with the corresponding RNA-Luc alone or in combination with RNA-L from the IVT reaction. Over the next few days, GBA activity was measured in the transfected cells and cell culture supernatant.

[0354] In addition, to measure interferon sensitivity in the presence and absence of NCT inhibitory proteins, cells were treated with IFN-β 6–24 hours post-transfection. For this purpose, recombinant IFN-β (Fisher Scientific) from mice (for C2C12 cells) or humans (for HEK293T, Vero E6, and BJ fibroblasts) was serially diluted 5-fold, and the diluted solution was added to cells 6 or 24 hours after transfection with RNA encoding either a single GBA protein or a combination of known NCT proteins. The proportion of GBA expression was normalized for untreated samples. IFN sensitivity EC 50 The values ​​are calculated in Prism (GraphPad) using a logarithmic (inhibitor) relative response-variable slope four-parameter nonlinear regression.

[0355] For interferon-producing cell lines (C2C12, BJ fibroblasts), interferon secretion, a marker of inflammation, was measured. To this end, cell supernatants were collected 24–72 hours post-transfection and clarified by centrifugation at 500g for 5 minutes. The presence of IFN-β in the clarified supernatant was then analyzed using ELISA (R&D Systems) according to the manufacturer's instructions.

[0356] For the GBA activity assay, cells were seeded and transfected with the appropriate RNA as described above. On day 3, the cell culture supernatant was collected, cell debris was removed by centrifugation, and the supernatant was preserved for the determination of secreted GBA activity. Adherent cells were separated from the plate using 500 μl of PBS and centrifuged to precipitate the cell pellet. The cell pellet was lysed using 1X GBA assay buffer (0.1 M sodium citrate, 0.2 M sodium phosphate, 0.25% Triton X-100, 0.25% sodium taurocholate, 1.25 mM EDTA, 5 mM DTT) and pre-equilibrated to 37°C before lysis. The protein concentration of the cell lysate was determined using the Pierce A660 protein assay according to the manufacturer's instructions, and then the cell lysate was diluted to a final protein concentration of 125 μg / ml using 1X GBA assay buffer. 40 μl of the pre-diluted cell lysate was pipetted in duplicate into each well of a clear 96-well plate. Two 20 μl aliquots of the previously collected cell culture supernatant were added to each well of a clear 96-well plate, and 20 μl of 2XGBA assay buffer was added to each well containing cell culture supernatant. Next, 20 μl of assay substrate (6 mM 4-MU-β-D-glucoside prepared in 1X GBA assay buffer) was added to each well containing cell lysate or cell culture supernatant. In adjacent wells, calibration curves were prepared using 4-methylumbelliferone in 1XGBA assay buffer. Samples were incubated at 37°C for 30 min–4 h in the presence of the assay substrate, followed by the addition of 100 μl of stop solution (0.5 M glycine, 0.3 M NaOH, pH 10). Subsequently, fluorescence of each sample and standard was measured using a Biotek Synergy LX plate reader at Ex / Em wavelengths of 360 / 445 nm. GBA activity in cell lysate samples was calculated using the following formula: Activity = [B / (T x V x P)] x D = pmol / min / mg = μU / mg, where B is the converted 4-MU amount (pmol) calculated using the standard curve, T is the reaction time (min), V is the sample volume (ml), P is the initial protein sample concentration, and D is the sample dilution factor (if applicable). GBA activity in cell culture supernatant was calculated using the following formula: Activity = [B / (T x V)] x D = pmol / min / ml = μU / ml, where B is the converted 4-MU amount (pmol) calculated using the standard curve, T is the reaction time (min), V is the sample volume (ml), and D is the sample dilution factor (if applicable).

[0357] Example 5: In vivo expression of luciferase from RNA

[0358] To evaluate the in vivo expression of nanoluciferase, five female C57BL / 6 mice aged 6-8 weeks were intramuscularly injected with RNA conjugated to a lipid nanoparticle formulation in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). The RNA was: (a) Figure 2A (a) RNA encoding luciferase or (b) RNA encoding luciferase in sequence with NCT inhibitor protein. For baseline measurements, PBS was administered intramuscularly to four mice only. The volume of the dose was administered using a syringe / needle.

[0359] Animals were randomly assigned to groups, and their weight was recorded on day 1, then every two weeks until the end of the study. Adverse events (RM, SD, RD) were recorded according to good laboratory standards. Any individual animal with a single observed weight loss >30% or three consecutive measurements of weight loss >25% was euthanized.

[0360] Whole blood was collected via the submandibular vein and processed to collect serum for analysis. Blood was collected on days 0 (before administration), 3, 7, 14, 21, 28, and 42. Serum luciferase concentration was analyzed by NanoLuc assay.

[0361] For the NanoLuc assay, 50 μl of diluted serum was loaded into an opaque white 96-well microplate. 50 μl of fresh 1X Nano-Glo assay reagent in the appropriate assay buffer (Promega) was added to each well, incubated for 2–5 minutes, and then luminescence measurement was performed using a Biotex Synergy LX plate reader.

[0362] Example 6: Immunogenicity of RNA-derived Sars-CoV2 spike protein in vivo

[0363] To assess the expression of the Sars-CoV2 spike protein in vivo, five 6-8 week old female C57BL / 6 mice were used in each group. Mice in each group were sensitized via intramuscular injection on day 0. On day 28, the sensitization was performed using the same method with RNA conjugated to a lipid nanoparticle formulation in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). The RNA was: (a) Figure 3A (a) RNA encoding the Sars-CoV2 spike protein, and (b) RNA encoding the Sars-CoV2 spike protein in sequence with the NCT inhibitor protein. For baseline measurements, four mice were administered PBS via intradermal injection only. The volume of the dose was administered using a syringe / needle.

