Compositions and methods for RNA-based protein expression

Abstract

The compositions and methods provided herein include ribonucleic acid (RNA) encoding a nucleocytoplasmic transport (NCT) inhibitor protein for improving target protein expression (e.g., target protein expression encoded by a DNA vector, mRNA, self-amplifying RNA, or RNA containing unmodified uridine nucleotides). The compositions and methods provided herein can be used to improve the expression of any target protein (e.g., viral protein antigens for use in vaccines).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 323,332, filed March 24, 2022, 63 / 329,050, filed April 8, 2022, 63 / 375,350, filed September 12, 2022, and 63 / 478,633, filed January 5, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING The contents of the electronic sequence listing (EXCI_004_04WO_SeqList_ST26.xml; size: 258,708 bytes; and creation date: March 23, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background The therapeutic application of target protein expression for the treatment and prevention of diseases has made considerable progress in recent years. For example, exogenous proteins have been designed to replace abnormal or defective proteins in many 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) induce cellular stress signals that directly inhibit protein production; RNA is rapidly degraded in cells, resulting in low protein expression levels; and DNA typically leads to protein overexpression. Therefore, there is a need for compositions and methods that overcome these challenges and improve target protein expression. Summary of the Invention [Means for solving the problem]

[0004] Abstract Provided herein are compositions comprising RNA encoding a nucleocytoplasmic transport (NCT) inhibitor protein and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a target protein. Also provided are methods for improving expression of a target protein using RNA encoding an NCT inhibitor protein.

[0005] In some embodiments, the compositions of the present disclosure comprise a self-amplifying ribonucleic acid (saRNA) encoding a nucleocytoplasmic transport (NCT) inhibitor protein. In some embodiments, the RNA encoding the target protein and / or the RNA encoding the NCT inhibitor protein is messenger RNA (mRNA).

[0006] In some embodiments, the saRNA encoding the NCT inhibitor protein encodes a target protein. In some embodiments, the composition comprising the saRNA encoding the NCT inhibitor protein comprises mRNA encoding the target protein.

[0007] In some embodiments, the mRNA or saRNA encoding the target protein comprises at least one unmodified uridine nucleotide.

[0008] In some embodiments, the saRNA comprises a sequence comprising any one 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 thereto.

[0009] In some embodiments, the NCT inhibitor 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.

[0010] In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is selected from Table 3.

[0011] In some embodiments, the composition comprises a delivery vehicle. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP).

[0012] In some aspects, provided herein are methods for improving expression of a target protein, comprising expressing a ribonucleic acid (RNA) encoding a nucleocytoplasmic transport (NCT) inhibitor protein, wherein said target protein is encoded by ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0013] In some embodiments of the method, the RNA encoding the target protein is a self-amplifying ribonucleic acid (saRNA). In some embodiments of the method, the RNA encoding the target protein is a messenger ribonucleic acid (mRNA). In some embodiments of the method, the RNA encoding the NCT inhibitor protein is a messenger RNA (mRNA). In some embodiments of the method, the saRNA encoding the target protein also encodes the NCT inhibitor protein.

[0014] In some embodiments of the method, the RNA encoding the target protein comprises at least one unmodified uridine nucleotide.

[0015] In some embodiments of the method, expression of the NCT inhibitor protein increases the target protein expression level 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, 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.

[0016] In some embodiments of the method, 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%.

[0017] In some embodiments of the method, 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.

[0018] In some embodiments of the method, expression of the NCT inhibitor protein reduces the EC50 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.

[0019] In some embodiments of the method, the NCT inhibitor protein and the target protein are expressed in a cell or a subject. In some embodiments of the method, the NCT inhibitor protein increases the duration that the target protein is found in the 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.

[0020] In some embodiments of the method, co-expression of the NCT inhibitor protein reduces the coefficient of variation (CV%) of the target protein in the tissue of the subject 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.

[0021] In some embodiments of the method, the NCT inhibitor 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.

[0022] In some embodiments of the method, the target protein is a viral antigen.

[0023] In some embodiments, provided herein are methods of reducing cell signaling in response to a stressor, comprising introducing into a cell a polynucleotide encoding an NCT inhibitor.

[0024] In some embodiments of the method, the cell signaling in response to the stressor is expression of interferon-beta, and introduction of the NCT inhibitor reduces interferon-beta expression in the cell by about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold, or about 100,000-fold. [Brief explanation of the drawings]

[0025] [Figure 1] 1A-1E show exemplary DNA templates for RNA preparation.

[0026] [Figure 2] Figures 2A and 2B show RNA constructs encoding luciferase. Figure 2A shows an exemplary RNA encoding luciferase. Figure 2B shows an exemplary RNA encoding an NCT inhibitory protein that can be co-expressed with the RNA of Figure 2A.

[0027] [Figure 3]Figures 3A and 3B show RNA constructs encoding the Sars-CoV2 spike protein. Figure 3A shows an exemplary RNA encoding the Sars-CoV2 spike protein. Figure 3B shows an exemplary RNA encoding an NCT inhibitory protein that can be co-expressed with the RNA of Figure 3A.

[0028] [Figure 4] Figures 4A and 4B show RNA constructs encoding glucosylceramidase (GBA). Figure 4A shows an exemplary RNA encoding a GBA protein. Figure 4B shows an exemplary RNA encoding an NCT inhibitory protein that can be co-expressed with the RNA of Figure 4A.

[0029] [Figure 5] Figures 5A and 5B show the layout of DNA constructs used for in vitro transcription (IVT) to generate a single mRNA construct encoding the L protein (L) and nanoluciferase (nLuc). Figure 5A shows the DNA construct used for IVT to generate a single mRNA construct encoding L and nLuc (L-NLuc) separated by a p2A separation element. Figure 5B shows the DNA construct used for IVT to generate a single mRNA construct encoding nLuc and L (NLuc-L) separated by a p2A separation element.

[0030] [Figure 6]Figures 6A and 6B show luciferase activity assays when luciferase was expressed from mammalian cells 24, 48, and 72 hours after transfection. Measurements were performed in quadruplicate to represent the mean. Error bars indicate standard deviation. The figures show total luciferase (nLuc) activity, measured in relative light units (RLU), using transfections of mRNA encoding either the L protein alone (L), nanoluciferase (nLuc) alone, or two configurations of a single mRNA encoding L and nLuc, as shown in Figures 5A (L-nLuc) and 5B (nLuc-L). These mRNAs contained approximately 50% natural uridine and approximately 50% pseudouridine. Figure 6A shows the results of transfections of mouse C2C12 cells. The presence of L protein in combination with nLuc showed increased activity as a result of higher expression of nLuc in the presence of L protein, up to 3.5-fold or 5.5-fold for L-nLuc and nLuc-L, respectively, after 72 hours. Figure 6B shows results from transfection of human BJ cells. The effect from the presence of L protein in combination with nLuc shows a more significant increase in activity as a result of higher expression of nLuc in the presence of L, up to 6.5-fold or 14-fold for L-nLuc and nLuc-L, respectively, after 72 hours in the presence of mRNA containing approximately 50% natural uridines.

[0031] [Figure 7]Figure 7 shows the activity assay of luciferase expressed from mammalian cells in the presence of an external source of interferon (IFN) beta. C2C12 cells were transfected with mRNA encoding either the L protein alone (L), nLuc alone, or a single mRNA encoding both L and nLuc, as shown in Figures 5A (L-nLuc) and 5B (nLuc-L). These mRNAs contained approximately 50% natural uridine and approximately 50% pseudouridine. Six hours after transfection, a 10-fold dilution series of IFN-beta was added to the cells. Luciferase activity was then measured 48 hours after IFN-beta stimulation. The graph shows the reduction in luciferase activity as a result of the reduction in luciferase expression in the presence of IFN-beta (as a percentage of the total expression of nLuc in the absence of IFN-beta). The approximate EC50 value of IFN-beta required to reduce luciferase expression for each construct is shown as a dotted line. The presence of the L protein protects cells from the presence of exogenous IFN-beta by 10-fold and 12-fold (for L-nLuc and nLuc-L, respectively), as demonstrated by the increase in approximate EC50 values.

[0032] [Figure 8] Figure 8 shows cellular IFN-beta production as an indicator of intracellular stress responses. Human BJ cells were transfected with mRNA encoding only the L protein (L), only nLuc, or two configurations of a single mRNA encoding both L and nLuc, as shown in Figures 5A (L-nLuc) and 5B (nLuc-L). These mRNAs contained approximately 50% natural uridine and approximately 50% pseudouridine. The presence of protein L inhibited IFN-beta production in the cells to baseline levels.

[0033] [Figure 9] Figure 9 shows the effect of mRNA-based L protein expression on poly I:C-induced downregulation of gene of interest expression.

[0034] [Figure 10] FIG. 10 shows the effect of mRNA-based L protein expression on IFN-beta-induced downregulation of gene of interest expression.

[0035] [Figure 11] Figures 11A-11C show the effect of mRNA-based L protein expression on natural nucleoside-induced downregulation of gene of interest expression. Figure 11A shows the effect of L protein on expression of a gene of interest from mRNA containing only natural nucleosides (including natural uridine) using different concentrations of L protein. Figure 11B shows the effect of L protein expression on modified mRNA containing only non-inflammatory nucleosides (including pseudo-uridine) using different concentrations of L protein. Figure 11C compares the effect of L protein on expression of a gene of interest from natural nucleoside-only mRNA and modified mRNA over a time course of 20 days post-transfection.

[0036] [Figure 12] Figures 12A and 12B show the effect of L protein expression on the expression of a gene of interest in self-amplifying RNA (saRNA). Figure 12A shows the effect of L protein expression on the expression of a gene of interest in saRNA using different concentrations of L protein. Figure 12B compares the effect of different concentrations of L protein on the expression of a gene of interest from saRNA over a time course of 20 days post-transfection.

[0037] [Figure 13] FIG. 13 shows the effect of L protein expression on the expression of a gene of interest in self-amplified RNA (saRNA) using mutant forms of L protein (SEQ ID NOs: 65, 66, 67, 68) at different concentrations 6 days after transfection.

[0038] [Figure 14]FIG. 14 shows the effect of L protein expression on the expression of a gene of interest in self-amplified RNA (saRNA) using mutant forms of L protein (SEQ ID NOs: 65, 66, 67, 68) at different concentrations 9 days after transfection.

[0039] [Figure 15] Figures 15A and 15B show the effect of L protein and self-amplifying RNA (saRNA) expressing a gene of interest on the relative expression of the inflammatory mediators IFN-beta (Figure 15A) and IP-10 (Figure 15B).

[0040] [Figure 16] Figure 16 shows the effect of L protein expression on self-amplifying RNA (saRNA)-based gene of interest expression over time in vivo. SaRNA expression of L protein significantly increased target protein expression by the saRNA construct.

[0041] [Figure 17] Figure 17 shows the effect of L protein expression on tetherin expression in the presence of IFN-beta. Tetherin is downstream of IFN-beta and is a negative regulator of VLP production.

[0042] [Figure 18] Figures 18A and 18B show mRNA expression vectors in which a gene of interest (GOI) was tagged with the NanoLuc reporter protein via a linker peptide. The coding sequences of G6PC1 (Figure 18A, SEQ ID NO: 99) and SERPINA1 (Figure 18B, SEQ ID NO: 104) were flanked by the hBG 5'UTR (SEQ ID NO: 98) and hBG 3'UTR (SEQ ID NO: 102). The mRNA expression vector contained a Cap1 mRNA cap structure, N1-methylpseudouridine modification, and a 120-nt polyA tail. mRNA was produced using in vitro transcription (IVT).

[0043] [Figure 19]Figures 19A-19D show the expression of G6PC1 and SERPINA1 mRNA in cell lysates. Each experiment was performed with three technical replicates. A total of five independent experiments (biological replicates) were performed. Figure 19A shows G6PC1 expression from L protein expression (EG Tech) in relative light units (RLU). Figure 19B shows G6PC1 expression from L protein expression (EG Tech) as fold change compared to no L protein. Figure 19C shows SERPINA1 expression from L protein expression (EG Tech) in relative light units (RLU). Figure 19D shows SERPINA1 expression from L protein expression (EG Tech) as fold change compared to no L protein.

[0044] [Figure 20] Figures 20A and 20B show plasmid DNA expression vectors encoding the lysosomal acid glucosylceramidase (GBA) gene (Figure 20A) and the erythropoietin (EPO) gene (Figure 20B), each tagged with a NanoLuc reporter protein via a peptide linker and driven by a CMV promoter.

[0045] [Figure 21] Figure 21 shows GBA expression 48 hours after transfection with the DNA expression vector of Figure 20A and mRNA encoding the L protein (EG Tech). Stimulation of cells with the pro-inflammatory molecules IFN-beta and poly I:C reduced GBA expression. However, in cells stimulated with IFN-beta and poly I:C, L protein mRNA expression increased GBA expression compared to cells without L protein.

[0046] [Figure 22] Figure 22 shows CCL2 induction in cells transfected with GBA expression plasmid DNA. CCL2 induction was reduced when L protein mRNA was also added to the cells.

[0047] [Figure 23-1]Figures 23A-23E show cells transfected with the GBA-encoding plasmid DNA of Figure 20A and stimulated with poly I:C to induce high levels of secretion of the inflammation-related molecules CCL2 (Figure 23A), CCL5 (Figure 23B), CXCL10 (Figure 24C), IFN-alpha (Figure 23D), and IFN-beta (Figure 23E). However, in the presence of L protein mRNA, the levels of each inflammatory molecule were reduced to the baseline levels of untransfected and unstimulated cells. [Figure 23-2] Same as above.

[0048] [Figure 24-1] Figures 24A-24E show cells transfected with the EPO-encoding plasmid DNA of Figure 20B and stimulated with poly I:C to induce high levels of secretion of the inflammation-related molecules CCL2 (Figure 24A), CCL5 (Figure 24B), CXCL10 (Figure 24C), IFN-alpha (Figure 24D), and IFN-beta (Figure 24E). However, in the presence of L protein mRNA, the levels of each inflammatory molecule were reduced to the baseline levels of untransfected and unstimulated cells. [Figure 24-2] Same as above.

[0049] [Figure 25] 25A-25C show self-amplifying RNA vector constructs encoding the nanoluciferase reporter gene along with the L protein coding sequence (FIG. 25A), the L protein mutant coding sequence (FIG. 25B), or the scrambled negative control sequence (FIG. 25C).

[0050] [Figure 26] Figure 26 shows the self-amplifying RNA construct-mediated expression levels of the L protein shown in Figures 25A-25C 2 and 5 days after transfection. The L protein significantly increased the saRNA-mediated expression of nanoluciferase.

[0051] [Figure 27]Figures 27A-27C show the effects of a single construct encoding both the L protein and luciferase gene (mRNA contained the pro-inflammatory ARCA cap and a 50%-50% mixture of natural uridine and pseudo-uridine) in human fibroblasts (Figure 27A), A549 cells (Figure 27B), and C2C12 mouse muscle cells (Figure 27C) 48 hours after transfection.

[0052] [Figure 28] Figures 28A-28C show that L protein mRNA expression, as measured by IFN-beta ELISA, was substantially abrogated by ARCA-capped and 50% native uridine-containing mRNA in human fibroblasts (Figure 28A), A549 cells (Figure 28B), and C2C12 mouse muscle cells (Figure 28C).

[0053] [Figure 29] Figures 29A-29D show that in the experiments described in Figures 27A-27C and Figures 28A-28D, induction of other pro-inflammatory modulators: IFN-beta Figure 29A; IFN-lambda 1 Figure 29B; IFN-lambda 2 / 3 Figure 29C; and IP-10 Figure 29D was also abrogated by L protein mRNA expression.

[0054] [Figure 30-1]Figures 30A-30H show exemplary arrangements of self-amplifying RNA constructs encoding the nonstructural protein (NSP1, NSP2, NSP3, and NSP4) subgenomic promoters (SGPs), gene of interest (GOI), internal ribosome entry site (IRES), P2A self-cleavage site, L protein (EG tech), and 5' and 3' UTRs, as well as an RNA cap and poly-adenosine tail. The saRNA constructs provided herein can be designed as cis-replicons in which the gene of interest (GOI), L protein, and saRNA replicase cassette are part of the same sequence of saRNA (Figures 30A-30C), as trans-replicons in which at least one RNA molecule containing the GOI and / or L protein does not contain the saRNA replicase cassette and the saRNA replicase cassette is provided on a different saRNA molecule (Figures 30D-30F), or as part of a combination of saRNA replicons in which at least one of the saRNA components (such as the replicase cassette, GOI, and / or L protein components) is provided as replication-incompetent RNA (e.g., as messenger RNA) (Figures 30G-30H). [Figure 30-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0055] Detailed Description Various compositions, methods, and kits for improving target protein expression are disclosed herein. In one embodiment, the disclosed compositions, methods, and kits provide ribonucleic acid (RNA) encoding a nucleocytoplasmic transport (NCT) inhibitor protein for improving target protein expression. As provided herein, the RNA encoding the NCT inhibitor protein can improve the duration, quality, and / or quantity of expression of a target protein encoded by RNA or DNA introduced into a cell. In some embodiments, the NCT inhibitor protein improves self-amplifying RNA (saRNA) expression of a target protein introduced into a cell. In some embodiments, the RNA encoding the NCT inhibitor protein improves expression of a target protein RNA containing at least one unmodified uridine nucleotide introduced into a cell.

