Systems and methods for protein expression

Abstract

The present disclosure provides methods and compositions for improved expression of target protein through co-expression of enhancer protein in subject.Provided herein is a method for expressing target protein in subject, comprising administering a vector system of one or more polynucleotides encoding target protein and enhancer protein, wherein the polynucleotides are operably linked, and the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or the enhancer protein is 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.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 160,672, filed March 12, 2021, the entire contents of which are incorporated herein by reference.

[0002] Incorporating sequence tables The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer Readable Copy of the Sequence Listing (Filename: EXCI_002WO_SeqList_ST25, file size 405 kilobytes). [Background technology]

[0003] Recombinant expression of proteins in eukaryotic cells grown in culture has applications in scientific research and medicine. Recombinantly produced proteins (such as antibodies, enzymes, G protein-coupled receptors (GPCRs), secreted proteins, ion channels, viral proteins, and growth factors) are used within the pharmaceutical industry as important assets for developing new drugs (e.g., small molecule discovery), therapeutic agents (e.g., antibodies and other biological drugs), and analytical methods. In addition to their use in the pharmaceutical industry, recombinantly produced mammalian proteins are increasingly used in the food industry (e.g., for so-called meat production). For many recombinant proteins, it remains difficult to achieve expression of the recombinant protein in a functional form.

[0004] Transgene expression in living organisms such as yeast, plants, animals and humans has applications in scientific research and medicine. The modern therapeutic field focuses on the production of biologics such as enzymes or antibodies. Currently, many therapeutics in this field are manufactured outside the target subject and then injected. This process comes with its own challenges such as finding a suitable expression system and producing the drug product stably and in high yield. Another emerging field in novel therapeutics is the expression of genes within living systems by administering polynucleotides and using the body's own cells to produce the final drug product. The advantages of producing biologics in the body have many advantages including, for example, natural post-translational modifications. Furthermore, by simply using polynucleotides as therapeutic agents, many shortfalls in the production and purification of biologics are eliminated, leaving a low-cost, scalable, safe and stable drug product. This form of drug delivery has been studied since the early 1990s with little therapeutic success. There are many hypotheses as to why these drugs have not been successfully produced in living animals or humans, but there is still no solution.

[0005] There remains an unmet need for compositions and methods useful in the in vivo production of recombinant proteins and biologics in animals or humans. [Brief description of the drawings]

[0006] [Figure 1] 6 exemplary methods of regulating gene expression in eukaryotic cells are presented. [Figure 2A]Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2B] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2C] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2D]Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2E] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2F] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2G]Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2H] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2I] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2J]Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2K] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2L] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2M]Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2N] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2O] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2P]Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2Q] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2R] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2S]Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2T] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2U] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2V]Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2W] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 