[0364] Animals were randomly assigned to groups, and their weight was recorded on day 1, then every two weeks until the end of the study. Adverse events (RM, SD, RD) were recorded according to good laboratory standards. Any individual animal with a single observed weight loss >30% or three consecutive measurements of weight loss >25% was euthanized.

[0365] Whole blood was collected via the submandibular vein and processed to collect serum for analysis. Blood was collected on days 0 (before administration), 3, 14, 28, and 42. Serum anti-SARS-CoV-2 spike protein antibodies (evoked humoral immune response) were analyzed by ELISA. Enzyme-linked immunosorbent assay (ELISA) was performed using purified SARS-CoV-2 spike protein RBD as the coating material. 75 μl of 2 μg / ml SARS-CoV-2 spike protein RBD (Creative...) was... The DAGC149 recombinant SARS-CoV-2 spike protein receptor-binding domain ([His]) was coated onto a high-binding 96-well plate, and the plate was incubated overnight at 4°C. After incubation, the plate was washed twice with PBS containing 0.05% Tween-20, and the wells were blocked using an EZ block (37°C for 2 hours). The plate was washed twice with PBS containing 0.05% Tween-20, and serum collected from mice was added to the wells (1:500 dilution for antibody binding detection, 1:100–1:781–2500 for endpoint titer measurement). The serum was incubated at room temperature for 1 hour, then washed three times with PBS containing 0.05% Tween-20, and 75 μl of secondary antibody (goat anti-rabbit, HRP-conjugate, 1:4,000 dilution) was added, and the plate was incubated at room temperature for 1 hour. The wells were thoroughly cleaned (washed 5 times with PBS containing 0.05% Tween-20) and developed using 75 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Surmodisc Inc. TMB single-component HRP microwell substrate). The reaction was allowed to proceed for 30 min, then terminated with 75 μl of stop solution (Surmodisc Inc. 450 NM LIQ stop reagent), and the absorbance at 450 nm was measured using a Biotex Synergy LX plate reader. The endpoint titer was calculated in Prism (GraphPad) using a log(inhibitor) relative response – variable slope four-parameter nonlinear regression interpolated from four times the background value.

[0366] Example 7: The effect of L protein expression on reporter gene expression when cells are stimulated by Poly I:C or IFN-β exogenous stimuli.

[0367] Poly I:C and IFN-β are activators of innate immune signaling pathways that reduce reporter gene expression in a dose-dependent manner. EMCV L protein, as an NCT inhibitor, protects reporter gene expression from downregulation.

[0368] Once the mRNA enters the cell, it may encounter host or administration-dependent induced inflammation, which can suppress the expression of the target gene (GOI) and subsequently inhibit its therapeutic effect. The effects of L protein were assessed when L protein was trans-added to human skin fibroblasts transfected with mRNA encoding the reporter gene GOI (firefly luciferase) and stimulated with Poly I:C (a synthetic double-stranded RNA analog used as a potent immunostimulant).

[0369] Human skin fibroblasts (BJ cells, ATCC) were cultured as described in Example 1. On day 0, BJ cells were seeded at 1e4 cells / well in complete DMEM (10% heat-inactivated FBS + 1x antibiotic-antifungal agent) in 96-well cell culture plates. Cells were incubated overnight at 37°C and 5% CO2. On day 1, cells were transfected with the indicated RNA construct using the Mirus TransIT mRNA transfection kit (Mirus) as described in Example 1. Within 5 minutes of adding the transfection complex to the cells, Poly I:C or IFN-β was added to the cells at the indicated final concentration. The reporter gene mRNA encoding firefly luciferase (Trilink) has a CleanCap mRNA cap structure, in which 100% of uridine in its sequence has been replaced by 5-methoxyuridine. This type of modified mRNA was used because it is a weak activator of the innate immune signaling pathway in the recipient cells, thus allowing us to focus primarily on the role of L protein mRNA in response to exogenous inflammatory stimuli. As described in Example 1, HiScribe was used according to the manufacturer's instructions. TM The T7 mRNA kit and CleanCap Reagent AG (NEB) were used to perform in vitro transcription of L protein-encoding mRNA and nLuc-filled mRNA, both containing 100% N1-methyl-pseudouridine to reduce their natural ability to activate pro-inflammatory cell signaling pathways. For transfection, 10 ng of reporter gene mRNA was mixed with an indicator amount of L protein-encoding mRNA and nLuc-filled mRNA to ensure that all transfections used equal amounts of mRNA. Two days post-transfection, firefly luciferase expression of the reporter gene was measured using the Bio-Glo luciferase assay (Promega) according to the manufacturer's instructions.

[0370] like Figure 9 and 10As shown, Poly I:C and IFN-β, as potent activators of the innate immune cell signaling pathway, inhibited firefly luciferase expression in a dose-dependent manner (diagonal stripes). However, in the presence of L NCT inhibitory protein-encoded mRNA (gray and black bars), Poly I:C and IFN-β (respectively...) showed... Figure 9 and Figure 10 The effect of reducing firefly luciferase is greatly diminished. Therefore, in the presence of inflammation modeled by the addition of Poly I:C and IFN-β at both excess and repressive levels, the LNCT repressor protein protects GOI expression. As expected from its anticipated mechanism of action of inhibiting nucleocytoplasmic transport, the LNCT repressor protein-encoded mRNA, in the absence of pro-inflammatory signals, partially suppresses overall reporter gene expression, as suppression of this pathway is generally associated with downregulation of protein translation. However, it is noteworthy that in the presence of Poly I:C and IFN-β, the LNCT repressor protein-encoded mRNA helps rescue reporter gene expression. Figure 9 and Figure 10 It has generally been observed that higher concentrations of pro-inflammatory stimuli, whether Poly I:C or IFN-β, result in a greater fold increase in reporter gene expression rescue. Interestingly, with Poly I:C as the stimulant, 1 ng L NCT inhibitor mRNA showed the strongest effect in rescuing reporter gene expression, while with IFN-β as the stimulant, 0.2 ng L NCT inhibitor mRNA showed the strongest effect. This suggests that, depending on the primary source and nature of the pro-inflammatory stimuli, different amounts of L NCT inhibitor mRNA (and more importantly, available) may be required to rescue target gene expression induced by activated innate immune cell signaling pathways.