[0056] In some embodiments of the present disclosure, the target protein is encoded by DNA and the NCT inhibitor protein is encoded by RNA (e.g., messenger RNA). In some embodiments, the target protein is encoded by RNA (e.g., messenger RNA) and the NCT inhibitor protein is encoded by the same or a separate messenger RNA. In some embodiments, the target protein is encoded by self-amplifying RNA (saRNA) and the NCT inhibitor protein is encoded by the same or a separate saRNA or mRNA. As provided herein, RNA encoding an NCT inhibitor protein can improve the duration, quality, and / or quantity of expression of a target protein encoded by DNA or RNA (e.g., saRNA or mRNA) introduced into a cell.

[0057] In some embodiments, the mRNA or saRNA of the present disclosure encoding a target protein comprises at least one unmodified uridine nucleotide. As provided herein, introduction of an RNA encoding an NCT inhibitor protein can improve the duration, quality, and / or quantity of expression of the target protein encoded by the RNA comprising one unmodified uridine nucleotide introduced into a cell.

[0058] Also provided herein are combinations of RNA-encoded target proteins and DNA-encoded NCT inhibitor proteins.

[0059] In some embodiments, the nucleocytoplasmic transport (NCT) inhibitor may be selected from the group consisting of picornavirus leader (L) protein, picornavirus 2A protease, rhinovirus 3C protease, herpes simplex virus (HSV) ICP27 protein, and rhabdovirus matrix (M) protein.

[0060] As discussed throughout this specification, RNA-mediated inhibition of nucleocytoplasmic transport by NCT ​​inhibitor proteins can inhibit cell signaling in response to cellular stress (e.g., interferon-beta) and can improve cellular responses to innate immune signaling, pro-inflammatory signaling, delivery vector-induced inflammation, natural nucleoside mRNA transfection, self-amplifying RNA transfection, and target protein expression. Without being bound by theory, it is believed that the compositions and methods of the present disclosure prevent cellular regulatory mechanisms from being activated in response to target protein expression, which improves the yield and / or functionality of the target protein.

[0061] In some embodiments, the disclosed methods and systems (e.g., introduction of RNA encoding an NCT inhibitor) are shown to improve control of cellular stress responses and prevent adverse effects on target protein expression due to in vivo inflammation and interferon induction associated with introduction of DNA and RNA into cells.

[0062] I. Definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with and techniques of chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0063] It should be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a drug candidate" refers to one protein or a mixture of such candidates, and reference to "the method" includes reference to equivalent steps and methods known to those of skill in the art, etc.

[0064] As used herein, the term "approximately" or "about" as applied to one or more reference values ​​refers to a value that is on the same order of magnitude and / or within a similar range to the stated reference value. In certain embodiments, unless otherwise stated or clear from the context, the term "approximately" or "about" refers to a range of values ​​that falls within 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 (greater than or less than the numerical value) of the stated reference value (except where such numerical value exceeds 100% of possible values).

[0065] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that stated range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated range or intervening value within that stated range, is included within the scope of the invention. The upper or lower limits of these smaller ranges may independently be included in the smaller ranges, and these are also encompassed within the scope of the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0066] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. These terms also encompass amino acid polymers that have been modified to include, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0067] As used herein, the term "native" or "wild-type" refers to a nucleotide sequence (e.g., a gene, or a gene product (e.g., an RNA or polypeptide)) that is present in a wild-type cell, tissue, organ, or organism. As used herein, the term "variant" refers to a variant of a reference polynucleotide or polypeptide sequence (e.g., a native polynucleotide or polypeptide sequence) (i.e., having less than 100% sequence identity to the reference polynucleotide or polypeptide sequence).

[0068] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any subject desiring treatment or therapy. The subject may be a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, e.g., a cynomolgus monkey. In some embodiments, the subject is a companion animal or a service animal (e.g., a cat or dog).

[0069] The term "treating" or "treatment" is used herein generally to mean using a therapeutic agent to obtain a desired pharmacological and / or physiological effect. The term treating or treatment can include treatment of a cell, a plurality of cells, a tissue, or a subject. The effect can be preventative, in terms of complete or partial prevention of a disease or its symptoms (e.g., reducing the likelihood that a disease or its symptoms will occur in a subject), and / or therapeutic, in terms of complete or partial reduction of symptoms, or partial or complete cure of a disease and / or adverse effects caused by a disease. As used herein, "treatment" includes any treatment of a disease in a mammal, including: (a) prevention of morbidity in a subject who may be susceptible to a disease but has not yet been diagnosed with the disease; (b) inhibition or delay of the onset or development of a disease; or (c) alleviation of a disease (e.g., amelioration of a disease or a symptom associated with a disease). A therapeutic agent can be administered before, during, or after the onset of a disease. Treatment of disease during disease progression may be particularly interesting if such treatment stabilizes or reduces undesirable clinical symptoms in the patient. In some embodiments, treatment occurs before complete loss of function in the affected tissue. In some embodiments, the treatment of the present invention will be administered before the symptomatic stage of the disease; in some embodiments, during the symptomatic stage of the disease; and in some embodiments, after the symptomatic stage of the disease.

[0070] It is to be understood that the invention is not limited to the particular methodology, products, devices, and factors described, as such methods, devices, and formulations may, of course, vary. It is also to 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 the invention, which will be limited only by the appended claims.

[0071] II. Target Protein Provided herein is a composition comprising RNA encoding a nucleocytoplasmic transport (NCT) inhibitor protein and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a target protein. Also provided herein is a method for improving the expression of a target protein using RNA encoding an NCT inhibitor protein. Examples of target protein sequences that can be introduced into cells with RNA encoding an NCT inhibitor protein include, but are not limited to, the sequences in Tables 1 and 2.

[0072] In some embodiments, the target protein is an antibody; an antibody-like molecule; a receptor; a monoclonal antibody; a portion or fragment of an antibody; 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, brentuximab vedotin, brodalumab, brolucizumab, burosumab, canakinumab, caplacizumab, capromab, catumaxomab, cemiplimab, certolizumab pegol, cetuximab, crizanlizumab, daclizumab , daratumumab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emapalumab, emicizumab, enfortumab vedotin, eptinezumab, erenumab, ertumaxomab, etaracizumab, evolocumab, fremanezumab, galcanezumab, gemtuzumab ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, idarucizumab, imciromab, infliximab, inotuzumab ozogamicin Zogamicin, ipilimumab, isatuximab, itolizumab, ixekizumab, lanadelumab, lokivetmab, mepolizumab, mogamulizumab, moxetumomab pasudotox, natalizumab, necitumumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab vedotin, racotumomab, ramucirumab , ranibizumab, raxibacumab, ravulizumab, reslizumab, risankizumab, rituximab, alumab, romosozumab, rovelizumab, ruplizumab, sacituzumab govitecan, sarilumab, secukinumab, siltuximab, talquetamab, teclistamab, teprotumumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, ustekinumab, and vedolizumab.Polypeptide sequences of such antibodies are publicly available - for example, the Thera-SAbDab database (opig.stats.ox.ac.uk) described in Raybould et al. (2020) Thera-SAbDab: the Therapeutic Structural Antibody Database. Nucleic Acids Res. 48(D1):gkz827.

[0073] 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 engager (BiTE) (such as blinatumomab (MT103) and solitomab). In some embodiments, the target protein is a protein-fold-based binding ligand or binder (such as an adnectin, anticalin, avimer, finomer, Kunitz domain, knottin, affibody, or DARPin).

[0074] In some embodiments, the target protein is a blood protein. Non-limiting examples of blood proteins include transferrin, t-PA, hirudin, C1 esterase inhibitor, antithrombin, plasma kallikrein inhibitor, plasmin, prothrombin complex, complement components, prealbumin (transthyretin), alpha 1 antitrypsin, alpha-1-acid glycoprotein, alpha-1-fetoprotein, alpha 2-macroglobulin, gamma globulin, beta-2 microglobulin, haptoglobin, ceruloplasmin, complement component 3, complement component 4, C-reactive protein (C RP), lipoproteins (chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL)), transferrin, prothrombin, mannose-binding lectin (MBL), albumin, globulins, fibrinogen, regulatory factors, and coagulation factors, such as factor I, factor II, factor III, factor IV, factor V, factor VI, factor VII, factor IX, factor X, factor XI, factor XII, factor XIII, von Willebrand factor (von Willeband factor, prekallikrein, Fitzgerald factor, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, alpha 2-antiplasmin, tissue plasminogen activator, urokinase, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and cancer procoagulant. In some embodiments, the target protein is a thrombolytic drug. Non-limiting examples of thrombolytic drugs include Eminase (anistreplase), Retavase (reteplase), Streptase (streptokinase, fungal kinase), alteplase, t-PA (a class of drugs that includes Activase), TNKase (tenecteplase), Abbokinase, and Kinlytic (rokinase).

[0075] In some embodiments, the target protein is a growth factor. Non-limiting examples of 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 cell motility stimulating factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor family, ciliary neurotrophic factor (CNF). NTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), colony-stimulating factors, macrophage colony-stimulating factor (M-CSF), epidermal growth factor (EGF), ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5, ephrin-B1, ephrin-B2, ephrin-B3, and fibroblast growth factors (FGF) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, GF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, fetal bovine somatotrophin (FBS), GDNF family ligand, glial cell-derived neurotrophic factor (GDNF), neurturin, persephin, artemin, growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor, 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, IL-7, keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP) (also known as hepatocyte growth factor-like protein (HGFLP)), myostatin (GDF-8), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4),Target proteins include placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), and the Wnt signaling pathway. In some embodiments, the target protein is a hormone. Non-limiting examples of hormones include glucagon-like peptide-1, insulin, human growth hormone, follicle-stimulating hormone, calcitonin, lutropin, glucagon-like peptide-2, leptin, parathyroid hormone, chorionic gonadotropin, thyroid-stimulating hormone, erythropoietin, and glucagon.

[0076] In some embodiments, the target protein is an enzyme, non-limiting examples of which include alpha-glycosidase, glucocerebrosidase, iduronate-2-sulfatase, alpha-galactosidase, urate oxidase, N-acetyl-galactosidase, carboxypeptidase, hyaluronidase, DNAse, asparaginase, uricase, adenosine deaminase and other enterokinases, cyclases, caspases, cathepsins, oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Target proteins for expression using the compositions and methods of the present invention can include proteins related to enzyme replacement, such as agalsidase beta, agalsidase alfa, imiglucerase, taliglucerase alfa, velaglucerase alfa, alglucerase, sebelipase alfa, laronidase, idursulfase, elosulfase alfa, galsulfase, alglucosidase alfa, C3 inhibitor, glucose-6-phosphatase catalytic subunit 1, phenylalanine hydroxylase, and Fuller-Hunter correction factor. In some embodiments, the compositions and methods of the present invention are used to produce enzymes. Such enzymes may be useful in the manufacture of clinical test kits or other diagnostic assays.

[0077] In some embodiments, the target protein is a membrane protein. Exemplary membrane proteins include ion channels, gap junctions, ionotropic receptors, transporters, integral membrane proteins (such as cell surface receptors), and proteins that shuttle between the membrane and the cytosol in response to signal transduction. In some embodiments, the cell surface receptor is a G protein-coupled receptor (GPCR), a tyrosine kinase receptor, and an integrin. 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, a seven-transmembrane (7-TM) receptor, a heptahelical receptor, a serpentine receptor, or a G protein-linked receptor (GPLR). In some embodiments, the target protein is a class A GPCR, a class B GPCR, a class C GPCR, a class D GPCR, a class E GPCR, or a class F GPCR. In some embodiments, the target protein is a class 1 GPCR, a class 2 GPCR, a class 3 GPCR, a class 4 GPCR, a class 5 GPCR, or a class 6 GPCR. In some embodiments, the target protein is a rhodopsin-like GPCR, a secretin receptor family GPCR, a metabotropic glutamate / pheromone GPCR, a fungal mating pheromone receptor, a cyclic AMP receptor, or a Frizzled / Smoothened 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 mimetic. In some embodiments, the target protein is a protein that shuttles between the membrane and the cytosol in response to signal transduction, such as a Ras protein, a Rac protein, a Raf protein, a Gα subunit, an arrestin, a Src protein, and other effector proteins.

[0078] In some embodiments, the target protein is a nucleosidase, an NAD+ nucleosidase, a hydrolase, a glucosylase, a glucosylase that hydrolyzes N-glycosyl compounds, an NAD+ glycohydrolase, an NADase, a DPNase, a DPN hydrolase, an NAD hydrolase, a diphosphopyridine nucleosidase, a nicotinamide adenine dinucleotide nucleosidase, an NAD glycohydrolase, an NAD nucleosidase, or a nicotinamide adenine dinucleotide glycohydrolase. In some embodiments, the target protein is an enzyme involved in nicotinate and nicotinamide metabolism and calcium signaling pathways.

[0079] In some embodiments, the target protein is selected from the group consisting of abatacept, aflibercept, agalsidase beta, albiglutide, aldesleukin, alefacept, alglucerase, alglucosidase alfa, aliskiren, alpha-1-proteinase inhibitors, alteplase, anakinra, ancestim, anistreplase, anthrax immune globulin (human), antihemophilic factor, antithrombin alpha, antithrombin III (human), antithymocyte globulin, antithymocyte globulin (horse), antithymocyte globulin (rabbit), aprotinin, arcitumomab, asfotase alfa, asparaginase, asparaginase (erwinia chrysanthemi), becaplermin, belatacept, belactant, bivalirudin, botulinum toxin type A, botulinum toxin type B, buserelin, C1 esterase inhibitor (human), C1 esterase inhibitor, choriogonadotropin alfa, chorionic gonadotropin (human), chorionic gonadotropin, coagulation factor IX, coagulation factor VIIa, coagulation factor X (human), coagulation factor XIII A-subunit, collagenase, conestat alfa, corticotropin, cosyntropin, daptomycin, darbepoetin alfa, defibrotide, denileukin diftitox, desirudin, dornase alfa, drotrecogin Alfa, dulaglutide, efalizumab, efmoloctocog alfa, elosulfase alfa, enfuvirtide, epoetin alfa, epoetin zeta, eptifibatide, etanercept, exenatide, factor IX complex (human), fibrinogen concentrate (human), fibrinolysin acaplasmin, filgrastim, filgrastim-sndz, follitropin alfa, follitropin beta, galsulfase, gastric intrinsic factor, glatiramer acetate, glucagon (recombinant), glucarpidase, gramicidin D, hepatitis A vaccine, hepatitis B immune globulin, human calcitonin, human clostridium tetani toxoid immunoglobulin, human rabies virus immunoglobulin, human Rho(D) immunoglobulin, human serum albumin, human varicella zoster immunoglobulin, hyaluronidase, hyaluronidase, ibritumomab, idursulfase, imiglucerase, immunoglobulin (human),Infliximab, insulin aspart, insulin (bovine), insulin gludec, insulin detemir, insulin glargine, insulin glulisine, insulin lispro, insulin (pork), regular insulin, regular insulin, insulin (porcine), insulin (isophane), interferon alpha-2a (genetical recombination), interferon alpha-2b, interferon alpha con-1, interferon alpha-n1, interferon alpha-n9, Interferon beta-1a, interferon beta-1b, interferon gamma-1b, intravenous immunoglobulin, ipilimumab, ixekizumab, laronidase, lenograstim, lepirudin, leuprolide, liraglutide, lucinactant, lutropin alfa, lutropin alfa, mecasermin, menotropins, epoetin beta, metreleptin, muromonab, alpha interferon, nesiritide, ocriplasmin, omalizumab, oprelvekin, OspA lipoprotein, oxytocin , palifermin, pancrelipase, boractant alfa, 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 (iodinated), simoctocog alfa, sipuleucel-T, somatotropin (genetical recombination), somatropin (genetical recombination) ), streptokinase, sulodexide, succotocog alfa, taliglucerase alfa, teduglutide, teicoplanin, tenecteplase, teriparatide, tesamorelin, thrombomodulin alfa, thymalfasin, thyroglobulin, thymotropin alfa, thymotropin alfa, tocilizumab, tositumomab, tuberculin purified protein derivative, turoctocog alfa, urofollitropin, urokinase, vasopressin, and velaglucerase alfa.