2X] Schematic diagrams of non-limiting exemplary constructs: EG1 (Figure 2A); EG2 (Figure 2B); EG3 and EG4 (Figure 2C); EG5 (Figure 2D); EG6 (Figure 2E); EG7 (Figure 2F); EG8 (Figure 2G); EG9 (Figure 2H); EG10 and EG11 (Figure 2I); EG12 and EG4 (Figure 2J); EG10 (Figure 2K); EG13 (Figure 2L); EG14 (Figure 2M); EG15 (Figure 2N); EG16 (Figure 2O); EG17 (Figure 2P); EG18 (Figure 2Q); EG19 (Figure 2R); EG20 (Figure 2S); EG21 (Figure 2T); EG22 (Figure 2U); EG23 (Figure 2V); EG24 (Figure 2W); and EG25 (Figure 2X). [Figure 3A]Optical and fluorescent microscopy of GFP expressed with construct EG2 (CMV-GFP-IRES-L) compared to control vector EG1. Figure 3A: Optical microscopy of cells containing EG1. Figure 3B: Fluorescence microscopy of cells containing EG1. Figure 3C: Optical microscopy of cells containing EG2. Figure 3D: Fluorescence microscopy of cells containing EG2. Expression of fluorescent GFP protein from the EG2 construct indicates the viability of the system. The reduction of deleterious overexpression in cells containing EG2 (Figure 3D) compared to cells containing EG1 (Figure 3B) demonstrates improved regulation of GFP expression by introduction of the L protein. Bars in Figures 3A-3D represent 400 microns. [Figure 3B] Optical and fluorescent microscopy of GFP expressed with construct EG2 (CMV-GFP-IRES-L) compared to control vector EG1. Figure 3A: Optical microscopy of cells containing EG1. Figure 3B: Fluorescent microscopy of cells containing EG1. Figure 3C: Optical microscopy of cells containing EG2. Figure 3D: Fluorescent microscopy of cells containing EG2. Expression of fluorescent GFP protein from the EG2 construct indicates the viability of the system. The reduction of deleterious overexpression in cells containing EG2 (Figure 3D) compared to cells containing EG1 (Figure 3B) demonstrates improved regulation of GFP expression by introduction of the L protein. Bars in Figures 3A-3D represent 400 microns. [Figure 3C] Optical and fluorescent microscopy of GFP expressed with construct EG2 (CMV-GFP-IRES-L) compared to control vector EG1. Figure 3A: Optical microscopy of cells containing EG1. Figure 3B: Fluorescent microscopy of cells containing EG1. Figure 3C: Optical microscopy of cells containing EG2. Figure 3D: Fluorescent microscopy of cells containing EG2. Expression of fluorescent GFP protein from the EG2 construct indicates the viability of the system. The reduction of deleterious overexpression in cells containing EG2 (Figure 3D) compared to cells containing EG1 (Figure 3B) demonstrates improved regulation of GFP expression by introduction of the L protein. Bars in Figures 3A-3D represent 400 microns. [Figure 3D]Optical and fluorescent microscopy of GFP expressed with construct EG2 (CMV-GFP-IRES-L) compared to control vector EG1. Figure 3A: Optical microscopy of cells containing EG1. Figure 3B: Fluorescent microscopy of cells containing EG1. Figure 3C: Optical microscopy of cells containing EG2. Figure 3D: Fluorescent microscopy of cells containing EG2. Expression of fluorescent GFP protein from the EG2 construct indicates the viability of the system. The reduction of deleterious overexpression in cells containing EG2 (Figure 3D) compared to cells containing EG1 (Figure 3B) demonstrates improved regulation of GFP expression by introduction of the L protein. Bars in Figures 3A-3D represent 400 microns. [Figure 4A] Light and fluorescence microscopy of GFP expressed with constructs EG3 and EG4 (T7-IRES-L-GFP and CMV-T7, respectively) compared to the control vector EG1. Figure 4A: Light microscopy of cells containing EG1. Figure 4B: Fluorescence microscopy of cells containing EG1. Figure 4C: Light microscopy of cells containing EG3+EG4. Figure 4D: Fluorescence microscopy of cells containing EG3+EG4. Expression of fluorescent GFP protein from the EG3+EG4 construct indicates the viability of the system. The reduction in expression in cells containing EG3+EG4 (Figure 4D) compared to cells containing EG1 (Figure 4B) demonstrates the improved regulation of GFP expression by introduction of the L protein. Bars in Figures 4A-4D represent 400 microns. [Figure 4B] Light and fluorescence microscopy of GFP expressed with