[0371] Example 8: The effect of L protein on reporter gene expression when cells are stimulated by endogenous delivery of pro-inflammatory mRNA transgenes (i.e., mRNA containing natural uridine).

[0372] It is known that in vitro transcribed mRNAs containing natural nucleosides can activate immune signaling pathways, leading to downregulation of intracellularly delivered mRNA expression (as shown in Example 7). It is known that mRNAs containing N1-methyl-pseudouridine induce immune activation at low levels, which is generally unrelated to the downregulation of intracellularly delivered mRNA expression. Consistent with the latter observation, the L NCT repressor protein-encoded mRNA enhanced GOI expression of strongly pro-inflammatory mRNAs containing natural uridine nucleosides, but did not enhance GOI expression of non-inflammatory mRNAs containing N1-methyl-pseudouridine.

[0373] Following the manufacturer's instructions, L NCT repressor protein-encoding mRNA was synthesized independently using the HiScribe T7 mRNA kit, the CleanCap Reagent AG kit (NEB), and 100% m1-pseudouridine (Trilink) and polyA tail (NEB). The mRNA expressing nanoluciferase was synthesized in the same manner, with or without the use of synthetic nucleoside substitutes, such as... Figure 11A-11C As shown. mRNA synthesis was performed according to the method described in Example 1 above.

[0374] For transfection, 10 ng of reporter gene mRNA was mixed with an indicator amount of L NCT repressor protein-encoded mRNA and irrelevant filler mRNA to ensure that all transfections were performed using equal amounts of mRNA. Two days post-transfection, nanoluciferase expression from the reporter gene was measured using the Nano-Glo luciferase assay (Promega) according to the manufacturer's instructions.

[0375] First, it was observed that on day 6 post-mRNA transfection, L NCT repressor protein-encoded mRNA increased reporter gene expression by 3.1–4.4-fold, depending on the amount of L NCT repressor protein-encoded mRNA co-transfected with the reporter gene, but this only occurred when the reporter gene-encoded mRNA was pro-inflammatory (i.e., containing only native nucleosides). Figure 11A When a reporter gene encoding a non-inflammatory mRNA containing pseudouridine was transfected into cells, the L NCT repressor protein did not increase transgene expression, as expected. Figure 11B ).

[0376] To further analyze the observed increase in gene expression from intrinsic inflammatory mRNAs, time-course experiments were conducted. BJ cells were co-transfected with either the aforementioned pro-inflammatory native mRNA or a non-inflammatory N1-methyl-pseudouridine-modified mRNA, as well as 1 ng / L of NCT repressor protein-encoding mRNA. Luciferase expression was measured on days 3, 6, 9, 13, 16, and 20 post-transfection. Similar to the previously mentioned results, when the reporter gene-encoding mRNA was pro-inflammatory (i.e., containing only native nucleosides), transgene expression was improved at all time points, with an average increase of 5.7-fold across all time points. Figure 11C However, when BJ cells were transfected with reporter mRNA containing non-inflammatory pseudouridine, the LNCT repressor protein had only a slight effect on the expression of the reporter gene mRNA at all time points. Figure 11C This is consistent with the results previously reported.

[0377] These data demonstrate the advantages of using L NCT repressor protein-encoded mRNA to increase gene expression in applications that require the use of natural or partially natural mRNA molecules.

[0378] Example 9: The effect of L protein on reporter gene expression when cells are endogenously stimulated by intracellularly delivered pro-inflammatory self-amplifying RNA (saRNA) transgenes.

[0379] Once mRNA enters the cell, it may encounter host or administration-dependent induced inflammation, which can suppress the expression of the target gene (GOI) and subsequently inhibit its therapeutic effect. One therapeutic modality that induces excessive inflammation is self-amplified mRNA (saRNA). The effect of EGT-404 on the expression of a reporter gene encoded by a saRNA cassette derived from Venezuelan equine encephalitis virus (VEEV) in human fibroblasts was evaluated. When delivered from independent mRNA molecules, the L NCT repressor protein-encoded mRNA enhanced the expression of saRNA in a dose-dependent manner at all measurement time points. Figure 12A and Figure 12B ).

[0380] Human skin fibroblasts (BJ cells) were cultured and transfected as described in Examples 7 and 8. L NCT repressor protein-encoding mRNA was produced using the HiScribe T7 mRNA kit and the CleanCap Reagent AG kit (NEB) with 100% m1-pseudouridine (Trilink) and polyA tail (NEB) according to the manufacturer's instructions. VEEV saRNA replicons encoding the reporter gene (nanoluciferase) were purchased from Aldevron. All transfections were performed using 100 ng of saRNA and an indicated amount of L NCT repressor protein-encoding mRNA. Unrelated mRNA was added as filler mRNA in appropriate volumes to maintain a constant total mRNA mass. Nanoluciferase expression was measured at indicated time points using the Nano-Glo luciferase assay system (Promega) according to the manufacturer's instructions.