[0080] In some embodiments, the target protein is a biosimilar. In some embodiments, the target protein is a therapeutic polypeptide (such as a biopharmaceutical, also known as a biologic); a potential biomarker polypeptide (such as a diagnostic, prognostic, or predictive biomarker); a prophylactic polypeptide (such as an adjuvant, soluble antigen, subviral particle, or virus-like particle); an accessory polypeptide (such as a polypeptide that supports the activity or binding of another molecule or inhibits another protein-protein interaction); or a polypeptide used in research (such as an antigen for generating novel monoclonal and polyclonal antibodies in animals, a reporter protein, or a tool polypeptide for studying physiological or pathological processes and the effects of drugs on these processes in animal models). In some embodiments, the target protein is a protein (such as a fluorescent protein) applied in microscopy and imaging. In some embodiments, the target protein is not a reporter protein (e.g., luciferase). In some embodiments, the target protein is a human protein.

[0081] In some embodiments, the target protein is an immunomodulatory agent. Non-limiting examples of immunomodulatory agents include cytokines, chemokines, interleukins, and interferons. In some embodiments, the target protein is an antigen for use as a vaccine or for research. In some embodiments, the target protein is a structural protein (such as a structural protein that functions in the assembly of a protein complex). In some embodiments, the target protein is an antimicrobial polypeptide; or an antiviral polypeptide. 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 pharmacokinetics-modulating protein, a small molecule-binding protein, an RNA-binding protein, or a protein-binding protein.

[0082] In some embodiments, the target protein is dopamine receptor 1 (DRD1), cystic fibrosis transmembrane conductance regulator (CFTR), C1 esterase inhibitor (C1-Inh), IL2-inducible T cell kinase (ITK), or NADase. In some embodiments, the target protein is firefly luciferase.

[0083] In some embodiments, the target protein is an antigen of a pathogenic organism (e.g., a virus or bacterium). In some embodiments, the target protein is a bacterial antigen - a protein antigen or a 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 a coronavirus, influenza virus, hepatitis B virus, 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 betacoronavirus. In some embodiments, the betacoronavirus is a severe acute respiratory syndrome (SARS) virus. In some embodiments, the SARS virus is a SARS-CoV-2 virus. In some embodiments, the betacoronavirus is a Middle East respiratory syndrome (MERS) virus.

[0084] Exemplary Target Protein Sequences In some embodiments, RNA encoding an NCT inhibitor protein (e.g., L protein) improves expression of a target protein, wherein the target protein is an antibody selected from the group including (see also Table 1): adalimumab, pembrolizumab, nivolumab, trastuzumab, bevacizumab, ustekinumab, ocrelizumab, secukinumab, vedolizumab, ibalizumab, nirsevimab, atortivimab, maftivimab, odesivimab, casirivimab, imdevimab, and brolucizumab. In some embodiments, RNA of the present disclosure encodes one or more sequences in Table 1, 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 to the aforementioned sequences. [Table 1]

[0085] In some embodiments, introduction of RNA encoding an NCT inhibitor protein (e.g., L protein) improves expression of a target protein, wherein said target protein is a blood protein or an immuno-oncology protein selected from the group including (see also Table 2): rFIX-Fc coagulation factor IX, taliglucerase, agalsidase beta, alglucosidase alpha, laronidase, idursulfase, HLA class I alpha chain (mouse K2-D1) and B2m (mouse), Nlrc5 (mouse), NLRC5 (human), scIL-12 (mouse), scIL-12 (human), and HLA class I alpha chain (human) and B2M (human). In some embodiments, an RNA of the present disclosure encodes one or more sequences of Table 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 to the foregoing sequences. [Table 2-1] [Table 2-2]

[0086] In some embodiments, the RNA encoding the NCT inhibitor protein (e.g., L protein) improves expression of a target protein, wherein the target protein is an antigen. In some embodiments, the target protein is a bacterial antigen-protein antigen or a 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 coronavirus, influenza virus, hepatitis B virus, 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 betacoronavirus. In some embodiments, the betacoronavirus is a severe acute respiratory syndrome (SARS) virus. In some embodiments, the SARS virus is a SARS-CoV-2 virus. In some embodiments, the betacoronavirus is a Middle East respiratory syndrome (MERS) virus.

[0087] In some embodiments, the viral protein is derived from West Nile virus, hi some embodiments, the viral protein is a precursor membrane protein (preM), an envelope glycoprotein (E), or a combination thereof.

[0088] In some embodiments, the viral antigen comprises one or more of the proteins from the viruses listed below in Table 3. In some embodiments, the RNA of the present disclosure encodes one or more sequences 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 to the foregoing sequences. [Table 3-1] [Table 3-2] [Table 3-3]

[0089] III. Nucleocytoplasmic Transport (NCT) Inhibitor Proteins Provided herein are compositions comprising RNA encoding a nucleocytoplasmic transport (NCT) inhibitor protein and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a target protein. Also provided herein are methods for improving target protein expression using RNA encoding an NCT inhibitor protein. Examples of NCT inhibitor proteins (e.g., L proteins) include, but are not limited to, the NCT inhibitor proteins 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 to the aforementioned NCT inhibitor proteins.

[0090] In some embodiments, the NCT inhibitor protein is a nuclear pore-blocking viral protein. In some embodiments, the NCT inhibitor protein is a native or synthetic peptide that can block nuclear pores, thereby inhibiting nucleocytoplasmic transport ("NCT"). In some embodiments, the NCT inhibitor protein is a viral protein. In some embodiments, the NCT inhibitor protein is selected from the group consisting of picornavirus leader (L) protein, picornavirus 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.

[0091] The NCT inhibitor protein may be a functional variant of any of the proteins disclosed herein. As used herein, the term "functional variant" refers to a protein that is homologous to the original protein and / or shares substantial sequence similarity with the original protein (e.g., 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 the original protein) and shares one or more functional characteristics of the original protein. For example, a functional variant of an NCT inhibitor protein retains the ability to inhibit NCT.

[0092] In some embodiments, the NCT inhibitor protein is a leader (L) protein from a picornavirus or a functional variant thereof. In some embodiments, the NCT inhibitor protein is a leader protein from the Cardiovirus, Hepatovirus, or Aphthovirus genus. For example, the NCT inhibitor protein can 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 himalayan hepatovirus, fopivirus, cardiovirus A, cardiovirus B, Theiler's murine encephalomyelitis virus (TMEV), Bilyuysk human encephalomyelitis virus (VHEV), Theiler-like rat virus (TRV), or Saffold virus (SAF-V).

[0093] In some embodiments, the NCT inhibitor protein is the L protein of Theiler's virus or a functional variant thereof. In some embodiments, the L protein comprises SEQ ID NO: 1. In some embodiments, the NCT inhibitor 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 to SEQ ID NO: 1.

[0094] In some embodiments, the L protein is an encephalomyocarditis virus (EMCV) L protein 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 thereto.

[0095] In some embodiments, the L protein is selected from the group consisting of a poliovirus L protein, an HRV16 L protein, a mengovirus L protein, and a Saffold virus 2 L protein, or a functional variant thereof.

[0096] In some embodiments, the NCT inhibitor protein is a picornavirus 2A protease or a functional variant thereof. In some embodiments, the NCT inhibitor protein is a 2A protease from an enterovirus, a rhinovirus, an aphthovirus, or a cardiovirus.

[0097] In some embodiments, the NCT inhibitor protein is a rhinovirus 3C protease or a functional variant thereof. In some embodiments, the NCT inhibitor protein is a picornavirus 3C protease. In some embodiments, the NCT inhibitor protein is a 3C protease derived from an enterovirus, a rhinovirus, an aphthovirus, or a cardiovirus. For example, in some non-limiting embodiments, the NCT inhibitor protein is a 3C protease derived from a poliovirus, a coxsackievirus, a rhinovirus, a foot-and-mouth disease virus, or a hepatovirus A.

[0098] In some embodiments, the NCT inhibitor protein is a coronavirus ORF6 protein or a functional variant thereof. In some embodiments, the NCT inhibitor protein is a viral protein that disrupts nuclear import complex formation and / or disrupts STAT1 transport into the nucleus.

[0099] In some embodiments, the NCT inhibitor protein is the Ebola virus VP24 protein or a functional variant thereof. In some embodiments, the NCT inhibitor protein is the Ebola virus VP40 protein or VP35 protein. In some embodiments, the NCT inhibitor protein is a viral protein that binds to the importin protein karyopherin-α (KPNA). In some embodiments, the NCT inhibitor protein is a viral protein that inhibits the binding of STAT1 to KPNA.

[0100] In some embodiments, the NCT inhibitor protein is a Venezuelan equine encephalitis virus (VEEV) capsid protein or a functional variant thereof. In some embodiments, the NCT inhibitor protein is a viral capsid protein that interacts with the nuclear pore complex.

[0101] In some embodiments, the NCT inhibitor protein is a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof. In some embodiments, the NCT inhibitor protein is an HSV ORF57 protein.

[0102] In some embodiments, the NCT inhibitor protein is a rhabdovirus matrix (M) protein or a functional variant thereof. In some embodiments, the NCT inhibitor protein is an M protein from a cytorhabdovirus, a dichorhavirus, an ephemerovirus, a lyssavirus, a novirhabdovirus, a nucleorhabdovirus, a perahabdovirus, a sigmavirus, a sprivivirus, a tibulovirus, a tupavirus, a varicosavirus, or a vesiculovirus.

[0103] In some embodiments, the NCT inhibitor protein may have an amino acid sequence comprising one or more of SEQ ID NOs: 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 to the foregoing sequences. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0104] In some embodiments, the NCT inhibitor (e.g., L protein) comprises a mutated sequence of any one of SEQ ID NOs: 161, 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 to the foregoing sequences.

[0105] In some embodiments, expression of the NCT inhibitor protein increases the target protein expression level 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, 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.

[0106] In some embodiments, 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%.

[0107] In some embodiments, 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.

[0108] In some embodiments, expression of the NCT inhibitor protein is associated with the EC of the target protein. 50 is reduced 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.

[0109] In some embodiments, the NCT inhibitor protein and the target protein are expressed in a cell or a subject.

[0110] In some embodiments, expression of the NCT inhibitor protein increases the duration that the target protein is found in a cell 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.

[0111] In some embodiments, expression of the NCT inhibitor protein reduces the coefficient of variation (CV%) of the target protein in the tissue of the subject 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.

[0112] In some embodiments, expression of an NCT inhibitor protein reduces cell signaling in response to a stressor. In some embodiments, the cell signaling in response to a stressor is expression of interferon-beta. In some embodiments, introduction of an NCT inhibitor reduces interferon-beta expression in cells by about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold, or about 100,000-fold.

[0113] In some embodiments, the NCT inhibitor protein improves characteristics of target protein expression, including, but not limited to, yield, quality, folding, post-translational modification, activity, localization, and downstream activity. In some embodiments, the NCT inhibitor protein may reduce one or more of misfolding, altered activity, incorrect post-translational modification, and / or toxicity. In some embodiments, expression of the NCT inhibitor protein increases the duration, quality, and / or quantity of the target protein.

[0114] In some embodiments, the RNA encoding the NCT inhibitor protein reduces interferon sensitivity and inflammation associated with target protein expression.

[0115] It is also contemplated herein that antibodies against NCT proteins and small interfering RNA (siRNA) of NCT proteins can improve target protein expression.For example, mRNA encoding anti-Xpo7 nanobody, mRNA encoding anti-NFT2 antibody, mRNA encoding Ran Q69L mutant protein, mRNA encoding MxB K131A mutant protein, mRNA encoding myristoylated ERK-derived phosphomimetic peptide, mRNA encoding myristoylated SMAD-derived phosphomimetic peptide, siRNA targeted to Ran mRNA, siRNA targeted to NTF2 mRNA, siRNA targeted to MxB mRNA, siRNA targeted to Xpo7 mRNA, or siRNA targeted to Nup93 mRNA can be used.NCT inhibitory siRNA is commercially available, for example, as a chemically synthesized sequence (IDT Technologies).

[0116] IV.RNA composition Provided herein are compositions comprising RNA encoding a nucleocytoplasmic transport (NCT) inhibitor protein and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a target protein. In some embodiments, the RNA encoding the NCT inhibitor protein and the RNA encoding the target protein are messenger RNA. In some embodiments, the RNA encoding the NCT inhibitor protein and the RNA encoding the target protein are self-amplifying RNA. In some embodiments, the RNA encoding the NCT inhibitor protein is messenger RNA and the RNA encoding the target protein is self-amplifying RNA. In some embodiments, the RNA encoding the NCT inhibitor protein is self-amplifying RNA and the RNA encoding the target protein is messenger RNA.

[0117] In some embodiments, a composition of the present disclosure comprises a messenger RNA encoding both an NCT inhibitor protein and a target protein, hi some embodiments, a composition comprises a first messenger RNA encoding an NCT inhibitor protein and a second messenger RNA encoding a target protein.

[0118] In some embodiments, the RNA of the present disclosure (e.g., saRNA or mRNA encoding an NCT inhibitor) is delivered to a cell simultaneously or on the same day as the saRNA, mRNA, or DNA encoding a target protein. In some embodiments, the RNA of the present disclosure (e.g., saRNA or mRNA encoding an NCT inhibitor) is delivered to a cell separately from the saRNA, mRNA, or DNA encoding a target protein or on a different day.

[0119] In some embodiments, RNA (e.g., saRNA or mRNA encoding an NCT inhibitor) is delivered to cells before saRNA, mRNA, or DNA encoding a target protein. In some embodiments, RNA (e.g., saRNA or mRNA encoding an NCT inhibitor) is delivered to cells about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 14 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 1 week, about 2 weeks, or about 3 weeks before saRNA, mRNA, or DNA encoding a target protein is delivered to cells.

[0120] In some embodiments, the RNA (e.g., saRNA or mRNA encoding an NCT inhibitor) is delivered to the cell after the saRNA, mRNA, or DNA encoding the target protein. In some embodiments, the RNA (e.g., saRNA or mRNA encoding an NCT inhibitor) is delivered to the cell about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 14 hours, about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, about 1 week, about 2 weeks, or about 3 weeks after the saRNA, mRNA, or DNA encoding the target protein is delivered to the cell.

[0121] In some embodiments, the L protein and / or gene of interest (GOI) are expressed using one or more saRNA vectors. Any saRNA vector known in the art may be used, including those based on alphaviruses and flaviviruses, 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), and classical swine fever virus (CSFV). The saRNA expression vector can be designed as a cis-replicon (the GOI, L protein, and saRNA replicase cassette are part of the same RNA sequence as the saRNA), as a trans-replicon (at least one RNA molecule containing the GOI and / or L protein does not contain the saRNA replicase cassette, while the saRNA replicase cassette is provided on a different saRNA molecule), or as a partial saRNA replicon combination (at least one of the saRNA components (such as the replicase cassette, GOI, and / or L protein components) is provided as a replication-incompetent RNA (e.g., as a regular mRNA)). Illustrations of selected cis-, trans-, and partial saRNA replicons are provided in Figures 30A-30H.

[0122] Exemplary NCT ​​inhibitor mRNA sequences In some embodiments, the messenger RNA (mRNA) encoding the 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 thereto.

[0123] In some embodiments, the messenger RNA (mRNA) encoding the mutant NCT inhibitor (e.g., an L protein having an E39A mutation) is encoded by SEQ ID NO: 162, 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 thereto.

[0124] In some embodiments, the messenger RNA (mRNA) encoding the mutant NCT inhibitor (e.g., an L protein having a K35Q mutation) is encoded by SEQ ID NO: 164, 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 thereto.

[0125] In some embodiments, the messenger RNA (mRNA) encoding the mutant NCT inhibitor (e.g., an L protein having a Y36F mutation) is encoded by SEQ ID NO: 166, 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 thereto.

[0126] In some embodiments, the messenger RNA (mRNA) encoding the mutant NCT inhibitor (e.g., an L protein having a D37A mutation) is encoded by SEQ ID NO: 168, 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 thereto.

[0127] In some embodiments, the messenger RNA (mRNA) encoding the 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.