constructs EG3 and EG4 (T7-IRES-L-GFP and CMV-T7, respectively) compared to the control vector EG1. Figure 4A: Light microscopy of cells containing EG1. Figure 4B: Fluorescence microscopy of cells containing EG1. Figure 4C: Light microscopy of cells containing EG3+EG4. Figure 4D: Fluorescence microscopy of cells containing EG3+EG4. Expression of fluorescent GFP protein from the EG3+EG4 construct indicates the viability of the system. The reduction in expression in cells containing EG3+EG4 (Figure 4D) compared to cells containing EG1 (Figure 4B) demonstrates the improved regulation of GFP expression by introduction of the L protein. Bars in Figures 4A-4D represent 400 microns. [Figure 4C]Light and fluorescence microscopy of GFP expressed with constructs EG3 and EG4 (T7-IRES-L-GFP and CMV-T7, respectively) compared to the control vector EG1. Figure 4A: Light microscopy of cells containing EG1. Figure 4B: Fluorescence microscopy of cells containing EG1. Figure 4C: Light microscopy of cells containing EG3+EG4. Figure 4D: Fluorescence microscopy of cells containing EG3+EG4. Expression of fluorescent GFP protein from the EG3+EG4 construct indicates the viability of the system. The reduction in expression in cells containing EG3+EG4 (Figure 4D) compared to cells containing EG1 (Figure 4B) demonstrates the improved regulation of GFP expression by introduction of the L protein. Bars in Figures 4A-4D represent 400 microns. [Figure 4D] Light and fluorescence microscopy of GFP expressed with constructs EG3 and EG4 (T7-IRES-L-GFP and CMV-T7, respectively) compared to the control vector EG1. Figure 4A: Light microscopy of cells containing EG1. Figure 4B: Fluorescence microscopy of cells containing EG1. Figure 4C: Light microscopy of cells containing EG3+EG4. Figure 4D: Fluorescence microscopy of cells containing EG3+EG4. Expression of fluorescent GFP protein from the EG3+EG4 construct indicates the viability of the system. The reduction in expression in cells containing EG3+EG4 (Figure 4D) compared to cells containing EG1 (Figure 4B) demonstrates the improved regulation of GFP expression by introduction of the L protein. Bars in Figures 4A-4D represent 400 microns. [Figure 5A] Fluorescence microscopy of DRD1-GFP fusions from constructs EG10 (CMV-[DRD1-GFP]) (Figure 5A) or EG8 (CMV-[DRD1-GFP]-IRES-L) (Figure 5C) is shown. DRD1-GFP using construct EG10 is expressed but is unable to transport the receptor to the outer membrane, resulting in the formation of inclusions (Figure 5B, arrows). DRD1-GFP using construct EG8 is expressed and robustly transported to the membrane, resulting in high quality and high yield of GPCR on the outer membrane (Figure 5D). [Figure 5B]Fluorescence microscopy of DRD1-GFP fusions from constructs EG10 (CMV-[DRD1-GFP]) (Figure 5A) or EG8 (CMV-[DRD1-GFP]-IRES-L) (Figure 5C) is shown. DRD1-GFP using construct EG10 is expressed but is unable to transport the receptor to the outer membrane, resulting in the formation of inclusions (Figure 5B, arrows). DRD1-GFP using construct EG8 is expressed and robustly transported to the membrane, resulting in high quality and high yield of GPCR on the outer membrane (Figure 5D). [Figure 5C] Fluorescence microscopy of DRD1-GFP fusions from constructs EG10 (CMV-[DRD1-GFP]) (Figure 5A) or EG8 (CMV-[DRD1-GFP]-IRES-L) (Figure 5C) is shown. DRD1-GFP using construct EG10 is expressed but is unable to transport the receptor to the outer membrane, resulting in the formation of inclusions (Figure 5B, arrows). DRD1-GFP using construct EG8 is expressed and robustly transported to the membrane, resulting in high quality and high yield of GPCR on the outer membrane (Figure 5D). [Figure 5D] Fluorescence microscopy of DRD1-GFP fusions from constructs EG10 (CMV-[DRD1-GFP]) (Figure 5A) or EG8 (CMV-[DRD1-GFP]-IRES-L) (Figure 5C) is shown. DRD1-GFP using construct EG10 is expressed but is unable to transport the receptor to the outer membrane, resulting in the formation of inclusions (Figure 5B, arrows). DRD1-GFP using construct EG8 is expressed and robustly transported to the membrane, resulting in high quality and high yield of GPCR on the outer membrane (Figure 5D). [Figure 6A] Fluorescence microscopy of DRD1-GFP fusions from constructs EG10 (CMV-[DRD1-GFP]) (Figure 6A) or EG12 and EG4 (T7-IRES-L-DRD1-GFP and CMV-T7, respectively) (Figure 6B) is shown. DRD1-GFP using EG10 is expressed but is unable to properly transport