[0381] On day 6 post-transfection, the expression of nanoluciferase encoded by saRNA was first analyzed. L NCT repressor protein enhanced luciferase expression in a concentration-dependent manner, increasing it by approximately 11-fold compared to the case without L NCT repressor protein-encoded mRNA. Figure 12A The optimal amount of L NCT inhibitor mRNA is 1 ng. Compared with the case without L NCT inhibitor protein-encoded mRNA, the relative effect of L NCT inhibitor mRNA in increasing saRNA reporter gene expression increased over time, reaching up to 26-fold at days 16 and 20 post-transfection. Figure 12BAt each time point, the optimal amount of L NCT inhibitor mRNA was 1 ng, similar to the results observed on day 6. These results indicate that when using self-amplifying RNA, L NCT inhibitor mRNA contributes to increasing transgene expression levels and duration, and that the self-amplifying RNA is a known strong inducer of innate immune signaling pathways in cells during its self-replication. This suggests that L NCT inhibitor protein-encoded mRNA increases transgene expression levels when innate immune signaling pathways are activated by exogenous or endogenous stimuli, consistent with the results of Examples 8-8.

[0382] Example 10: Alternative mechanisms of nucleocytoplasmic transport inhibition to enhance protein expression

[0383] Nucleocytoplasmic transport (NCT) inhibition can also be achieved through the alternative mechanisms described in Examples 1-9, such as using non-viral proteins that bind to NCT pathway proteins, or using RNAi to knock down the expression of NCT pathway genes. For example, overexpression of dominant-negative mutants of NCT pathway proteins, such as the Ran Q69L mutant or the MxB K131A protein, can block NCT. Similarly, myristyl peptides derived from ERK and SMAD proteins can inhibit Nup93-dependent NCT of transcription factors and RNA. In this example, a method for knocking down NCT proteins (e.g., Ran, NTF2, MxB, Xpo7, and Nup93) using siRNA is described.

[0384] As described in Example 7, Poly I:C and IFN-β are known activators of innate immune signaling pathways that can reduce the expression of delivered mRNA molecules. Inhibition of these signaling pathways using NCT inhibitory proteins can increase the expression of target proteins. As described herein, siRNA inhibition of NCT pathway proteins can also inhibit Poly I:C and IFN-β signaling to increase target protein expression.

[0385] Methods to knock down NCT inhibitory protein with siRNA

[0386] In short, on day 0, BJ cells were seeded at 1E4 cells / well in 96-well cell culture plates in complete DMEM (i.e., containing 10% heat-inactivated FBS + 1X antibiotic-antifungal agent). Cells were incubated overnight at 37°C and 5% CO2. On day 1, cells were co-transfected with reporter gene mRNA and NCT repressor RNA. As described in Example 1, transfection was performed using the MirusTransIT mRNA transfection kit (Mirus) with 10 ng of reporter gene mRNA and an appropriate amount of NCT repressor RNA. Within 5 minutes of adding the transfection complex to the cells, Poly I:C or IFN-β was added to the cells at the indicated final concentration.

[0387] The reporter gene mRNA encodes firefly luciferase, possessing a CleanCap mRNA cap structure, with 100% of uridine replaced by N1-methylpseudouridine in the sequence. The luciferase mRNA was co-transfected with any of the following NCT repressive RNAs: (a) mRNA encoding an anti-Xpo7 nanobody, (b) mRNA encoding an anti-NFT2 antibody, (c) mRNA encoding a Ran Q69L mutant protein, (d) mRNA encoding an MxB K131A mutant protein, (e) mRNA encoding a myristylated ERK-derived phosphomimicrypeptide, (f) mRNA encoding a myristylated SMAD-derived phosphomimicrypeptide, (g) siRNA targeting Ran mRNA, (h) siRNA targeting NTF2 mRNA, (i) siRNA targeting MxB mRNA, (j) siRNA targeting Xpo7 mRNA, or (j) siRNA targeting Nup93 mRNA. HiScribe was used according to the manufacturer's instructions, as described in Example 1. TM T7 mRNA kit and CleanCap Reagent AG (NEB) in vitro transcription of NCT repressive mRNA containing 100% N1-methyl-pseudouridine. NCT repressive siRNA was commercially available as a chemically synthesized sequence (IDT Technologies).

[0388] On day 3 (48 hours post-transfection), firefly luciferase expression from the reporter gene was measured using the Bio-Glo luciferase assay (Promega) according to the manufacturer's instructions. The effect of NCT repressive RNA on enhancing luciferase expression at any given concentration of Poly I:C and IFN-β was determined by calculating the fold change in luciferase expression in the presence and absence of the corresponding NCT repressive RNA.

[0389] Example 11: Mutant forms of the L protein regulate target protein expression levels.

[0390] As described in Example 9, co-expression of L protein mRNA and reporter gene luciferase saRNA was performed. In this example, mutant forms of the EMCV L protein (SEQ ID NO: 65, 66, 67, 68) differentially regulated saRNA-based reporter gene expression. The comparable wild-type EMCV L protein sequence is SEQ ID NO: 69. Reporter gene expression was also regulated by the concentration of the transfected L protein mutant (…). Figure 13 and Figure 14 ).

[0391] The same system, namely L protein mRNA and saRNA-based reporter gene luciferase expression, was used to detect the expression of related inflammatory mediators. Measurements were taken 6 days post-transfection. With the introduction of the aforementioned L protein saRNA system, IP-10 ( Figure 15B ) and IFN-β Figure 15A The expression of all samples decreased. The data shown are from N=3 experiments.