[0128] In some embodiments, the messenger RNA (mRNA) encoding the NCT inhibitor (e.g., L protein) is encoded by the sequence of SEQ ID NO:177. Exemplary target protein mRNA sequences

[0129] In some embodiments, a messenger RNA (mRNA) of the disclosure is encoded by the following expression vector in the order depicted in Figures 18A and 18B, 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 to the foregoing sequences: In some embodiments, a mRNA of the disclosure is encoded by the following expression vector with a different coding sequence (e.g., SERPINA1 (SEQ ID NO: 104) or other coding sequence), 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 to the foregoing sequences:

[0130] hBG 5'UTR sequence: (SEQ ID NO: 98),

[0131] G6PC1 coding sequence: (SEQ ID NO: 99),

[0132] Linker sequence: (SEQ ID NO: 100),

[0133] NanoLuc reporter coding sequence: (SEQ ID NO: 101), and

[0134] hBg 3'UTR sequence: (SEQ ID NO: 102)

[0135] Exemplary saRNA sequences In some embodiments, the self-amplifying RNA (saRNA) of the present disclosure is encoded by the following expression vectors or sequences 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 to the aforementioned sequences: In some embodiments, the saRNA of the present disclosure is encoded by the following expression vectors having different gene of interest (GOI) sequences after assembly as depicted in Figure 30A as a Venezuelan equine encephalitis virus (VEEV)-based replicon, or sequences 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 to the aforementioned sequences: In some embodiments, the saRNA of the present disclosure is encoded by the following expression vectors with different gene of interest (GOI) sequences after assembly as depicted in FIG. 30A as a Venezuelan Equine Encephalitis Virus (VEEV)-based replicon, or sequences with 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 to the foregoing sequences:

[0136] 5'CSE: (SEQ ID NO: 79),

[0137] NSP1-4 and SGP cassette: (SEQ ID NO: 80),

[0138] GOI / antigen cassette (e.g., firefly luciferase): (SEQ ID NO: 81),

[0139] Internal ribosome entry site (IRES): (SEQ ID NO: 82),

[0140] EG Tech L protein coding sequence: (SEQ ID NO: 83), and

[0141] 3'CSE and polyA: (SEQ ID NO: 84).

[0142] In some embodiments, the saRNA of the disclosure is encoded by the following expression vectors or sequences 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 to the aforementioned sequences: In some embodiments, the saRNA of the disclosure is encoded by the following expression vectors having different gene of interest (GOI) sequences after assembly as depicted in Figure 30A as a Semliki Forest virus (SFV)-based replicon, or sequences 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 to the aforementioned sequences:

[0143] 5'CSE: (SEQ ID NO: 85),

[0144] NSP and SGP cassette: (SEQ ID NO: 86),

[0145] GOI / antigen cassette (e.g. mKate2): (SEQ ID NO: 87),

[0146] Internal ribosome entry site (IRES): (SEQ ID NO: 88),

[0147] EG Tech L protein coding sequence: (SEQ ID NO: 89),

[0148] SFV 26S RNA part A: (SEQ ID NO: 90), and

[0149] SFV 26S RNA part B, 3'CSE, and polyA: (SEQ ID NO: 91).

[0150] In some embodiments, the saRNA of the disclosure is encoded by the following expression vectors or sequences 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 to the aforementioned sequences: In some embodiments, the saRNA of the disclosure is encoded by the following expression vectors having different gene of interest (GOI) sequences after assembly as depicted in Figure 30A as a Venezuelan Equine Encephalitis Virus (VEEV)-based replicon, or sequences 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 to the aforementioned sequences:

[0151] 5'CSE: (SEQ ID NO: 92),

[0152] NSP1-4 and SGP cassette: (SEQ ID NO: 93),

[0153] GOI / antigen cassette (e.g., full-length influenza HA as antigen): (SEQ ID NO: 94),

[0154] Internal ribosome entry site (IRES): (SEQ ID NO: 95),

[0155] EG Tech L protein coding sequence: (SEQ ID NO: 96), and

[0156] 3'CSE and polyA: (SEQ ID NO: 97).

[0157] RNA modification The RNAs provided herein (e.g., messenger RNAs or self-amplifying RNAs for expression of target proteins and NCT inhibitor proteins) can be modified as described herein. For example, the RNAs can be modified to reduce immunogenicity in cells or tissues. The RNAs provided herein can include one or more of an untranslated region (UTR), a 5' cap, and a poly-adenosine tail.

[0158] In some embodiments, RNA can be synthesized as unmodified or modified RNA. RNA can be modified to enhance stability and / or avoid immune detection and degradation. Modified RNA can include, for example, one or more of nucleotide modifications, nucleoside modifications, backbone modifications, sugar modifications, and / or base modifications. In some embodiments, the modified nucleoside is pseudouridine or a pseudouridine analog. In some embodiments, the pseudouridine analog is N-1-methylpseudouridine.

[0159] In some embodiments, the RNA is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, 95%, 99%, or 100% depleted of uracil nucleosides and substituted with a uracil nucleoside analog (e.g., pseudouridine, 5-methoxyuridine, or N-1-methyl-pseudouridine).

[0160] In some embodiments, RNA can comprise RNA backbone modification.Typically, backbone modification is the modification that the phosphate of the backbone of the nucleotide contained in RNA is chemically modified.Exemplary backbone modifications can include, but are not limited to, the modification that phosphodiester bond is replaced with a member from the group consisting of peptide, methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (for example, cytidine 5'-0-(1-thiophosphate)), boranophosphate, and / or positively charged guanidinium group, or other phosphodiester bond replacement means.

[0161] Cap Structure In some embodiments, the RNA may include a 5' cap structure. The 5' cap may include, for example, a triphosphate linkage and a guanine nucleotide methylated at the 7-nitrogen. In some embodiments, the 5' cap is 5' 7-methylguanosine (m7G). A wide variety of m7G cap analogs are known in the art, many of which are commercially available. These include the m7GpppG described above, as well as the ARCA 3'-OCH3 and 2'-OCH3 cap analogs (Jemielity, J. et al., RNA, 9:1108-1122 (2003)). Additional cap analogs for use in embodiments of the present invention include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10:1479-1487 (2004)), phosphorothioate 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. Pat. Nos. 8,093,367 and 8,304,529 (incorporated herein by reference).

[0162] In some embodiments, the 5' cap comprises or consists of an internal ribosome entry site (IRES). In some embodiments, the IRES is within the 5' UTR. In some embodiments, the 5' cap comprises or consists of a 2A self-cleaving peptide (e.g., one or more of P2A, T2A, E2A, and F2A).

[0163] Tail Structure The presence of a "tail" may function to protect RNA from exonuclease degradation. Poly-A tails are thought to stabilize natural messenger and synthetic sense RNA. Thus, in certain embodiments, a long poly-A tail can be added to an RNA molecule, thereby making the RNA more stable. Poly-A tails can be added using a variety of techniques recognized in the art. For example, long poly-A tails can be added to synthetic RNA or in vitro transcribed RNA using poly-A polymerase (Yokoe, et al. Nature Biotechnology. 1996;14:1252-1256). Alternatively, the transcription template can encode a long poly-A tail. Furthermore, poly-A tails can be added directly to PCR products by transcription. Alternatively, poly-A can be ligated to the 3' end of the sense RNA using RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

[0164] In some embodiments, the RNA can include a 3' poly(A) tail structure. The length of the poly-A tail can 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 in length.

[0165] 5' and 3' untranslated regions (UTRs) 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 5' UTR may be between about 50 and about 100, or from about 50 to about 500 nucleotides in length. In some embodiments, the 3' UTR includes one or more of a poly-A signal, a binding site for a protein that can affect RNA stability or localization, or one or more binding sites for an miRNA. In some embodiments, the 3' UTR may be between about 0 and about 50, or from about 50 to about 100 nucleotides in length.

[0166] Exemplary 3' and 5' UTR sequences may be derived from RNAs with relatively long half-lives (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase the stability of the sense RNA molecule. For example, the 5' UTR sequence may include a subsequence of the cytomegalovirus (CMV) immediate early 1 (IE1) gene, or a fragment thereof, to improve nuclease resistance and / or improve the half-life of the polynucleotide. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide compared to their unmodified counterparts, including modifications to improve the resistance of such polynucleotides to in vivo nuclease digestion. In some embodiments, the UTRs may improve tissue-specific expression. Exemplary UTRs include hBG 5' UTR (SEQ ID NO: 98) and hBG 3' UTR (SEQ ID NO: 102).

[0167] DNA templates for in vitro RNA production For example, RNA of the present disclosure encoding an NCT inhibitor protein or a target protein can be produced by in vitro transcription (IVT) of one or more DNA templates having a polynucleotide sequence(s) encoding the desired RNA. The DNA template can include one or more promoters that enable transcription. In some embodiments, for example, the template can include a T7 promoter configured to transcribe the target protein-encoding nucleotide sequence and / or the NCT inhibitor protein-encoding nucleotide sequence. Alternatively, the RNA can be prepared synthetically.

[0168] The template may include a separation element for expressing proteins individually. In various embodiments, the template is a bicistronic template or a polycistronic template. The separation element may be an internal ribosome entry site (IRES) or a 2A element. In some embodiments, the template may include a nucleic acid encoding a 2A self-cleaving peptide. Exemplary 2A self-cleaving peptides include P2A, E2A, F2A, and T2A.

[0169] In some embodiments, the first polynucleotide or the second polynucleotide, or both, are operably linked to an internal ribosome entry site (IRES).

[0170] In some embodiments, the first polynucleotide or the second polynucleotide, or both, are operably linked to a 2A element.

[0171] In some embodiments, the first polynucleotide, the second polynucleotide, or both are operably linked by a stem-loop structure that induces translation termination codon readthrough. 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).

[0172] In some embodiments, a DNA template encoding an RNA of the present disclosure may comprise or encode the following sequence, in the order depicted in any one of Figures 1A-1E, 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 to the foregoing sequences: In some embodiments, a DNA template encoding an RNA of the present disclosure may comprise or encode the following sequence, in the order depicted in any one of Figures 1A-1E, 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 to the foregoing sequences, but encoding a different target protein sequence:

[0173] T7 promoter SEQ ID NO: 70,

[0174] 5'UTR SEQ ID NO: 71,

[0175] 3'UTR SEQ ID NO: 72,

[0176] PolyAtract SEQ ID NO: 73,

[0177] Nano-luciferase-nucleotide target protein sequence SEQ ID NO: 74,

[0178] Nano-luciferase-amino acid sequence SEQ ID NO: 75,

[0179] SARS-CoV-2 spike-nucleotide target protein sequence SEQ ID NO: 76,

[0180] SARS-CoV-2 spike - amino acid sequence SEQ ID NO: 15,

[0181] GBA signal peptide-nucleotide target protein sequence SEQ ID NO: 77, and

[0182] GBA mature protein-nucleotide target protein sequence SEQ ID NO:78.

[0183] V. DNA Composition Any DNA sequence that encodes a target protein can be used with the methods and compositions described herein (ie, RNA encoding an NCT inhibitor protein).

[0184] In some embodiments, the plasmid expression vector may comprise the following sequence, in the order depicted in Figures 20A and 20B, 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 to the foregoing sequences: In some embodiments, the plasmid expression vector may comprise the following sequence, in the order depicted in Figures 20A and 20B, 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 to the foregoing sequences, but encoding a different NCT inhibitor protein or target protein sequence:

[0185] GBA-NanoLuc / EPO Nano-Luc expression plasmid sequence, expression cassette:

[0186] CMV enhancer and promoter sequence: (SEQ ID NO: 108),

[0187] Multiple cloning site (MCS) sequence: (SEQ ID NO: 109), GBA coding sequence: (SEQ ID NO: 110) or EPO coding sequence: (SEQ ID NO: 116),

[0188] Linker sequence: (SEQ ID NO: 111),

[0189] Nanoluciferase reporter sequence: (SEQ ID NO: 112), and

[0190] 3'UTR and SV40 poly(A) signal sequence: (SEQ ID NO: 113).

[0191] VI. Delivery Vehicles In some embodiments, one or more RNAs and / or can be delivered to cells or tissues via a delivery vehicle. In some embodiments, the delivery vehicle is a viral delivery vehicle. In some embodiments, the delivery vehicle is a non-viral delivery vehicle.

[0192] Non-viral delivery vehicles In some embodiments, the delivery vehicle may be a nanoparticle. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP) (including, but not limited to, nanoparticles comprising lipids and / or polymers, liposomes, liposomal nanoparticles, cationic lipids, or exosomes). In other embodiments, the delivery vehicle may comprise calcium phosphate nucleotides, aptamers, cell-penetrating peptides, or other vector tags.

[0193] In some embodiments, the nanoparticles are polymeric nanoparticles. In some embodiments, the nanoparticles are metal nanoparticles.

[0194] In some embodiments, RNA can be delivered via lipid nanoparticles (LNPs). Exemplary LNPs can include one or more different lipids and / or polymers. In some embodiments, LNPs can include one or more of ionic or cationic lipids, neutral lipids, cholesterol, and / or stabilizing lipids (e.g., PEGylated lipids).

[0195] In some embodiments, the LNP may comprise an ionizable lipid. Ionic lipids may refer to any of several lipid species that have a net positive charge at a selected pH (such as physiological pH). Ionic lipids may also refer to, for example, lipids in an ionized state, such as cationic lipids, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl N-Dioleyl-N,N-dimethylammonium-propane (DODAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or (DLenDMA), N-Dioleyl-N,N-dimethylammonium chloride (DODAC), or one or more of these variations.

[0196] In some embodiments, the LNPs may comprise a neutral ionic or zwitterionic lipid. In some embodiments, the neutral lipid is selected from one or more of the following: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine ( POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), or variations thereof.

[0197] In some embodiments, the LNPs comprise a PEGylated lipid, cholesterol, and one or more ionic lipids. An exemplary LNP formulation comprises approximately 1.5% DMG-PEG(2000), 38.5% cholesterol, 10% DSPC, and 50% DLin-MC3-DMA.

[0198] 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, and then purifying the resulting nanoparticles by filtration. In some embodiments, LNPs or liposomes can be produced using a microfluidic device to rapidly mix reagents and form monodisperse particles of controlled size. In some embodiments, LNPs or liposomes can encapsulate RNA and / or associate with one or more RNAs through electrostatic interactions.

[0199] Viral Delivery Vehicles In other embodiments, the delivery vehicle comprises a recombinant virus or virus-like particle, such as an adenovirus, adeno-associated virus (AAV), herpesvirus, or retrovirus, such as a lentivirus. In some embodiments, the delivery vehicle comprises a modified viral vector, such as an adenovirus dodecahedron or a recombinant adenovirus assembly.

[0200] VII. Treatment Methods The treatment methods described herein refer to the treatment of a disease or disorder in a subject in need thereof by administering one or more RNAs for expression of a target protein together with an NCT inhibitor protein (e.g., an L protein). In some embodiments, the RNA encoding the NCT inhibitor protein is messenger RNA. In some embodiments, the RNA encoding the NCT inhibitor protein is self-amplifying RNA.

[0201] In some embodiments, the RNA or DNA provided herein for expression of a target protein with RNA encoding an NCT inhibitor protein (e.g., L protein) encodes an adalimumab protein, CDR sequence, or variable sequence of a light or heavy chain. In some embodiments, the RNA or DNA encoding an adalimumab protein or sequence and the RNA encoding an NCT inhibitor 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.

[0202] In some embodiments, the RNA of the present 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 a disease or infection associated with or resulting from the pathogen from which the antigenic protein is derived. In some embodiments, the antigenic protein is a viral antigenic protein. As provided herein, the RNA or DNA of the present disclosure encoding a target protein may encode any of the viral antigenic proteins in Table 3 to be administered together with the RNA encoding the NCT inhibitor protein as a vaccine. In some embodiments, the RNA or DNA encoding a target protein of the present disclosure provides a vaccine against coronaviruses, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), or West Nile virus. In some embodiments, the RNA or DNA encoding a target protein of the present disclosure provides a vaccine against influenza virus, hepatitis B virus, human papillomavirus (HPV), West Nile virus, or human immunodeficiency virus (HIV).

[0203] In some embodiments, RNA encoding the L protein (e.g., messenger RNA or self-amplifying RNA) is administered to 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 an equivalent concentration thereof.

[0204] In some embodiments, expression of the NCT inhibitor protein and the target protein increases the expression level of the target protein in a cell or a subject. In some embodiments, expression of the NCT inhibitor protein and the target protein increases the expression level of the target protein in a cell 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 20-fold, about 30-fold, about 40-fold, 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.

[0205] In some embodiments, the RNA encoding the NCT inhibitor protein reduces the expression level of the target protein in a cell or a subject. In some embodiments, expression of the NCT inhibitor protein and the target protein reduces the expression level of the target protein in a cell or a subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0206] In some embodiments, the RNA encoding the NCT inhibitor protein increases the expression level of the target protein in a cell or subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, or about 1000%.

[0207] In some embodiments of the present disclosure, the RNA encoding the NCT inhibitor protein increases the activity of a target protein in a cell of a subject or in the subject. In some embodiments, expression of the NCT inhibitor protein and the target protein increases the activity of the target protein in a cell of a subject 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.

[0208] In some embodiments of the present disclosure, the RNA encoding the NCT inhibitor protein increases the duration that the target protein is found in the cells of a subject or in the subject. In some embodiments, the RNA encoding the NCT inhibitor protein increases the duration that the target protein is found in the cells of a subject 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.

[0209] In some embodiments of the present disclosure, the RNA encoding the NCT inhibitor protein reduces the coefficient of variation (CV%) of the target protein in a subject's tissue or in the subject. In some embodiments, the RNA encoding the NCT inhibitor protein reduces the coefficient of variation (CV%) of the target protein in a subject's tissue or in the subject 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.

[0210] In some embodiments of the present disclosure, the RNA encoding the NCT inhibitor protein reduces degradation of the target protein, ie, 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.