the receptor to the outer membrane, resulting in the formation of inclusions (Figure 6A, arrows). DRD1-GFP using EG12 in combination with EG4 is expressed and robustly transported to the membrane, resulting in high quality and high yield of GPCR on the outer membrane (Figure 6B). [Figure 6B]Fluorescence microscopy of DRD1-GFP fusions from constructs EG10 (CMV-[DRD1-GFP]) (Figure 6A) or EG12 and EG4 (T7-IRES-L-DRD1-GFP and CMV-T7, respectively) (Figure 6B) is shown. DRD1-GFP using EG10 is expressed but is unable to properly transport the receptor to the outer membrane, resulting in the formation of inclusions (Figure 6A, arrows). DRD1-GFP using EG12 in combination with EG4 is expressed and robustly transported to the membrane, resulting in high quality and high yield of GPCR on the outer membrane (Figure 6B). [Figure 7] Anti-CFTR Western blots are shown. Co-expression of the L protein and CFTR delivered as a PCR product or vector (left of dashed line) results in reduced yields but more homogenous samples compared to control expression of CFTR without co-expression of the L protein (right of dashed line). [Figure 8A] Results of His-tag purification of ITK are shown. Figure 8A shows SDS-PAGE of ITK affinity purified using His-tag. Lanes: lane 1, SeeBlue2+ prestained; lane 2, 500 ng GFP; lane 3, 2 μg ITK; lane 4, 5 μg ITK; lane 5, 10 μg ITK. Figure 8B shows Western blot analysis after SDS-PAGE of Figure 8A, with arrows pointing to ITK monomers and dimers. [Figure 8B] Results of His-tag purification of ITK are shown. Figure 8A shows SDS-PAGE of ITK affinity purified using His-tag. Lanes: lane 1, SeeBlue2+ prestained; lane 2, 500 ng GFP; lane 3, 2 μg ITK; lane 4, 5 μg ITK; lane 5, 10 μg ITK. Figure 8B shows Western blot analysis after SDS-PAGE of Figure 8A, with arrows pointing to ITK monomers and dimers. [Figure 9A] Schematic diagram of luciferase gene construct under CMV promoter. [Figure 9B] A map of the plasmid carrying the construct shown in Figure 9A is shown. [Figure 9C]Schematic diagram of the luciferase reporter gene and EMCV L1 protein linked by an IRES sequence under a shared CMV promoter. [Figure 9D] A map of the plasmid carrying the construct shown in Figure 9C is shown. [Figure 10] Luciferase expression measured by bioluminescence reading is shown. The use of the plasmid in Figure 9B results in higher luciferase expression initially, but expression decreases after day 18. The use of the plasmid disclosed herein (Figure 9D) allows stable expression of the reporter gene for a long period of time. [Figure 11] Bioluminescence images over time are shown. Note: The injection of animal 4 in the test group failed during the experiment, so this animal was excluded from data analysis. Use of the plasmid in Figure 9B results in highly variable luciferase expression, including loss of expression in two individual animals (animals 1 and 2) beyond day 18. Use of the plasmid disclosed herein (Figure 9D) allows for stable expression of the reporter gene over an extended period of time with low inter-animal variability. [Figure 12] Bioluminescence images over time are shown for a representative mouse expressing luciferase alone and a representative mouse expressing luciferase in the presence of an enhancer protein. The use of the plasmids disclosed herein (FIG. 9D) allows for stable expression of reporter genes over long periods of time. [Figure 13A] 1 shows a map of a plasmid carrying a nucleic acid sequence encoding the adalimumab antibody under a CMV promoter. [Figure 13B] 1 shows a map of a plasmid carrying a nucleic acid sequence encoding the adalimumab antibody and a gene encoding the EMCV L1 protein linked by a nucleic acid sequence encoding an IRES under a shared CMV promoter. [Figure 14A] Shown are images from a light microscope (top) and an immunofluorescence experiment (bottom) of HEK293T cells expressing adalimumab from an EG140 control plasmid. [Figure 14B]Shown are images from a light microscope (top) and from an immunofluorescence experiment of HEK293T cells expressing adalimumab in combination with the L1 enhancer protein from the EG141 plasmid. [Figure 15] 1 shows the results from an ELISA experiment performed to detect the presence of adalimumab in the supernatants of HEK293T cells transiently transfected with either EG140 or EG141. A purified recombinant human anti-TNFα antibody (NBP2-62567 Novus Biologicals) was used as a positive