[0392] Example 12. Effect of L protein on reporter gene expression in vivo when using self-amplified RNA (saRNA) to express the reporter gene.

[0393] To demonstrate the beneficial effect of the L protein on the in vivo expression of the reporter gene encoded by the saRNA, the following experiments were performed. As described in Example 9, the mRNA encoding the L protein (or the corresponding scrambled negative control sequence) was produced internally. As described in Example 9, the saRNA vector expressing nanoluciferase was purchased from Aldevron. The nanoluciferase saRNA and L protein mRNA (or nanoluciferase saRNA and the corresponding scrambled negative control mRNA) were mixed and formulated into lipid nanoparticles (LNPs) containing SM-102, 1,2-DSPC, cholesterol, and DMG-PEG(2000) lipids (SM-102LNP), and stored in 20 mM Tris pH 7.5 8.7% sucrose buffer. C57BL6 mice (female, 10-12 weeks old) were treated with SM-102LNP via intramuscular injection at a total dose of 2 μg / mouse, with N = 4 mice per group. On indicated dates, in vivo imaging was performed using IVIS to measure nanoluciferase expression, as shown below. The nano-luciferase substrate fluorofurimazine (FFz) was prepared according to the manufacturer's instructions (Promega) and administered to mice via intraperitoneal (ip) injection, 50 μl per mouse. IVIS imaging was performed 10 minutes after intraperitoneal injection of FFz.

[0394] Figure 16 This study demonstrated the effect of L protein expression on the expression of the target gene in self-amplified RNA (saRNA) in C57BL6 mice transfected with lipid nanoparticles carrying the L protein and saRNA system. Mice were injected with 2 μg of SM-102LNP, with four mice per experimental group. As shown, in vivo expression of the saRNA-based luciferase was 2–4 times higher in the presence of L protein mRNA.

[0395] Example 13. Effect of L protein mRNA expression on the mRNA expression of treatment-related proteins.

[0396] In this embodiment, the effect of L protein (EG Tech) mRNA on the expression of treatment-related gene mRNA vectors when co-delivered to target cells was investigated. The mRNA vectors of the following treatment-related genes were studied: G6PC1 and SERPINA1. Mutations in the G6PC1 gene in glycogen storage disease type I (GSDI) (also known as von Gierke disease) lead to a reduced ability of the body to break down glycogen, resulting in hypoglycemia, hepatomegaly, and renal enlargement. Expression of functional G6PC1 in a gene therapy setting can provide a therapeutic benefit for GSDI. Mutations in SERPINA1 contribute to the development of α-1 antitrypsin deficiency (A1AD), causing lung and liver damage in affected individuals. Expression of functional SERPINA1 in a gene therapy setting can provide a therapeutic benefit for A1AD.

[0397] To assess the expression of G6PC1 and SERPINA1, an mRNA vector was designed in which the treatment-related target gene (GOI) was fused to the NanoLuc reporter protein via an adaptor peptide. Figure 18A and Figure 18B The coding sequences of these GOIs are flanked by hBG 5'UTR and hBG 3'UTR. The mRNA expression vector contains a Cap1 mRNA cap structure, N1-methylpseudouridine modification, and a 120-nt polyA tail. The GOI mRNA expression vectors were generated by a commercial third-party supplier using in vitro transcription (IVT). The L protein (EG Tech) mRNA also contains a Cap1 mRNA cap structure and N1-methylpseudouridine modification, and was generated by a commercial third-party supplier using in vitro transcription (IVT).

[0398] NIH / 3T3 mouse fibroblast cells were obtained from ATCC and cultured according to the cell bank's instructions. In short, cells were cultured in a humidified environment of 37°C with 5% CO2 using Dubelcco modified Eagle medium (DMEM) supplemented with high glucose and GlutaMAX, containing 10% fetal bovine serum (FBS) and 1X antifungal / antibiotic. On day 0, NIH / 3T3 cells were seeded into 96-well plates. On day 1, cells were transfected using the MIRUS TransIT mRNA transfection kit according to the manufacturer's instructions, with 10 ng GOI mRNA per well, or a mixture of 10 ng GOI mRNA and 0.16 ng EG Tech mRNA per well, or a mixture of 10 ng GOI mRNA and 1.25 ng L protein (EG Tech) mRNA per well. On day 3, 48 hours post-transfection, the cell supernatant was removed, and the activity of the fused NanoLuc reporter gene was measured using the Promega Nano-Glo luciferase assay system according to the manufacturer's instructions to determine the expression of treatment-related GOIs (G6PC1 and SERPINA1) in the cell lysates. Each experiment was repeated three times. A total of five independent experiments (biological replicates) were performed. Results showed... Figures 19A-19D middle.

[0399] In the absence of L protein (EG Tech) mRNA, the expression level of the G6PC1 mRNA vector in NIH / 3T3 cells was moderate (<10,000 RLU); however, when G6PC1 mRNA was co-transfected with L protein (EG Tech) mRNA, G6PC1 expression increased (>100,000 RLU). See [link to relevant documentation]. Figure 19A This corresponds to a 20-fold or 34-fold increase in G6PC1 expression, depending on the amount of L protein (EG Tech) used for co-transfection; see [link to relevant documentation]. Figure 19B Even in the absence of L protein (EG Tech) mRNA, the basal expression level of the SERPINA1 mRNA vector was relatively high (>100,000 RLU). See [link to documentation]. Figure 19C However, co-transfection with L protein (EG Tech) mRNA led to a further approximately 2-fold increase in SERPINA1 expression. Figure 19D These results indicate that L protein mRNA expression can have significant benefits in increasing gene expression in treatment-associated GOI mRNA vectors, thereby expanding the applicability of L protein to gene replacement therapy and gene therapy in general.