[0211] Route of administration In some embodiments, one or more RNAs encoding an NCT inhibitor protein (e.g., an L protein) can be delivered orally, subcutaneously, intravenously, intranasally, intradermally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In exemplary embodiments, the RNA can be administered intramuscularly or subcutaneously.

[0212] VIII. Kit Also provided herein is a kit containing RNA encoding the disclosed NCT inhibitor protein for improving target protein expression. The kit may include, for example, a suitable container (e.g., a vial or tube) and instructions for its use.

[0213] In some embodiments, the kits of the present disclosure contain RNA encoding an NCT inhibitor (e.g., an L protein) comprising a sequence in Table 4, the sequence of SEQ ID NO: 161, a mutant sequence of any of 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 to the foregoing sequences.

[0214] In some embodiments, the kits of the present disclosure include a delivery vehicle (e.g., LNP) or RNA encoding an NCT inhibitor packaged in or together with a delivery vehicle. In some embodiments of the present disclosure, the kits including RNA encoding an L protein (e.g., messenger RNA or self-amplifying RNA) are present in a vial or tube in an amount 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, or an equivalent amount, for use per well of a 96-well plate.

[0215] Enumeration of Embodiments Embodiment I-1. A method of improving expression of a target protein in a cell or a subject, comprising administering one or more ribonucleic acids (RNAs) encoding the target protein and an NCT inhibitor protein.

[0216] Embodiment I-2. The method of embodiment I-1, wherein said NCT inhibitor protein has at least 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 to the amino acid sequence of an NCT inhibitor protein in Table 4.

[0217] Embodiment I-3. The method of embodiment I-1, wherein said 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 to the L protein sequence of SEQ ID NO:2.

[0218] Embodiment I-4. The method of any one of embodiments I-1 to I-3, wherein said target protein is selected from any of the sequences of Tables 1, 2, and 3, 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 to said sequences.

[0219] Embodiment I-5. The method of any one of embodiments I-1 to I-4, wherein said target protein is an antibody.

[0220] Embodiment I-6. The method of embodiment I-5, wherein said target protein is adalimumab, and wherein, optionally, said 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 to said sequence; 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 to said sequence.

[0221] Embodiment I-7. The method of any one of embodiments I-1 to I-4, wherein said target protein is an antigen of a pathogen, optionally the SARS-CoV-2 spike (S) protein, and wherein, optionally, said 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 thereto.

[0222] Embodiment I-8. The method of any one of embodiments I-1 to I-7, wherein said method comprises delivery of one or more RNAs using lipid nanoparticles (LNPs).

[0223] Embodiment I-9. The method of embodiment I-8, wherein said LNP comprises a PEGylated lipid, cholesterol, and a neutral lipid, and an ionic lipid.

[0224] Embodiment I-10. The method of embodiment I-8, wherein said LNPs comprise 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.

[0225] Embodiment I-11. A method of treating a subject in need of treatment for a disease or disorder, comprising administering to said subject one or more messenger ribonucleic acid (RNA) encoding a target protein and an NCT inhibitor protein.

[0226] Embodiment I-12. The method of embodiment I-11, wherein said NCT inhibitor protein has at least 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 to the amino acid sequence of an NCT inhibitor protein in Table 4.

[0227] Embodiment I-13. The method of embodiment I-11, wherein said 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 to the L protein sequence of SEQ ID NO:2.

[0228] Embodiment I-14. The method of any one of embodiments I-11 to I-13, wherein said target protein is selected from any of the sequences of Tables 1, 2, and 3, 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 to said sequences.

[0229] Embodiment I-15. A method of vaccinating a subject in need thereof, comprising administering to said subject one or more messenger ribonucleic acid (RNA) encoding a target protein and an NCT inhibitor protein, wherein said target protein is an antigen protein.

[0230] Embodiment I-16. The method of embodiment I-15, wherein said NCT inhibitor protein has at least 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 to the amino acid sequence of an NCT inhibitor protein in Table 4.

[0231] Embodiment I-17. The method of embodiment I-15 or I-16, wherein the antigenic protein is selected from any of the sequences 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 to the aforementioned sequences.

[0232] Embodiment I-18. The method of any preceding embodiment, wherein co-expression of said NCT inhibitor protein and said target protein increases the expression level 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 20-fold, about 30-fold, about 40-fold, 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.

[0233] Embodiment I-19. The method of any preceding embodiment, wherein co-expression of said NCT inhibitor protein and said target protein reduces the expression level of the target protein in a cell or subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0234] Embodiment I-20. The method of any preceding embodiment, wherein co-expression of said NCT inhibitor protein and said target protein increases the activity of the target protein in a cell of 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.

[0235] Embodiment I-21. The method of any preceding embodiment, wherein co-expression of said NCT inhibitor protein and said target protein increases the duration that said target protein is found in the cells of the subject 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, 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.

[0236] Embodiment I-22. The method of any preceding embodiment, wherein co-expression of said NCT inhibitor protein and said target protein reduces the coefficient of variation (CV%) of said target protein in said tissue of said subject or in said subject 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.

[0237] Embodiment I-23. The method of any preceding embodiment, wherein co-expression of said NCT inhibitor protein and said target protein reduces degradation of said 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.

[0238] Embodiment I-24. Co-expression of said NCT inhibitor protein and said target protein enhances EC of said target 5010-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 reduction in

[0239] Embodiment I-25. A composition comprising one or more ribonucleic acids (RNAs) encoding a target protein and an NCT inhibitor protein.

[0240] Embodiment I-26. The composition of embodiment I-24, wherein said NCT inhibitor protein has at least 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 to the amino acid sequence of an NCT inhibitor protein in Table 4.

[0241] Embodiment I-27. The composition of embodiment I-24, wherein said 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 to the L protein sequence of SEQ ID NO:2.

[0242] Embodiment I-28. The composition of any one of embodiments I-24 to I-27, wherein said target protein is selected from any of the sequences of Tables 1, 2, and 3, 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 to said sequences.

[0243] Embodiment I-29. The composition of any one of embodiments I-24 to I-27, wherein said target protein is an antibody.

[0244] Embodiment I-30. The composition of embodiment I-28, wherein the target protein is adalimumab, and 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 thereto; 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 thereto.

[0245] Embodiment I-31. The composition of any one of embodiments I-24 to I-27, wherein the target protein is an antigen of a pathogen, optionally the SARS-CoV-2 spike (S) protein, and 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 thereto.

[0246] Embodiment I-32. The composition of any one of embodiments I-24 to I-30, wherein said target protein is selected from any of the sequences of Tables 1, 2, and 3, 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 to said sequences.

[0247] Embodiment I-33. The composition of any one of embodiments I-24 to I-31, wherein said method comprises delivery of one or more RNAs using lipid nanoparticles (LNPs).

[0248] Embodiment I-34 The composition of embodiment I-32, wherein said LNP comprises a PEGylated lipid, cholesterol, and a neutral lipid, and an ionic lipid.

[0249] Embodiment I-35. The composition of embodiment I-32, wherein said LNPs comprise 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.

[0250] Embodiment I-36. (i) one or more polynucleotides encoding an NCT inhibitor; and (ii) Delivery Vehicle Including, the system.

[0251] Embodiment I-37. The system of embodiment I-36, wherein the polynucleotide encoding said NCT inhibitor is RNA.

[0252] Embodiment I-38. The system of embodiment I-36, wherein said delivery vehicle is a non-viral delivery vehicle.

[0253] Embodiment I-39. The system of embodiment I-38, wherein said non-viral delivery vehicle is an LNP.

[0254] Embodiment I-40. The system of embodiment I-36, wherein the delivery vehicle is a viral delivery vehicle.

[0255] Embodiment I-41. The system of embodiment I-36, comprising RNA encoding a target protein.

[0256] Embodiment I-42. The system of embodiment I-41, wherein said RNA comprises at least one unmodified uridine nucleotide.

[0257] Embodiment I-43. The system of embodiment I-41, wherein said RNA is a self-amplifying RNA (saRNA).

[0258] Embodiment I-44. A method of reducing cell signaling in response to a stressor, comprising administering to a cell a polynucleotide encoding an NCT inhibitor.

[0259] Embodiment I-45. The method of embodiment I-44, wherein said stressor is selected from the group consisting of innate immune signaling; pro-inflammatory signaling; delivery vector-induced inflammation; natural nucleoside mRNA transfection; self-amplifying RNA transfection; and target protein expression.

[0260] Embodiment I-46 The method of embodiment I-44, wherein the polynucleotide encoding said NCT inhibitor is RNA.

[0261] Embodiment I-47 The system of embodiment I-36, wherein the polynucleotide encoding said NCT inhibitor comprises the sequence of SEQ ID NO: 65, 66, 67, or 68.

[0262] Embodiment I-48 The method of embodiment I-11, wherein said target protein is an antigen for use in a vaccine.

[0263] Embodiment I-49. The system of any one of embodiments I-41 to I-43, wherein said target protein is an antigen for use in a vaccine.

[0264] Embodiment I-50. The method of embodiment I-44, comprising co-expressing a target protein, wherein said target protein is an antigen for use in a vaccine. [Example]

[0265] Example Example 1: Method for producing RNA The compositions and methods of the present disclosure provide RNA encoding target proteins (e.g., therapeutic proteins, viral proteins, antibodies, and reporter proteins) and nucleocytoplasmic transport (NCT) inhibitor proteins (e.g., NCT inhibitor L protein). Encapsulation of RNA encoding an NCT inhibitor protein can provide one or more of the following: a) extended duration of target protein expression, b) reduced cellular inflammation in vitro, c) reduced inflammation in vivo, d) higher quality and / or quantity of target protein expression in vivo and in vitro, and / or e) improved cytokine secretion.

[0266] Construction of RNA molecules All assemblies were created into a plasmid backbone capable of propagation in E. coli, containing a promoter controlling a 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 isolated from the rest of the plasmid template by a second terminator (a transcription terminator derived from lambda phage). Genes containing the backbone elements were synthesized using phosphoramidite chemistry. Figures 1A-1E show that the gene of interest (GOI) can be delivered on a separate RNA from the NCT inhibitor (e.g., NCT inhibitor L protein) (Figure 1A) and co-transfected, or can be delivered on the same RNA (Figure 1B-1E). The NCT inhibitor (e.g., L protein) on the same RNA can be either before or after the gene of interest in the 5' to 3' direction. A protease cleavage site (e.g., a furin site) can be present (Figure 1B and Figure 1C). An internal ribosome entry site (IRES) may also be present (FIGS. 1D and 1E).

[0267] Structural genes used for plasmid construction were synthesized by phosphoramidite chemistry, chemically amplified, and cloned into vectors as described above using isothermal assembly reactions such as NEB HI-FI or Gibson Assembly.

[0268] Figures 5A and 5B show the layout of DNA template constructs used for in vitro transcription (IVT) to generate a single mRNA construct encoding the L protein (L) and nanoluciferase (nLuc). Figure 5A shows the DNA template construct used for IVT to generate a single mRNA construct encoding L and nLuc (L-NLuc) separated by a p2A separation element. Figure 5B shows the DNA template construct used for IVT to generate a single mRNA construct encoding nLuc and L (NLuc-L) separated by a p2A separation element.

[0269] Preparation of DNA templates The fully cloned DNA template contained a 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 using an equal volume of 1:1 phenol / chloroform. The DNA was precipitated by adding 1 / 10 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. The DNA was incubated at -20°C for 30 minutes, after which the DNA was pelleted in a microcentrifuge for 15 minutes. The DNA pellet was washed with 500 μl of 70% ethanol and centrifuged at maximum speed for 15 minutes. The ethanol was carefully removed, and the pellet was air-dried and resuspended in nuclease-free water. In some cases, DNA templates for RNA synthesis were generated using PCR instead of linearizing fully cloned DNA using BSAI restriction enzyme as described above. For these PCR reactions, the forward primer was designed to hybridize with the T7 promoter and 3' UTR regions of the fully cloned plasmid DNA template, ensuring that the 5' end of the PCR amplicon would be compatible with CleanCap reagent (the RNA capping reagent used downstream). The reverse primer was designed to hybridize with the 3' UTR region of the fully cloned plasmid DNA template, ensuring that the 3' UTR of the DNA template would be retained within the amplicon. The PCR-generated DNA templates were gel-purified using agarose gel electrophoresis, Zymoclean Gel DNA Recovery Kit (Zymo Research), and Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer's instructions. The DNA templates generated by either method were then used as templates for RNA synthesis as described below.

[0270] RNA synthesis To generate in vitro transcription-derived RNA, we used the HiScribe™ T7 ARCA RNA Kit, which generates a 5' 7-methylguanosine (m7G) cap by co-transcription. In some cases, in vitro transcription of RNA was performed using the HiScribe T7 mRNA Kit (NEB) with CleanCap Reagent AG Kit using 100% N1-pseudouridine (Trilink) according to the manufacturer's instructions, using the respective gel-purified PCR products as templates for the RNA synthesis reaction. All components were thawed on ice before reaction assembly. Reactions were assembled on ice to the following specifications: [Table 5A] [Table 5B]

[0271] The reaction was mixed, pulse-spun, and incubated at 37°C for 1-2 hours. To remove the input DNA template, 2 uL of DNase I was added, mixed thoroughly, and incubated at 37°C for 15 minutes. The synthesized RNA was purified using the Monarch RNA Cleanup Kit (NEB). The poly(A) tailing reaction was then assembled on ice to the following specifications: [Table 6]

[0272] The reaction was mixed, pulse-spun, and incubated for 30 minutes at 37° C. Poly(A)-tailed RNA was purified using the Monarch RNA Cleanup kit (NEB).

[0273] RNA purification As described above, RNA was purified after the in vitro transcription step and the poly(A)-tailing step using the Monarch RNA Cleanup Kit (NEB) according to the manufacturer's instructions. In some cases, RNA was purified using phenol-chloroform extraction and ethanol precipitation. The reaction was adjusted to 180 μl by adding nuclease water, and 20 μl of 3 M sodium acetate (pH 5.2) was added. The 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. The RNA was precipitated by adding 2 volumes of ethanol. The reaction was incubated at -20°C for at least 30 minutes, and the pellet was collected by centrifugation. The supernatant was removed, and the pellet was rinsed with 500 μl of cold 70% ethanol before being resuspended in 50 μl of 0.1 mM EDTA and stored at -20°C.

[0274] Cell lines - culture and transfection HEK293 cells are used to demonstrate the application of the present system, method, and composition in human eukaryotic cells. HEK293 adherent cells (CLS) were cultured in Dulbecco's Modified Eagle's Medium High Glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 Anti-Anti (Gibco). HEK293 cells were grown to 80% confluency at 37°C and 5% CO2 and then transiently transfected using Lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were analyzed 48 hours later as described in this example.

[0275] Vero cells are used to illustrate the application of the present system, method, and composition in animal eukaryotic cells. Vero E6 adherent cells (ATCC) were cultured in Dulbecco's Modified Eagle's Medium High Glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 U Anti-Anti (Gibco). Vero cells were grown to 80% confluency at 37°C and 5% CO2 and then transiently transfected using Lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were analyzed 48 hours later as described in this example.

[0276] C2C12 cells were used to demonstrate the cytokine sensitivity improvement of the present system, method, and composition in animal cells. C2C12 adherent cells (ATCC) were cultured in Dulbecco's Modified Eagle's Medium High Glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 Anti-Anti (Gibco). C2C12 cells were grown to 80% confluency at 37°C and 5% CO2 and then transiently transfected using Lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were analyzed 48 hours later as described in this example.

[0277] BJ fibroblast cells were used to demonstrate the improved cytokine sensitivity of the systems, methods, and compositions of the present invention in human cells. BJ fibroblast adherent cells (ATCC) were cultured in Dulbecco's Modified Eagle's Medium High Glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000 Anti-Anti (Gibco). C2C12 cells were grown to 80% confluency at 37°C and 5% CO2 and then transiently transfected using Lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were analyzed 48 hours later as described in this example.

[0278] In vitro RNA transfections were generally performed using the Mirus TransIT mRNA Transfection Kit (Mirus) according to the manufacturer's instructions unless otherwise indicated. For cotransfection experiments (i.e., dose titration studies using multiple RNA molecules in trans), all indicated RNA molecules were first mixed in Opti-MEM. In these experiments, an inert filler RNA was used to ensure that the total RNA concentration for each transfection was identical. The amount of RNA used is indicated in each example. Mirus TransIT Boost Reagent was then added to the undiluted RNA sample. Mirus TransIT Transfection Reagent was then added, and the tube was gently tapped and inverted to mix thoroughly. The tube was incubated at room temperature for a maximum of 5 minutes, after which the reaction was "quenched" by adding pre-warmed complete DMEM medium (Gibco). The transfection complex was then added to the cells. For experiments in which cells were stimulated with poly I:C or IFN-beta, the stimulator was added to the cells at the indicated final concentration within 5 minutes of treating the cells with the transfection complex.

[0279] Production of LNP-RNA An exemplary LNP formulation provided herein is as follows: 1.5% DMG-PEG(2000), 38.5% cholesterol, 10% DSPC, and 50% DLin-MC3-DMA. To produce the LNP-mRNA formulation used in this experiment, lipids were dissolved in ethanol. RNA was dissolved in an aqueous acidified buffer (e.g., 50 mM sodium citrate (pH 4)). For example, the lipids were combined with the RNA solution by rapid mixing at a volume ratio of 1:3 (lipid:RNA) and a nitrogen to phosphate ratio of 6:1 (ratio of nitrogen of ionic lipid to phosphate of mRNA). After rapid mixing, the mixture was further diluted 1:1 with pH 6 buffer and then diafiltered against 15 mL PBS using an Amicon Ultraspin filter with a molecular weight cutoff of 10 kDa.

[0280] Nanoluciferase assay For the NanoLuc assay, cell culture supernatants were collected into microcentrifuge tubes and cleared from cell debris by centrifugation. Adherent cells on 24-well plates were lysed at room temperature using 250 μl of 0.2% Triton® X-100 in PBS. 50 μl of clarified cell culture supernatant or 50 μl of cell lysate was loaded into an opaque 96-well microplate. Collected supernatant and cell lysate samples were analyzed for NanoLuc expression using the Nano-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. Briefly, 50 μl of fresh 1x Nano-Glo Assay Reagent in the respective assay buffer (Promega) was added to each well, and after 2–5 minutes of incubation, luminescence was measured using a Biotex Synergy LX plate reader. Cell lysates were further analyzed for their protein concentration using the A660 Reagent (Thermo Scientific) according to the manufacturer's instructions. Briefly, 40 μl of sample or standard was mixed with 150 μl of A660 assay reagent, the absorbance at 660 nm was measured using a Biotek Synergy LX plate reader, and protein concentrations were quantified based on a standard curve. Luminescence of cell lysates was normalized per μg of cell lysate protein (i.e., giving luminescence values ​​in units of RLU / μg protein), while luminescence of cell culture supernatants was normalized using the volume of supernatant used in the assay (i.e., giving luminescence values ​​in units of RLU / ml of supernatant).

[0281] animal For animal studies, C57BL / 6 (Charles River Laboratories) or corresponding disease models were used. Ages are indicated in the examples. Animals of the same sex were group-housed in polycarbonate cages containing appropriate bedding. Mice were identified by either a visible tattoo on the tail or implantation of an electronic identification chip. Mice were allowed to acclimate for at least 5 days before treatment to become accustomed to the experimental environment. Housing was set up as described in the Guide for the Care and Use of Laboratory Animals, with social housing and chews for animal enrichment. Targeted environmental conditions were a temperature of 19-25°C, humidity between 30% and 70%, and a light / dark cycle of 12 hours light and 12 hours dark. Pelleted food (Lab Diet Certified CR Rodent Food 5CR4) was provided ad libitum. Water was available ad libitum in the form of municipal water treated by reverse osmosis and ultraviolet irradiation.

[0282] Example 2: In vitro expression of a reporter gene (luciferase) from RNA bearing the L protein To test the effect of the L protein in the mRNA construct, interferon-producing mouse C2C12 cells and human BJ fibroblasts were transfected with a single mRNA construct encoding both luciferase and L protein (NCT inhibitor) proteins, separated by a p2A element (Figures 5A and 5B). These mRNA constructs contained an ARCA cap and a 50%-50% mixture of natural uridine and pseudo-uridine. Due to the use of natural uridine in the mRNA, these mRNA constructs are inherently pro-inflammatory, as they are able to activate innate immune signaling pathways in transfected cells, as is known in the art. Both positions of the NCT inhibitor relative to the gene of interest (5' to the gene and 3' to the gene) were tested, demonstrating the flexibility of the approach regarding the location of the NCT inhibitor. Interestingly, the position of the L protein also modulated the effect of the L protein on the expression of the gene of interest.

[0283] On day 0, C2C12 cells and BJ fibroblasts were seeded in 200 μl DMEM (C2C12) or 200 μl MEM (BJ fibroblasts) containing 10% FBS in 96-well culture microplates. On day 1, cells were transfected with the RNA constructs encoded by Figures 5A and 5B using Lipofectamine™ MessengerMAX™ Transfection Reagent (Invitrogen) according to the manufacturer's instructions. On subsequent days, transfected cells and cell culture supernatants were assayed for NanoLuc luciferase activity 24, 48, and 72 hours after transfection. Figures 6A and 6B show luciferase activity measured directly by the expressed enzyme in mouse C2C12 cells and human BJ cells. Surprisingly, coexpression of the L protein (a NCT inhibitor) increased luciferase expression by more than 10-fold. This demonstrates the ability of the L protein to protect the expression of the gene of interest (GOI) from internal stress and inflammatory signals induced by mRNA transfection.

[0284] To further investigate the role of the L protein in protecting against stress and inflammatory signals, exogenous IFN-beta (a stress pathway stimulator) was added to cells. It is known that protein expression is inhibited when exogenous IFN-beta reaches a threshold concentration. To measure cellular sensitivity to exogenous IFN-beta in the presence and absence of the L protein, C2C12 cells were transfected with mRNA encoding either the L protein alone (L), nLuc alone, or a single mRNA encoding both L and nLuc, as shown in Figures 5A (L-nLuc) and 5B (nLuc-L). Six hours after transfection, 10-fold serial dilutions of mouse recombinant IFN-beta were added to C2C12 cells. Luciferase activity was then measured 48 hours after IFN-beta stimulation. The approximate EC of IFN-beta required for the reduction of luciferase protein expression was calculated. 50is measured as the concentration at which luciferase expression (as indicated by activity normalized to no IFN-beta) is reduced by 50% (dotted line).

[0285] Figure 7 shows the reduction in luciferase expression in the presence of IFN-beta. The approximate EC of IFN-beta required to reduce luciferase protein expression for each construct. 50 The approximate EC 50 The presence of L protein protected cells from exogenous IFN-beta stimulation by 10-fold and 12-fold (for L-nLuc and nLuc-L, respectively), as demonstrated by a decrease in IFN-beta values. Thus, L protein protected protein expression from the external stress stimulus of IFN-beta.

[0286] IFN-sensitive ECs 50 Values ​​are calculated using log(inhibitor) vs. response-variable slope 4-parameter nonlinear regression in Prism (GraphPad).

[0287] IFN-beta secretion was used as a marker of inflammation. To this end, IFN-beta-producing human BJ cells were transfected with mRNA encoded by the constructs shown in Figures 5A and 5B. Supernatants were collected 24 to 72 hours after transfection and clarified by centrifugation at 500 g for 5 minutes. The clarified supernatants were then analyzed for the presence of IFN-beta by ELISA (R&D Systems) according to the manufacturer's instructions.

[0288] Figure 8 shows the levels of IFN-beta produced by BJ cells after transfection with the mRNA construct. Notably, the presence of the L protein prevented BJ cells from producing IFN-beta in response to mRNA transfection. In the presence of L protein mRNA, RNA-specific stress induced with 50% natural uridine-containing pro-inflammatory mRNA was not propagated by the cells, as exemplified by abrogating the cells' ability to upregulate IFN-beta expression and secretion as part of a stress response.

[0289] Example 3: In vitro expression of viral proteins (spike Covid19) from RNA To test whether the Sars-CoV2 spike protein was expressed in HEK293T, Vero E6, C2C12, and / or BJ fibroblasts, the following experiment was performed: HEK293T, Vero E6, C2C12, and / or BJ fibroblasts were transfected with RNA expressing only the Sars-CoV2 spike protein (Figure 3A) as a control and co-transfected in parallel with two RNA constructs derived from the IVT reaction, one expressing the Sars-CoV2 spike protein and the other expressing an NCT inhibitory protein (Figures 3A and 3B), as described below.

[0290] On day 0, HEK293T, Vero E6, C2C12, and / or BJ fibroblast cells were seeded in 500 μl DMEM (HEK293T, Vero E6, C2C12) or 500 μl MEM (BJ fibroblasts) containing 10% FBS in 24-well cell culture microplates. On day 1, cells were transfected with the respective RNA-spike alone or in combination with RNA-L from the IVT reaction using Lipofectamine™ MessengerMAX™ Transfection Reagent (Invitrogen) according to the manufacturer's instructions. At a later date, transfected cells and cell culture supernatants were assayed for NanoLuc luciferase activity.

[0291] To further measure interferon sensitivity with and without NCT inhibitor protein, cells were treated with IFN-beta 6–24 h after transfection. For mouse (C2C12) or human (HEK293T, Vero E6, BJ fibroblast) cells, recombinant IFN-beta (Fisher Scientific) was serially diluted 5-fold and added to cells 6 or 24 h after transfection with RNA encoding the SARS-CoV2 spike protein alone or in combination with the NCT inhibitor protein. The ratio of SARS-CoV2 spike expression was normalized to the untreated sample. EC50 values ​​for IFN sensitivity were calculated using a four-parameter nonlinear regression of log(inhibitor) versus response with variable slope in Prism (GraphPad).

[0292] For interferon-producing cell lines (C2C12, BJ fibroblasts), interferon secretion as a marker of inflammation was measured. For this, cell supernatants were collected 24-72 hours after transfection and clarified by centrifugation at 500 g for 5 minutes. The clarified supernatants were then analyzed for the presence of IFN-beta by ELISA (R&D Systems) according to the manufacturer's instructions.

[0293] Furthermore, an ELISA assay was used to demonstrate 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. The following 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 with primary antibody (rabbit anti-RBD, diluted 1:500 in EZ Block, 75 μl per well) at room temperature for 1 hour. The wells were then washed three times with PBST and incubated with goat anti-rabbit HRP secondary antibody (75 μl per well) diluted 1:1000 in EZ Block reagent for 1 hour at room temperature. The wells are then washed five times with PBST and 75 ul of TMB substrate is added to each well, and after 30 minutes of incubation at room temperature, 75 ul of stop solution is added and the absorbance at 450 nm is measured using a plate reader.

[0294] Example 4: In vitro expression of GBA To test whether GBA is expressed as a secreted protein in HEK293T, Vero E6, C2C12, and / or BJ fibroblasts, the following experiment was performed. HEK293T, Vero E6, C2C12, and / or BJ fibroblasts were transfected with RNA expressing GBA alone (Figure 4A) as a control and co-transfected in parallel with two RNA constructs derived from the IVT reaction, one expressing GBA protein and the other expressing an NCT inhibitory protein (Figures 4A and 4B), as described below. On day 0, HEK293T, Vero E6, C2C12, and / or BJ fibroblasts were seeded in 500 μl DMEM (HEK293T, Vero E6, C2C12) or 500 μl MEM (BJ fibroblasts) containing 10% FBS in 24-well cell culture microplates. On day 1, cells are transfected with either RNA-Luc alone or in combination with RNA-L from the IVT reaction using Lipofectamine™ MessengerMAX™ Transfection Reagent (Invitrogen) according to the manufacturer's instructions. At a later date, transfected cells and cell culture supernatants are assayed for GBA activity.

[0295] To further measure interferon sensitivity with and without NCT inhibitor protein, cells were treated with IFN-beta 6–24 h after transfection. For mouse (C2C12) or human (HEK293T, Vero E6, and BJ fibroblast) cells, recombinant IFN-beta (Fisher Scientific) was serially diluted 5-fold and added to cells 6 or 24 h after transfection with RNA encoding GBA protein alone or in combination with NCT inhibitor protein. The ratio of GBA expression was normalized to the untreated sample. EC50 values ​​for IFN sensitivity were calculated using a four-parameter nonlinear regression of log(inhibitor) versus response with variable slope in Prism (GraphPad).

[0296] For interferon-producing cell lines (C2C12, BJ fibroblasts), interferon secretion as a marker of inflammation is measured. For this, cell supernatants are collected 24-72 hours after transfection and clarified by centrifugation at 500 g for 5 minutes. The clarified supernatants are then analyzed for the presence of IFN-beta by ELISA (R&D Systems) according to the manufacturer's instructions.

[0297] For GBA activity assays, cells were seeded and transfected with the respective RNAs as described above. On day 3, cell culture supernatants were collected, cleared from cell debris by centrifugation, and retained for assay of secreted GBA activity. Adherent cells were detached from the plate using 500 μl PBS and pelleted by centrifugation. Cell pellets were lysed using 1× 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) pre-equilibrated to 37°C prior to lysis. The protein concentration of the cell lysate was determined using a Pierce A660 protein assay according to the manufacturer's instructions, and the cell lysate was then diluted to a final protein concentration of 125 μg / ml using 1× GBA assay buffer. 40 μl of prediluted cell lysate was pipetted in duplicate into each well of a clear 96-well plate. 20 μl of cell culture supernatant, previously collected, was added in duplicate to each well of a clear 96-well plate, and 20 μl of 2× GBA assay buffer was added to each well containing cell culture supernatant. 20 μl of assay substrate (6 mM 4-MU-beta-D-glucopranosid prepared in 1× GBA assay buffer) was then added to each well containing cell lysate or cell culture supernatant. A standard curve was prepared in adjacent wells using 4-methylumbelliferone in 1× GBA assay buffer. Samples were incubated in the presence of the assay substrate at 37°C for 30 minutes to 4 hours, after which 100 μl of stop solution (0.5 M glycine, 0.3 M NaOH (pH 10)) was added. The fluorescence of each sample and standard is then measured using a Biotek Synergy LX plate reader at Ex / Em wavelengths of 360 / 445 nm.The GBA activity in the cell lysate samples is calculated using the following formula: activity = [B / (T × V × P)] × D = pmol / min / mg = μU / mg, where B is the reciprocal of the amount of 4-MU (pmol) calculated using the standard curve, T is the reaction time (min), V is the sample volume (ml), P is the initial concentration of the protein sample, and D is the dilution factor of the sample (if applicable). The GBA activity in the cell culture supernatant is calculated using the following formula: activity = [B / (T × V)] × D = pmol / min / ml = μU / ml, where B is the reciprocal of the amount of 4-MU (pmol) calculated using the standard curve, T is the reaction time (min), V is the sample volume (ml), and D is the dilution factor of the sample (if applicable).

[0298] Example 5: In vivo expression of luciferase from RNA To evaluate nanoluciferase expression in vivo, groups of five female C57BL / 6 mice each, 6-8 weeks of age, are injected intramuscularly with either (a) the luciferase-encoding RNA shown in Figure 2A or (b) the luciferase-encoding RNA in series with the NCT inhibitory protein complexed with a lipid nanoparticle formulation in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO (pH 7.4)). For baseline measurements, four mice are injected intramuscularly with PBS alone. The dose is administered using a syringe / needle.

[0299] Animals were randomly assigned to groups, and body weights were recorded on day 1 and then every two weeks until the end of the study. Adverse events (RM, SD, RD) were recorded according to appropriate laboratory criteria. Any individual animal that experienced a single observed weight loss of more than 30% or three consecutive measurements of more than 25% weight loss was euthanized.

[0300] Whole blood was collected via the submandibular vein and processed to obtain serum for analysis. Blood was collected on days 0 (pre-dose), 3, 7, 14, 21, 28, and 42. Serum was analyzed for luciferase concentration by NanoLuc assay.

[0301] For the NanoLuc assay, 50 μl of diluted serum was loaded into an opaque 96-well microplate. 50 μl of fresh 1× Nano-Glo assay reagent in the respective assay buffer (Promega) was added to each well, and after 2–5 min of incubation, luminescence was measured using a Biotex Synergy LX plate reader.

[0302] Example 6: Immunogenicity of RNA-derived Sars-CoV2 spike in vivo To evaluate the expression of the Sars-CoV2 spike in vivo, groups of five female C57BL / 6 mice, 6–8 weeks of age, were used. The groups were injected intramuscularly with either (a) RNA encoding the Sars-CoV2 spike shown in Figure 3A or (b) RNA encoding the Sars-CoV2 spike contiguous with an NCT inhibitor protein, complexed with a lipid nanoparticle formulation in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO (pH 7.4)) on day 0 (priming) and boosted in the same manner on day 28. For baseline measurements, four mice were injected intradermally with PBS alone. The dose was administered using a syringe / needle.

[0303] Animals were randomly assigned to groups, and body weights were recorded on day 1 and then every 2 weeks until the end of the study. Adverse events (RM, SD, RD) were recorded according to appropriate laboratory criteria. Any individual animal that experienced a single observed weight loss of more than 30% or three consecutive measurements of more than 25% weight loss was euthanized.