[0400] Example 14: The effect of L protein mRNA expression on the expression of target protein in therapy-related plasmid DNA.

[0401] In this embodiment, the effect of L protein (EG Tech) mRNA on the expression of treatment-related genes in the pDNA vector was evaluated when L protein (EG Tech) mRNA was co-delivered with the pDNA vector to target cells. The treatment-related gene GBA (lysosomal acid glucoceramitinase) is important for cell membrane turnover, degradation of complex lipids, and cholesterol processing. Mutations in this gene can lead to the development of a lysosomal storage disease called Gaucher disease, in which lipid accumulation of glucoserebrosides occurs at abnormal levels in various organs, leading to multiple functional disorders. Gene replacement therapy using a fully functional GBA gene may offer therapeutic benefits.

[0402] To evaluate the ability of L protein (EG Tech) mRNA to express GBA protein in a plasmid DNA (pDNA) expression vector, a pDNA expression vector was first designed in which GBA was fused to the NanoLuc reporter protein via a peptide linker, and the expression of the GBA-NanoLuc fusion protein was driven by the CMV promoter. Figure 20A GBA pDNA was transfected into cells along with L protein (EG Tech) mRNA, as shown below.

[0403] NIH / 3T3 mouse fibroblast cells were obtained from ATCC and cultured according to the cell bank's instructions. Briefly, cells were cultured in a humidified environment of 37°C with 5% CO2 using Dubelcco modified Eagle medium (DMEM) supplemented with high glucose and GlutaMAX, containing 10% fetal bovine serum (FBS) and 1X antifungal / antibiotic. On day 0, NIH / 3T3 cells were seeded into 96-well plates. On day 1, 0.3 ng of L protein (EG Tech) mRNA was transfected into the cells using the MIRUS TransIT mRNA transfection kit according to the manufacturer's instructions. Immediately after adding the L protein (EG Tech) mRNA complex to the cells, cells were further transfected with GBA-NanoLuc pDNA (50 ng / well) using the MIRUS TransIT-Lenti transfection kit according to the manufacturer's instructions.

[0404] Because Gaucher disease is associated with increased inflammation in affected tissues, and increased inflammation is known to suppress pDNA expression, in some cases, IFN-β (1000 pg / ml) or Poly I:C (1000 ng / ml) was added to cells 4 hours after transfection to model the inflammatory environment and investigate the effect of L protein (EG Tech) mRNA on GOI expression in pDNA vectors in these settings.

[0405] On day 3, 48 hours post-transfection, the cell supernatant was removed, and the activity of the fused NanoLuc reporter protein was measured using the Promega Nano-Glo luciferase assay system according to the manufacturer's instructions to determine the expression of GBA protein in the cell lysate. Results showed... Figure 21 middle.

[0406] It was observed that when 0.3 ng L protein (EG Tech) mRNA was used per well, GBA expression of the pDNA vector in unstimulated cells increased by 23%. Figure 21 (Left bar). Stimulation of cells with the pro-inflammatory model molecules IFN-β and Poly I:C resulted in a reduction of GBA expression by approximately 80%. Notably, in cells stimulated with IFN-β and Poly I:C, L protein (EG Tech) mRNA was able to rescue 79% and 51% of GBA expression in the pDNA vector, respectively. Figure 21 (middle and right bars). These results demonstrate that L protein (EG Tech) mRNA can enhance gene expression of pDNA vectors under baseline conditions and in strongly pro-inflammatory environments, which is particularly important for diseases with a range of inflammatory components.

[0407] Example 15: The effect of L protein mRNA expression on plasmid DNA-induced cytokine induction.

[0408] Delivery of cytoplasmic plasmid DNA (pDNA) can lead to the activation of cellular response pathways (e.g., cGAS-STING and others), which in turn can lead to the activation of excessive inflammatory pathways in cells, resulting in decreased GOI expression and / or the induction of excessive inflammation in transduced cells. The latter can limit the applicability of DNA vectors for therapeutic use, as excessive inflammation may be associated with adverse reactivity and other side effects. Therefore, in this embodiment, the role of L protein (EG Tech) mRNA in inhibiting the activation of pro-inflammatory mediators induced by intracellularly delivered pDNA, with or without additional pro-inflammatory stimulation, was investigated.

[0409] The pDNA expressing GBA (as previously used) Figure 20AThe L protein (EG Tech) mRNA was transfected into cells as shown below. Mouse fibroblast NIH / 3T3 cells were obtained from ATCC and cultured according to the cell bank's instructions. Briefly, cells were cultured in a humidified environment of 37°C with 5% CO2 using Dubelcco modified Eagle medium (DMEM) supplemented with high glucose and GlutaMAX, containing 10% fetal bovine serum (FBS) and 1X antifungal / antibiotic. On day 0, NIH / 3T3 cells were seeded into 96-well plates. On day 1, cells were transfected with 0.3 ng or 1.25 ng of L protein (EG Tech) mRNA per well using the MIRUS TransIT mRNA transfection kit, according to the manufacturer's instructions. Immediately after adding the L protein (EG Tech) mRNA complex to the cells, cells were further transfected with GBA-NanoLuc pDNA (50 ng / well) using the MIRUS TransIT-Lenti transfection reagent, according to the manufacturer's instructions. On day 3, 48 hours after transfection, the cell supernatant was removed, cell debris was removed by centrifugation, and CCL2 secretion was analyzed using the BiolegendLegendPlex mouse antiviral response detector, following the manufacturer’s instructions. CCL2 is an inflammatory molecule induced by cytoplasmic double-stranded DNA through the cGAS-STING pathway.