[0304] Whole blood was collected via the submandibular vein and processed to obtain serum for analysis. Blood was collected on days 0 (pre-dose), 3, 14, 28, and 42. Serum was analyzed for anti-SARS-CoV-2 spike antibodies (elicitation of a humoral immune response) by ELISA assay. An enzyme-linked immunosorbent assay (ELISA) was performed using purified SARS-CoV-2 spike RBD protein as the coating material. High-binding 96-well plates were coated with 75 μl of 2 μg / ml SARS-CoV-2 spike RBD (Creative Diagnostics® DAGC149 recombinant SARS-CoV-2 spike protein receptor-binding domain [His]), and the plates were incubated overnight at 4°C. After incubation, the plates were washed twice with 0.05% Twen-20 in PBS, and the wells were blocked using EZ-block (37°C for 2 hours). Plates were washed twice with 0.05% Tween®-20 in PBS, and serum collected from mice was added to the wells (1:500 dilution for bound antibody detection, 1:100 to 1:7812500 for endpoint titration). After incubation for 1 hour at room temperature, plates were washed three times with 0.05% Tween-20 in PBS, and 75 μl of secondary antibody (goat anti-rabbit, HRP conjugate, 1:4,000 dilution) was added and incubated for 1 hour at room temperature. The wells were thoroughly washed (five times with 0.05% Tween-20 in PBS), and binding was developed using 75 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Surmodisc Inc. TMB One-Component HRP Microwell Substrate). The reaction is carried out for 30 minutes, after which it is stopped with 75 μl of stop solution (450NM LIQ Stop Reagent from Surmodisc Inc.) and the absorbance at 450 nm is measured using a Biotex Synergy LX plate reader. Endpoint titers are calculated in Prism (GraphPad) using variable slope 4-parameter nonlinear regression from values ​​interpolated from log(inhibitor) vs. reaction minus 4 times background.

[0305] Example 7: Effect of L protein expression on reporter gene expression when cells are exogenously stimulated with poly I:C or IFN-beta.

[0306] Poly I:C and IFN-beta (activators or innate immune signaling pathways) reduce reporter gene expression in a dose-dependent manner. EMCV L protein, as an NCT inhibitor protein, protects reporter gene expression from downregulation.

[0307] Once mRNA enters the cells, host- or administration-dependent induced inflammation can occur, thereby inhibiting the expression of the encoded gene of interest (GOI) and subsequent therapeutic effects. The effects of protein L were assessed when added in trans to human dermal fibroblasts transfected with mRNA encoding a reporter GOI (firefly luciferase) and stimulated with poly I:C, a synthetic double-stranded RNA analog that acts as a potent immunostimulant.

[0308] Human dermal fibroblasts (BJ cells, ATCC) were grown 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-antimycotic) in 96-well cell culture plates. The cells were incubated overnight at 37°C and 5% CO2. On day 1, cells were transfected with the indicated RNA constructs 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-beta was added to the cells at the indicated final concentrations. A reporter mRNA (Trilink) encoding firefly luciferase features the CleanCap mRNA cap structure, in which 100% of the uridines in its sequence are replaced with 5-methoxyuridine. This type of modified mRNA was used because it is a weak activator of innate immune signaling pathways in recipient cells, allowing us to primarily focus on the effects of L protein mRNA in situations where inflammatory stimuli are exogenously applied. L protein-encoding mRNA and nLuc filler mRNA (both of which contain 100% N1-methyl-pseudouridine to reduce their ability to naturally activate pro-inflammatory cell signaling pathways) were in vitro transcribed using the HiScribe™ T7 mRNA Kit with CleanCap Reagent AG (NEB) according to the manufacturer's instructions, as described in Example 1. For transfections, 10 ng of reporter mRNA was mixed with the indicated amounts of L protein-encoding mRNA and filler nLuc mRNA to ensure that all transfections were performed using equal amounts of mRNA. Two days after transfection, firefly luciferase expression from the reporter gene was measured using the Bio-Glo Luciferase Assay (Promega) according to the manufacturer's instructions.

[0309] As shown in Figures 9 and 10, poly I:C and IFN-beta, respectively, as potent activators of the innate immune cell signaling pathway, inhibit firefly luciferase expression in a dose-dependent manner (diagonally striped bars). However, in the presence of L NCT inhibitor protein-encoding mRNA (gray and black bars), the effect of reducing firefly luciferase by poly I:C and IFN-beta (Figures 9 and 10, respectively) is significantly reduced. Thus, L NCT inhibitor protein protects GOI expression in the presence of excessive and inhibitory levels of inflammation, as modeled by the addition of poly I:C and IFN-beta. As predicted by its predicted mechanism of action of inhibiting nucleocytoplasmic transport, L NCT inhibitor protein-encoding mRNA inhibited global reporter gene expression to some extent in the absence of pro-inflammatory signals. This is because inhibition of that pathway is generally associated with some degree of downregulation of protein translation. Notably, however, in the presence of poly I:C and IFN-beta, LNCT inhibitor protein-encoding mRNA was useful in restoring reporter gene expression (Figures 9 and 10). A general finding was that in both cases, the fold increase in reporter gene expression recovery was greater with higher concentrations of the pro-inflammatory stimulus. Interestingly, in the case of poly I:C as a stimulus, 1 ng of LNCT inhibitor mRNA showed the strongest effect on restoring reporter gene expression, whereas in the case of IFN-beta, 0.2 ng of LNCT inhibitor mRNA was the strongest effect. This suggests that, depending on the primary source and nature of the pro-inflammatory stimulus, different amounts of LNCT inhibitor mRNA may be required (and, more importantly, can be used) to restore the expression of target genes induced by activated innate immune cell signaling pathways as needed.

[0310] Example 8: Effect of L protein on reporter gene expression when cells are endogenously stimulated with intracellularly delivered pro-inflammatory mRNA transgenes (i.e., mRNA containing natural uridines).

[0311] In vitro transcribed mRNA containing natural nucleosides is known to activate immune signaling pathways, which can result in downregulation of intracellularly delivered mRNA expression (as shown in Example 7). mRNA containing N1-methyl-pseudouridine is known to induce immune activation at low levels, which is generally not associated with downregulation of intracellularly delivered mRNA expression. Consistent with the latter observation, L NCT inhibitor protein-encoding mRNA enhances GOI expression derived from potent pro-inflammatory mRNA containing natural uridine nucleosides, while failing to form non-inflammatory mRNA containing N1-methyl-pseudouridine.

[0312] The mRNA encoding the LNCT inhibitor protein was produced in-house using the HiScribe T7 mRNA Kit with CleanCap Reagent AG Kit (NEB) with 100% ml-pseudouridine (Trilink) and polyA tailing (NEB) according to the manufacturer's instructions. Nanoluciferase-expressing mRNA was synthesized in an identical manner, except for the presence or absence of synthetic nucleoside substitutions, as shown in Figures 11A-11C. mRNA synthesis was performed as described in Example 1 above.

[0313] For transfection, 10 ng of reporter mRNA was mixed with the indicated amounts of L NCT inhibitor protein-encoding mRNA and irrelevant filler mRNA to ensure that all transfections were performed using equal amounts of mRNA. Two days after transfection, reporter gene-derived nanoluciferase expression was measured using the Nano-Glo Luciferase Assay (Promega) according to the manufacturer's instructions.

[0314] First, we observed that at day 6 after mRNA transfection, LNCT inhibitor protein-encoding mRNA increased reporter gene expression by 3.1-4.4-fold, depending on the amount of LNCT inhibitor protein-encoding mRNA co-transfected with the reporter gene, but this was only observed when the reporter gene-encoding mRNA was pro-inflammatory (i.e., contained only natural nucleosides) (Figure 11A). When cells were transfected with reporter gene-encoding non-inflammatory mRNA containing pseudo-uridine, LNCT inhibitor protein did not increase transgene expression, as expected (Figure 11B).

[0315] To further analyze the observed phenomenon of increased gene expression driven by inherently inflammatory mRNAs, a time-course experiment was performed. BJ cells were co-transfected with the above-mentioned pro-inflammatory natural mRNAs or non-inflammatory N1-methyl-pseudouridine-modified mRNAs and 1 ng of LNCT inhibitor protein-encoding mRNA, and luciferase expression was measured at days 3, 6, 9, 13, 16, and 20 post-transfection. Similar to the previous results, when the reporter gene-encoding mRNA was pro-inflammatory (i.e., contained only natural nucleosides), transgene expression was improved at all time points, with an average improvement of 5.7-fold across all time points (Figure 11C). However, when BJ cells were transfected with non-inflammatory pseudouridine-containing reporter mRNA, LNCT inhibitor protein only slightly affected reporter mRNA-driven expression at all time points (Figure 11C), which is consistent with previously reported results.

[0316] These data demonstrate the benefits of using L NCT inhibitor protein-encoding mRNA to increase gene expression in applications requiring the use of natural or partially natural mRNA molecules.

[0317] Example 9: Effect of L protein on reporter gene expression when cells are endogenously stimulated with intracellularly delivered pro-inflammatory self-amplifying RNA (saRNA) transgenes.

[0318] Once mRNA enters the cell, induced inflammation can occur, depending on the host or administration, thereby inhibiting the expression of the encoded gene of interest (GOI) and subsequent therapeutic effects. One therapeutic modality that induces excessive inflammation is self-amplifying mRNA (saRNA). We evaluated the role of EGT-404 on reporter gene expression encoded on a saRNA cassette derived from Venezuelan equine encephalitis virus (VEEV) in human fibroblasts. When delivered from a separate mRNA molecule, the LNCT inhibitor protein-encoding mRNA enhanced saRNA in a dose-dependent manner at all measured time points (Figures 12A and 12B).

[0319] Human dermal fibroblasts (BJ cells) were cultured and transfected as in Examples 7 and 8. mRNA encoding the LNCT inhibitor protein was produced using the HiScribe T7 mRNA kit with CleanCap Reagent AG kit (NEB) with 100% ml-pseudouridine (Trilink) and polyA tailing (NEB) according to the manufacturer's instructions. A reporter gene (nano-luciferase) encoding a VEEV saRNA replicon was purchased from Aldevron. All transfections were performed using 100 ng of saRNA and the indicated amount of mRNA encoding the LNCT inhibitor protein. To maintain a constant total mRNA mass, an appropriate amount of unrelated mRNA was added to the mixture as filler mRNA. At the indicated time points, nano-luciferase expression was measured using the Nano-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions.

[0320] The expression of the saRNA-encoded nano-luciferase was first analyzed 6 days after transfection. The LNCT inhibitor protein enhanced luciferase expression in a concentration-dependent manner by approximately 11-fold compared to conditions in which LNCT inhibitor protein-encoding mRNA was not used (Figure 12A), with the optimal amount of LNCT inhibitor mRNA being 1 ng. The relative effect of LNCT inhibitor mRNA on improving saRNA reporter gene expression increased over time by up to 26-fold on days 16 and 20 after transfection compared to conditions in which LNCT inhibitor protein-encoding mRNA was not used (Figure 12B). At each time point, the optimal amount of LNCT inhibitor mRNA was 1 ng, similar to the amount observed on day 6. These results suggest that LNCT inhibitor mRNA is useful for increasing the level and duration of transgene expression when using self-amplifying RNA, a known potent inducer of innate immune signaling pathways in cells during the self-amplifying process. This demonstrates that L NCT inhibitor protein-encoding mRNA increased transgene expression when the innate immune signaling pathway was activated by exogenous or endogenous stimuli, which is consistent with the results in Example 8-8.

[0321] Example 10: Alternative mechanisms to nucleocytoplasmic shuttling inhibition to enhance protein expression.

[0322] Nucleocytoplasmic transport (NCT) inhibition can also be achieved by alternative mechanisms to those described in Examples 1-9 (e.g., nonviral proteins that bind to NCT pathway proteins or RNAi to knock down expression of NCT pathway genes). For example, overexpression of dominant-negative mutants of NCT pathway proteins (e.g., Ran Q69L mutant or MxB K131A protein) can block NCT. Similarly, myristoylated peptides derived from ERK and SMAD proteins inhibit Nup93-dependent NCT of transcription factors and RNA. This example describes a method for knocking down NCT proteins (e.g., Ran, NTF2, MxB, Xpo7, and Nup93) with siRNA.

[0323] As described in Example 7, poly I:C and IFN-beta are known activators of innate immune signaling pathways, reducing the expression of delivered mRNA molecules. Inhibition of these signaling pathways with NCT inhibitor proteins allowed for increased target protein expression. As described herein, siRNA inhibition of NCT pathway proteins can also inhibit poly I:C and IFN-beta signaling to increase target protein expression.

[0324] Method for knocking down NCT inhibitor proteins with siRNA Briefly, on day 0, BJ cells are seeded at 1E4 cells / well in complete DMEM (i.e., containing 10% heat-inactivated FBS + 1x antibiotic-antimycotic solution) in 96-well cell culture plates. The cells are incubated overnight at 37°C and 5% CO2. On day 1, the cells are co-transfected with reporter mRNA and NCT inhibitory RNA. Transfection is performed using the Mirus TransIT mRNA transfection kit (Mirus) described in Example 1 with 10 ng of reporter mRNA and an appropriate amount of NCT inhibitory RNA. Within 5 minutes of adding the transfection complex to the cells, poly I:C or IFN-beta is added to the cells at the final concentrations shown.

[0325] The reporter mRNA encodes firefly luciferase and features the CleanCap mRNA cap structure, with 100% of the uridines in the sequence replaced with N1-methylpseudouridine. The luciferase mRNA was cotransfected with one of the following NCT inhibitor RNAs: (a) mRNA encoding an anti-Xpo7 nanobody, (b) mRNA encoding an anti-NFT2 antibody, (c) mRNA encoding the Ran Q69L mutant protein, (d) mRNA encoding the MxB K131A mutant protein, (e) mRNA encoding a myristoylated ERK-derived phosphomimetic peptide, (f) mRNA encoding a myristoylated SMAD-derived phosphomimetic peptide, (f) siRNA targeted to Ran mRNA, (g) siRNA targeted to NTF2 mRNA, (h) siRNA targeted to MxB mRNA, (i) siRNA targeted to Xpo7 mRNA, or (j) siRNA targeted to Nup93 mRNA. NCT inhibitory mRNA is transcribed in vitro using the HiScribe™ T7 mRNA Kit (NEB) with CleanCap Reagent AG containing 100% N1-methyl-pseudouridine according to the manufacturer's instructions, as described in Example 1. NCT inhibitory siRNA is purchased as a chemically synthesized sequence (IDT Technologies).

[0326] On day 3 (i.e., 48 hours after transfection), firefly luciferase expression from the reporter gene is measured using the Bio-Glo Luciferase Assay (Promega) according to the manufacturer's instructions. The effect of NCT inhibitory RNAs on the enhancement of luciferase expression at any given poly I:C and IFN-beta concentration is determined by calculating the fold difference in luciferase expression in the presence and absence of each NCT inhibitory RNA.

[0327] Example 11: Mutant forms of the L protein control target protein expression levels.

[0328] Co-expression of L protein mRNA and saRNA expression of the reporter gene luciferase was performed as described in Example 9. Here, mutant forms of the EMCV L protein (SEQ ID NOs: 65, 66, 67, and 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 mutants (Figures 13 and 14).

[0329] Using the same system, saRNA-based expression of L protein mRNA and the reporter gene luciferase, we examined the expression of related inflammatory mediators. Expression of both IP-10 (Figure 15B) and IFN-beta (Figure 15A) was reduced by introduction of the L protein saRNA system described above, measured 6 days after transfection. Data shown are from N=3 experiments.

[0330] Example 12. Effect of L protein on reporter gene expression in vivo when reporter genes are expressed using self-amplifying RNA (saRNA).

[0331] To demonstrate the beneficial effect of L protein on saRNA-encoded reporter gene expression in vivo, the following experiment was performed. L protein-encoding mRNA (or the respective scrambled negative control sequence) was produced in-house as described in Example 9. saRNA vectors expressing nanoluciferase were purchased from Aldevron as described in Example 9. Nanoluciferase saRNA and L protein mRNA (or nanoluciferase saRNA and the respective scrambled negative control mRNA) were mixed and then formulated into lipid nanoparticles (LNPs) containing SM-102, 1,2-DSPC, cholesterol, and DMG-PEG(2000) lipids (SM-102 LNPs) and stored in 20 mM Tris (pH 7.5) 8.7% sucrose buffer. C57BL6 mice (female and 10–12 weeks old) were treated with SM-102 LNPs (total dose of 2 μg / mouse and N = 4 mice / group) via intramuscular injection. On the indicated days, nanoluciferase expression was measured by in vivo imaging using IVIS as follows: The nanolucifase substrate fluorofurimazine (FFz) was prepared according to the manufacturer's instructions (Promega) and administered to mice via intraperitoneal (ip) injection at 50 μl / mouse. IVIS imaging was performed 10 minutes after FFz ip injection.

[0332] Figure 16 shows the effect of L protein expression on the expression of a gene of interest via self-amplifying RNA (saRNA) in C57BL6 mice transfected with lipid nanoparticles carrying the L protein and saRNA system. Mice were injected with 2 μg of SM-102 LNP, with four mice per experimental group. As shown, in vivo saRNA-based luciferase expression was between 2- and 4-fold higher in the presence of L protein mRNA.

[0333] Example 13. Effect of L protein mRNA expression on the mRNA expression of therapeutically relevant proteins.

[0334] In this example, the effect of L protein (EG Tech) mRNA on the expression of therapeutically relevant gene mRNA vectors when co-delivered to target cells was investigated. The following therapeutically relevant gene mRNA vectors were studied: G6PC1 and SERPINA1. The G6PC1 gene is mutated in glycogen storage disorder type 1 (GSDI), also known as von Gierke disease, which results in the body breaking down glycogen, potentially leading to hypoglycemia, hepatomegaly, and kidney enlargement. Expression of functional G6PC1 in a gene therapy setting can provide therapeutic benefits for GSDI. SERPINA1 mutations lead to the development of alpha-1 antitrypsin deficiency (A1AD), which causes lung and liver damage in affected individuals. Expression of functional SERPINA1 in a gene therapy setting can provide therapeutic benefits for A1AD.

[0335] To assess the expression of G6PC1 and SERPINA1, we designed mRNA vectors in which therapeutically relevant genes of interest (GOIs) were fused to the NanoLuc reporter protein via a linker peptide (Figures 18A and 18B). The coding sequences of these GOIs were flanked by the hBG 5'UTR and hBG 3'UTR. The mRNA expression vectors contained a Cap1 mRNA cap structure, an N1-methylpseudouridine modification, and a 120-nt polyA tail. The GOI mRNA expression vectors were produced using in vitro transcription (IVT) by a third-party commercial supplier. Additionally, the L protein (EG Tech) mRNA, containing a Cap1 mRNA cap structure and an N1-methylpseudouridine modification, was produced using in vitro transcription (IVT) by a third-party commercial supplier.

[0336] Mouse fibroblast NIH / 3T3 cells were obtained from ATCC and grown according to the cell bank's instructions. Briefly, cells were grown in a humidified environment at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with high glucose and GlutaMAX, containing 10% fetal bovine serum (FBS) and 1x antimycotic / antibiotic solution. On day 0, NIH / 3T3 cells were seeded into 96-well plates. On day 1, cells were transfected with 10 ng of GOI mRNA per well, or a mixture of 10 ng of GOI mRNA and 0.16 ng of EG Tech mRNA per well, or a mixture of 10 ng of GOI mRNA and 1.25 ng of L protein (EG Tech) mRNA per well using the MIRUS TransIT mRNA transfection kit according to the manufacturer's instructions. On day 3 (i.e., 48 hours after transfection), cell supernatants were removed, and expression of the therapeutically relevant GOIs (G6PC1 and SERPINA1) in the cell lysates was measured by measuring fusion NanoLuc reporter activity using the Promega Nano-Glo Luciferase Assay System according to the manufacturer's instructions. Each experiment was performed with three technical replicates. A total of five independent experiments (biological replicates) were performed. The results are shown in Figures 19A-19D.

[0337] The expression level of the G6PC1 mRNA vector in NIH / 3T3 cells was moderate (less than 10,000 RLU) in the absence of L protein (EG Tech) mRNA; however, cotransfection of G6PC1 mRNA with L protein (EG Tech) mRNA increased G6PC1 expression (more than 100,000 RLU) (Figure 19A). This corresponded to a 20- or 34-fold increase in G6PC1 expression, depending on the amount of L protein (EG Tech) used for cotransfection (Figure 19B). Although the basal expression level of the SERPINA1 mRNA vector was relatively high (more than 100,000 RLU) in the absence of L protein (EG Tech) mRNA (Figure 19C), cotransfection of L protein (EG Tech) mRNA further increased SERPINA1 expression by approximately 2-fold (Figure 19D). These results indicated that mRNA expression of L protein could be of great benefit in increasing gene expression of mRNA vectors of therapeutically relevant GOIs, expanding the application area of ​​L protein to gene replacement therapy and gene therapy in general.

[0338] Example 14: Effect of L protein mRNA expression on therapeutically relevant plasmid DNA expression of target proteins.

[0339] In this example, we evaluated the effect of L protein (EG Tech) mRNA on the expression of therapeutically relevant genes in pDNA vectors when co-delivered to target cells. The therapeutically relevant gene, GBA (lysosomal acid glucosylceramidase), 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 disorder called Gaucher disease, in which glucose-reducing lipids accumulate at abnormal levels in different organs, potentially causing a wide range of functional disorders. Gene replacement therapy using a fully functional GBA gene may have therapeutic benefits.

[0340] To evaluate the ability of L protein (EG Tech) mRNA to express GBA protein from a plasmid DNA (pDNA) expression vector, we first designed a pDNA expression vector in which GBA was fused to the NanoLuc reporter protein via a peptide linker, while expression of the GBA-NanoLuc fusion protein was driven by a CMV promoter (Figure 20A). GBA pDNA was transfected into cells together with L protein (EG Tech) mRNA as follows.

[0341] Mouse fibroblast NIH / 3T3 cells were obtained from ATCC and grown according to the cell bank's instructions. Briefly, cells were grown in a humidified environment at 37°C with 5% CO2 using Dulbecco's modified Eagle's medium (DMEM) supplemented with high glucose and GlutaMAX, containing 10% fetal bovine serum (FBS) and 1x antimycotic / antibiotic solution. On day 0, NIH / 3T3 cells were seeded into 96-well plates. On day 1, cells were transfected with 0.3 ng L protein (EG Tech) mRNA / 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, the cells were further transfected with GBA-NanoLuc pDNA (50 ng / well) using the MIRUS TransIT-Lenti transfection reagent according to the manufacturer's instructions.

[0342] Because Gaucher disease is associated with elevated inflammation in affected tissues and elevated inflammation is known to suppress expression from pDNA, in some situations IFN-beta (1000 pg / ml) or poly I:C (1000 ng / ml) were added to cells 4 hours after transfection to model an inflammatory environment, and the effect of L protein (EG Tech) mRNA on GOI expression from pDNA vectors was studied in those situations.

[0343] On day 3 (i.e., 48 hours after transfection), the cell supernatant was removed, and the expression of GBA protein in the cell lysate was determined by measuring the fusion NanoLuc reporter activity using the Promega Nano-Glo Luciferase Assay System according to the manufacturer's instructions. The results are shown in Figure 21.

[0344] We observed that pDNA vector-derived GBA expression in unstimulated cells increased by 23% when 0.3 ng / well of L protein (EG Tech) mRNA was used (Figure 21, left bar). Stimulation of cells with the pro-inflammatory model molecules IFN-beta and poly I:C reduced GBA expression by approximately 80%. Notably, in IFN-beta and poly I:C-stimulated cells, L protein (EG Tech) mRNA was able to restore pDNA vector-derived GBA expression by 79% and 51%, respectively (Figure 21, center bar and right bar). These results demonstrate that L protein (EG Tech) mRNA can enhance pDNA vector-derived gene expression both under baseline conditions and in a strongly pro-inflammatory environment, which is particularly important in diseases with a range of inflammatory components.

[0345] Example 15: The effect of L protein mRNA expression on plasmid DNA induced cytokine induction.

[0346] Cytosolic plasmid DNA (pDNA) delivery can activate cellular response pathways (e.g., cGAS-STING and other pathways), which in turn can activate excessive inflammatory pathways in cells, resulting in reduced GOI expression in transduced cells and / or the induction of excessive inflammation. The latter may limit the applicability of DNA vectors for therapeutic use, as excessive inflammation may be associated with unfavorable reactogenicity and other side effect profiles. Therefore, in this example, we investigated the effect of L protein (EG Tech) mRNA in suppressing the activation of pro-inflammatory mediators induced by pDNA delivered into cells in the presence or absence of additional pro-inflammatory stimuli.

[0347] GBA-expressing pDNA (previously used and shown in Figure 20A) was transfected into cells along with L protein (EG Tech) mRNA as follows. Mouse fibroblast NIH / 3T3 cells were obtained from ATCC and grown according to the cell bank's instructions. Briefly, cells were grown in a humidified environment at 37°C with 5% CO2 using Dulbecco's modified Eagle's medium (DMEM) supplemented with high glucose and GlutaMAX, containing 10% fetal bovine serum (FBS) and 1x antimycotic / antibiotic solution. 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 / 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, the cells were further transfected with GBA-NanoLuc pDNA (50 ng / well) using MIRUS TransIT-Lenti transfection reagent according to the manufacturer's instructions. On day 3 (i.e., 48 h posttransfection), cell supernatants were removed, cleared from cellular debris by centrifugation, and analyzed for secretion of CCL2 (an inflammatory molecule induced by cytosolic double-stranded DNA via the cGAS-STING pathway) using the Biolegend LegendPlex Mouse Antiviral Response Panel according to the manufacturer's instructions.

[0348] The induction of CCL2 in cells transfected with GBA-expressing pDNA was reduced when pDNA was co-transfected with L protein (EG Tech) mRNA (Figure 22). These results demonstrate that L protein (EG Tech) mRNA can reduce excessive inflammatory pathway activation in pDNA-transfected cells, offering further promise for advancing the application of pDNA technology in vivo.

[0349] Example 16: Effect of L protein mRNA expression on inflammation induction in cells transfected with plasmid DNA.

[0350] In this example, we extended our analysis of the effects of L protein (EG Tech) mRNA to help suppress the induction of excessive inflammation in pDNA-transfected cells. In addition to the GBA-expressing pDNA, we designed an additional therapeutically relevant pDNA expression vector in which the NanoLuc reporter protein sequence was fused to the coding sequence for erythropoietin (EPO) downstream of a CMV promoter (Figure 20B).

[0351] NIH / 3T3 cells were grown and transfected with L protein (EG Tech) mRNA and GBA- and EPO-encoding pDNA as in the previous example. However, to study the effect of L protein (EG Tech) mRNA in suppressing pDNA-induced inflammation under more highly proinflammatory conditions, the cells were additionally stimulated with 1000 ng / ml poly I:C. This experimental setup allowed us to study the effect of L protein (EG Tech) mRNA in suppressing excessive proinflammatory molecules when inflammatory signaling is induced by pDNA (e.g., the cGAS-STING pathway) and other orthogonal pathways. Forty-eight hours after transfection, cell supernatants were removed, cleared from cell debris by centrifugation, and analyzed for secretion of CCL2, CCL5, CXCL10, IFN-alpha, and IFN-beta using the Biolegend LegendPlex Mouse Antiviral Response Panel according to the manufacturer's instructions.

[0352] Transfection of 3T3 cells with both GBA-encoding pDNA (Figures 23A-23E) and EPO-encoding pDNA (Figures 24A-24E) and further stimulation of the cells with poly I:C was observed to induce high levels of secretion of the inflammation-related molecules CCL2, CCL5, CXCL5, IFN-alpha, and IFN-beta. The additive effect of pDNA- and poly I:C-induced CCL2 was clearly evident when comparing the results with Figure 22. Importantly, in the presence of L protein (EG Tech) mRNA, the levels of CCL2, CCL5, CXCL5, IFN-alpha, and IFN-beta were reduced to the baseline levels of untransfected and poly I:C-unstimulated cells. These results further highlight the desirability of using L protein (EG Tech) mRNA in DNA-based therapeutic approaches, where proinflammatory signals from multiple sources must be simultaneously suppressed to achieve the desired therapeutic effect.

[0353] Example 17: Effect of L protein on saRNA expression of reporter protein.

[0354] To evaluate the effect of L protein (EG Tech) RNA on enhancing GOI expression from saRNA vectors, in contrast to previous examples herein in which L protein (EG Tech)-encoding mRNA was cotransfected in trans with a VEEV-based saRNA vector, we designed constructs in which the L protein (EG Tech)-encoding RNA sequence, L protein (EG Tech) mutant-encoding RNA sequence, or scrambled negative control sequence was included in the replicon cassette of a self-amplifying (saRNA) vector (Figures 25A-25C, respectively). The vectors used in these examples were constructed by cloning a secreted nanoluciferase (NanoLuc) and subsequent IRES-driven L protein (EG Tech) expression cassette into a commercially available VEEV-based saRNA vector (Simplicon SCR724, Millipore Sigma). L protein (EG Tech) WT encodes the wild-type protein sequence, L protein (EG Tech) mutant encodes a version of the L protein (EG Tech) protein with the indicated amino acid mutations, and scrambled negative control corresponds to an RNA sequence in which an early stop codon has been introduced to ensure functional L protein (EG Tech) protein cannot be expressed. Different amino acid mutants of the L protein (EG Tech) protein are used to modulate the activity of the L protein (EG Tech) protein and thereby modulate NCT.

[0355] BJ cells were grown and transfected with saRNA constructs designed as in the previous example (Example 11) using 200 ng of saRNA / well. Two and five days after transfection, Fluc expression in the transfected cells was measured using the Promega Nano-Glo Luciferase Assay System according to the manufacturer's instructions and normalized to the scrambled negative control sequence. The results are shown in Figures 27A-27C. When included in a VEEV-based saRNA replicon, L protein (EG Tech)-encoding RNA was observed to increase saRNA-derived NanoLuc expression by 33-fold, 26-fold, or 18-fold at day 2 posttransfection, depending on whether WT, E39A (SEQ ID NO: 138), or K35Q L protein (SEQ ID NO: 148) (EG Tech) protein mutants were used, respectively. By day 5 posttransfection, the effect of the L protein (EG Tech) in enhancing GOI expression became stronger, resulting in a 63-, 35-, or 17-fold increase in NanoLuc expression, depending on whether the WT or mutant version of the L protein (EG Tech) was used, respectively.

[0356] In summary, these results indicate that the effect of L protein (EG Tech) RNA is universal and can be used to enhance GOI expression in different configurations (including the use of L protein (EG Tech) mRNA to enhance expression of GOI mRNA 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 an integral part of a saRNA replicon).

[0357] Example 18: Effect of L protein mRNA expression on natural nucleoside inflammatory signals.

[0358] To further demonstrate that L protein (EG Tech) mRNA universally enhances GOI expression and reduces the activation of pro-inflammatory signals, 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 muscle cells. Experiments were performed as described in Example 2. Briefly, cells were transfected with a single mRNA construct encoding both luciferase and L protein (NCT inhibitor) proteins, separated by a p2A element. These mRNA constructs contained an ARCA cap and a 50%-50% mixture of 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 in the art. Forty-eight hours after transfection, luciferase activity was measured using a Promega NanoGlo Luciferase Assay System. Activation of innate immune signaling pathways was measured using IFN-beta ELISA (R&D Biosystems) and Biolegend LegendPlex human antiviral panel according to the manufacturer's instructions.

[0359] Results showed that inclusion of the L protein (EG Tech) RNA sequence in the mRNA expression vector increased GOI expression by 3.5- to 7.6-fold, depending on the cell line used (Figures 27A-27C). Furthermore, use of L protein (EG Tech) virtually eliminated induction of IFN-beta expression by ARCA-capped mRNA containing 50% natural uridines in all cell lines tested, as measured by IFN-beta ELISA (Figures 28A-28C). Furthermore, induction of several other pro-inflammatory modulators was also eliminated, as assessed by the LegendPlex human antiviral panel in BJ cells (Figures 29A-29D).

[0360] These results demonstrate that L protein (EG Tech) can be applied to improve GOI expression while suppressing activation of undesirable inflammatory pathways across cell types.

Claims

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

2. A composition comprising a self-amplifying ribonucleic acid (saRNA) encoding a nuclear cytoplasmic transport (NCT) inhibitor protein.

3. The composition of claim 1 , wherein the RNA encoding the target protein and / or the RNA encoding the NCT inhibitor 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 an mRNA encoding a target protein.

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

7. 3. 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 thereto.

8. 8. The composition of any one of claims 1 to 7, wherein the NCT inhibitor 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 according to any one of claims 1 to 8, wherein the target protein is a viral antigen.

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

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

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

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

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

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

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

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

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

19. 19. The method of any one of claims 13 to 18, wherein expression of the NCT inhibitor protein increases the target protein expression level 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.

20. 19. The method of any one of claims 13 to 18, wherein 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%.

21. 21. The method of any one of claims 13 to 20, wherein 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.

22. The expression of the NCT inhibitor protein is ５０ 22. The method of any one of claims 13 to 21, wherein the amount of the IL-16 gene is reduced 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.

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

24. 24. The method of claim 23, wherein expression of the NCT inhibitor protein increases the duration that the target protein is found in the 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. 25. The method of claim 23 or claim 24, wherein co-expression of the NCT inhibitor protein reduces the coefficient of variation (CV%) of the target protein in the tissue of the subject 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. 26. The method of any one of claims 13 to 25, wherein the NCT inhibitor 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 to 26, wherein the target protein is a viral antigen.

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

29. 29. The method of claim 28, wherein the cell signaling in response to a stressor is expression of interferon-beta, and introduction of the NCT inhibitor reduces interferon-beta expression 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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