[0410] When pDNA is co-transfected with L protein (EG Tech) mRNA, CCL2 induction is reduced in cells transfected with pDNA expressing GBA. Figure 22 These results demonstrate that L protein (EG Tech) mRNA can reduce the activation of excessive inflammatory pathways in pDNA-transfected cells, showing further promise for accelerating the in vivo application of pDNA technology.

[0411] Example 16: The effect of L protein mRNA expression on inflammation induced in plasmid DNA transfected cells.

[0412] In this embodiment, the analysis of the effect on L protein (EG Tech) mRNA was expanded to help suppress the induction of excessive inflammation in pDNA-transfected cells. In addition to pDNA expressing GBA, another treatment-related pDNA expression vector was designed, in which the NanoLuc reporter protein sequence is fused with an erythropoietin (EPO) coding sequence downstream of the CMV promoter. Figure 20B ).

[0413] NIH / 3T3 cells were grown and transfected with L protein (EG Tech) mRNA and GBA and EPO-encoded pDNA as described in the previous example. However, to investigate the effect of L protein (EG Tech) mRNA on suppressing pDNA-induced inflammation under more pro-inflammatory conditions, cells were further stimulated with 1000 ng / ml Poly I:C. This experimental setup allowed us to investigate the role of L protein (EG Tech) mRNA in suppressing excessive pro-inflammatory molecules when inflammatory signaling is induced by pDNA (e.g., the cGAS-STING pathway) and other orthogonal pathways. Forty-eight hours post-transfection, the cell supernatant was removed, cell debris was removed by centrifugation, and the secretion of CCL2, CCL5, CXCL10, IFN-α, and IFN-β was analyzed using the Biolegend LegendPlex mouse antiviral response detector according to the manufacturer's instructions.

[0414] According to observations, pDNA encoded by GBA ( Figures 23A-23E ) and EPO encode pDNA ( Figures 24A-24E Both transfected 3T3 cells and further stimulated with Poly I:C to induce high levels of secretion of inflammation-related molecules CCL2, CCL5, CXCL5, IFN-α, and IFN-β. When the results were compared with... Figure 22 When compared, the additive effect of pDNA-induced CCL2 and Poly I:C-induced CCL2 was evident. Importantly, in the presence of L protein (EG Tech) mRNA, the levels of CCL2, CCL5, CXCL5, IFN-α, and IFN-β decreased to baseline levels in untransfected and Poly I:C-unstimulated cells. These results further highlight the desirability of using L protein (EG Tech) mRNA in DNA-based therapies where the desired therapeutic effect must be achieved by simultaneously inhibiting pro-inflammatory signaling from multiple sources.

[0415] Example 17: The effect of L protein on saRNA expression reporter protein.

[0416] To evaluate the effect of L protein (EG Tech) RNA on enhancing GOI expression in the saRNA vector, a construct was designed in which the L protein (EG Tech) encoding RNA sequence, the L protein (EG Tech) mutant encoding RNA sequence, or the disordered negative control sequence were contained in the replication cassette of the self-amplified (saRNA) vector (respectively...). Figures 25A-25CThis differs from the embodiments described previously herein, where the L protein (EG Tech) encoding mRNA was trans-co-transfected with a VEEV-based saRNA vector. The vector used in this embodiment was constructed by cloning a secreted nanoluciferase (NanoLuc) and subsequently an IRES-driven L protein (EG Tech) expression cassette into a commercially available VEEV-based saRNA vector (Simplicon SCR724, Millipore Sigma). The L protein (EG Tech) WT encodes the wild-type protein sequence, the L protein (EG Tech) mutant encodes a version of the L protein (EG Tech) with specified amino acid mutations, and the scrambled negative control corresponds to an RNA sequence introducing an early stop codon to ensure that the functional L protein (EG Tech) is not expressed. Different amino acid mutants of the L protein (EG Tech) are used to modulate the activity of the L protein (EG Tech), thereby regulating NCT.

[0417] Following the method described in the previous example (Example 11), BJ cells were grown and transfected with a designed saRNA construct, using 200 ng saRNA / well. Two and five days post-transfection, Fluc expression in transfected cells was measured using the Promega Nano-Glo luciferase assay system according to the manufacturer's instructions, and normalized relative to the disordered negative control sequence conditions. Results are as follows... Figures 27A-27C As shown. It was observed that when the L protein (EG Tech) encoding RNA was contained in a VEEV-based saRNA replicon, on day 2 post-transfection, NanoLuc expression from the saRNA increased by 33-fold, 26-fold, or 18-fold, depending on whether the WT, E39A (SEQ ID NO:138), or K35Q L protein (SEQ ID NO:148) (EG Tech) mutant was used. On day 5 post-transfection, the effect of the L protein (EG Tech) in enhancing GOI expression became even stronger, with NanoLuc expression increasing by 63-fold, 35-fold, or 17-fold, depending on whether the WT or mutant version of the L protein (EG Tech) was used.

[0418] In summary, these results demonstrate that the role of L protein (EG Tech) RNA is universal and can be used to enhance GOI expression in different conformations: including when L protein (EG Tech) mRNA and GOI mRNA are co-transfected, when L protein (EG Tech) mRNA and GOI mRNA are part of a bicistronic mRNA expression vector, when L protein (EG Tech) mRNA is co-transfected with a saRNA expression vector, and when L protein (EG Tech) RNA is part of a saRNA replicon, using L protein (EG Tech) mRNA to enhance GOI mRNA expression.

[0419] Example 18: The effect of L protein mRNA expression on natural nucleoside inflammatory signaling.

[0420] To further demonstrate the universality of L protein (EG Tech) mRNA in enhancing GOI expression and reducing pro-inflammatory signaling activation, L protein (EG Tech) was tested in parallel in different cell lines. The selected cell lines were BJ human fibroblasts, A549 human lung epithelial cells, and C2C12 mouse myocytes. Experiments were performed as described in Example 2. Briefly, cells were transfected with single mRNA constructs encoding luciferase and L protein (NCT inhibitor) proteins separated by p2A elements. These mRNA constructs contained an ARCA cap and a mixture of 50%–50% natural uridine and pseudouridine. Due to the use of natural uridine in the mRNA, these mRNA constructs are inherently pro-inflammatory because they can activate innate immune signaling pathways in transfected cells, as is known to those skilled in the art. Luciferase activity was measured using the Promega NanoGlo luciferase assay system 48 hours post-transfection. Following the manufacturer's instructions, the activation of innate immune signaling pathways was measured using the IFN-β ELISA (R&D Biosystems) and the BiolegendLegendPlex human antiviral detector.

[0421] The results showed that including the L protein (EG Tech) RNA sequence in the mRNA expression vector increased GOI expression by 3.5-7.6 times, depending on the cell line used. Figures 27A-27C Furthermore, the use of L protein (EG Tech) virtually eliminated the induction of IFN-β expression by ARCA-capped mRNA containing 50% natural uridine in all test cell lines, as measured by IFN-β ELISA. Figures 28A-28C Furthermore, as assessed using the LegendPlex human antiviral detector in BJ cells, the induction of several other pro-inflammatory regulators was also eliminated. Figures 29A-29D ).

[0422] These results demonstrate that the L protein (EG Tech) can be used to improve GOI expression while inhibiting the activation of unwanted inflammatory pathways in various cell types.

Claims

1. A composition comprising: ribonucleic acid (RNA) encoding the target protein or deoxyribonucleic acid (DNA) encoding the target protein; and Ribonucleic acid (RNA) encoding a nucleocytoplasmic transport (NCT) inhibitory protein.

2. A composition comprising a self-amplifying ribonucleic acid (saRNA) encoding a nucleocytoplasmic transport (NCT) inhibitory protein.

3. The composition of claim 1, wherein the RNA encoding the target protein and / or the RNA encoding the NCT inhibitory protein is messenger RNA (mRNA).

4. The composition of claim 2, wherein the saRNA encodes a target protein.

5. The composition of claim 2, wherein the composition comprises mRNA encoding a target protein.

6. The composition of any one of claims 1-5, wherein the mRNA or saRNA encoding the target protein comprises at least one unmodified uridine nucleotide.

7. The composition of claim 2, wherein the saRNA is encoded by a sequence comprising one or more of SEQ ID NOs: 79-97, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereof.

8. The composition of any one of claims 1-7, wherein the NCT inhibitory protein comprises: a sequence of Table 4, a sequence of SEQ ID NO: 65, 66, 67 or 68, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

9. The composition of any one of claims 1-8, wherein the target protein is a viral antigen.

10. The composition of claim 9, wherein the viral antigen is selected from Table 3.

11. The composition of any one of claims 1-10, further comprising a delivery vehicle.

12. The composition of claim 11, wherein the delivery vehicle is a lipid nanoparticle (LNP).

13. A method for improving the expression of a target protein, the method comprising expressing a ribonucleic acid (RNA) encoding a nucleocytoplasmic transport (NCT) inhibitory protein, wherein the target protein is encoded by ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

14. The method of claim 13, wherein the RNA encoding the target protein is a self-amplifying ribonucleic acid (saRNA).

15. The method of claim 13, wherein the RNA encoding the target protein is messenger ribonucleic acid (mRNA).

16. The method of any one of claims 13-15, wherein the RNA encoding the NCT inhibitory protein is messenger RNA (mRNA).

17. The method of claim 14, wherein the saRNA encoding the target protein also encodes a NCT-inhibiting protein.

18. The method of any one of claims 13-17, wherein the RNA encoding the target protein comprises at least one unmodified uridine nucleotide.

19. The method of any one of claims 13-18, wherein the expression of the NCT inhibitory protein increases the expression level of the target protein by about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 200 times, about 500 times, or about 1000 times.

20. The method of any one of claims 13-18, wherein expression of the NCT inhibitory protein reduces the expression level of the target protein by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90%.

21. The method of any one of claims 13-20, wherein the expression of the NCT inhibitory protein increases the activity of the target protein by about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 150 times, about 200 times, or about 300 times.

22. The method of any one of claims 13-21, wherein the expression of the NCT inhibitory protein causes the EC of the target protein to 50 About 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold lower.

23. The method of any one of claims 13-22, wherein the NCT inhibitory protein and the target protein are expressed in a cell or a subject.

24. The method of claim 23, wherein expression of the NCT inhibitory protein increases the duration of presence of the target protein in a cell or subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold.

25. The method of claim 23 or claim 24, wherein co-expression of the NCT inhibitory protein reduces the coefficient of variation (CV%) of the target protein in the subject's tissue by about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, or about 3-fold.

26. The method of any one of claims 13-25, wherein the NCT inhibitory protein comprises: a sequence of Table 4, a sequence of SEQ ID NO: 65, 66, 67 or 68, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

27. The method of any one of claims 13-26, wherein the target protein is a viral antigen.

28. A method of reducing cellular signaling in response to a stressor, the method comprising introducing into a cell a polynucleotide encoding an NCT inhibitor.

29. The method of claim 28, wherein the cell signaling in response to a stressor is expression of interferon-β, and wherein introducing the NCT inhibitor reduces expression of interferon-β in the cell by about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold, or about 100,000-fold.

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