control in this experiment. [Figure 16] 1 shows the results from an ELISA experiment performed to detect binding of human TNF-α to adalimumab secreted by HEK293T cells transiently transfected with either EG140 or EG141. [Figure 17] 1 shows results from a luciferase reporter assay: Adalimumab in the supernatant of HEK293T cells transiently transfected with either EG140 or EG141 can suppress TNF-α-mediated activation of luciferase expression in reporter cells. [Figure 18A] Results from SDS PAGE are shown. [Figure 18B] 1 shows the results from a Western blot experiment demonstrating the heavy and light chains of adalimumab expressed from EG140 or EG-141 transfected cells. [Figure 19] Log quantification of bioluminescence imaging after subcutaneous injection of 30 μg of plasmid expressing firefly luciferase alone (Fluc Std) and Fluc in combination with the L enhancer protein (Fluc EG) is shown. [Figure 20] 1 shows the schematic design of pAAVtransfer_Adalimumab(Std) plasmid and enhancer protein plasmid pAAVtransfer_Adalimumab+L, showing the position of the adalimumab expression cassette and enhancer protein L relative to the 5' and 3' inverted terminal repeat (ITR) regions. [Figure 21]Figure 1 shows a map of the pAAVtransfer_Adalimumab(Std) plasmid. The adalimumab expression cassette is located between the 5' and 3' inverted terminal repeat (ITR) regions of the AAV transfer vector. [Figure 22] Figure 1 shows a map of the enhancer protein (EMCVgp1) pAAVtransfer_Adalimumab (EG) plasmid. The adalimumab expression cassette is located between the 5' and 3' inverted terminal repeat (ITR) regions of the AAV transfer vector. [Figure 23] Figure 1 shows the protein concentration (ng / ml) of adalimumab in cell culture supernatants of HEK293T cells transfected with pAdalimumab, pAdalimumab+enhancer L, pAAVtransfer adalimumab, and pAAVtransfer adalimumab+enhancer L plasmids. Adalimumab protein concentration in cell culture supernatants was measured using quantitative ELISA. [Figure 24] Secreted adalimumab protein EC50 values ​​measured in the HEK dual TNF-α reporter cell assay are shown. Top, adalimumab EC50 in cells transfected with pAdalimumab_STD plasmid and enhancer protein pAdalimumab_EG plasmid. Bottom, adalimumab EC50 in cells transfected with pAAVtransfer adalimumab_STD and enhancer L protein pAAVtransfer adalimumab_EG plasmid. Tables 4 and 5 summarize the results. [Diagram 25] The amount of secreted adalimumab concentration and the relative adalimumab activity normalized to the activity of the respective control vector lacking enhancer protein L are shown. [Figure 26A]Figure 26 shows the concentration of adalimumab in mouse serum after mice were treated with recombinant AAV vectors encoding adalimumab alone (AAV_Adalimumab_STD) and with enhancer protein L (AAV_Adalimumab_EG). Figure 26A shows the results of AAV vectors administered via intramuscular injection. Figure 26B shows the results of AAV vectors administered via subcutaneous injection. [Figure 26B] Figure 26 shows the concentration of adalimumab in mouse serum after mice were treated with recombinant AAV vectors encoding adalimumab alone (AAV_Adalimumab_STD) and with enhancer protein L (AAV_Adalimumab_EG). Figure 26A shows the results of AAV vectors administered via intramuscular injection. Figure 26B shows the results of AAV vectors administered via subcutaneous injection. [Figure 27] 1 shows the schematic design of pGBA-NanoLuc_STD and enhancer protein pGBA-NanoLuc_EG plasmids. [Figure 28A] The maps of pGBA-NanoLuc_STD plasmid and enhancer protein (EMCVgp1) pGBA-NanoLuc_EG plasmid are shown, respectively. [Figure 28B] The maps of pGBA-NanoLuc_STD plasmid and enhancer protein (EMCVgp1) pGBA-NanoLuc_EG plasmid are shown, respectively. [Figure 29] Figure 2 shows the Western blot results of pGBA-NanoLuc_STD and pGBA-NanoLuc_EG constructs expressed in HEK 293T cells. The predicted size of the pGBA-NanoLuc protein chimera is approximately 75 kDa. [Figure 30A]The expression of reporter protein and enzyme activity of GBA in HEK293T cell lysate (FIG. 30A and FIG. 30C, respectively) and supernatant (FIG. 30B and FIG. 30D, respectively) upon transfection with pGBA-NanoLuc_STD plasmid and enhancer protein pGBA-NanoLuc_EG plasmid are shown. The total expression of both NanoLuc and GBA activity is higher in the absence of enhancer protein L. [Figure 30B] The expression of reporter protein and enzyme activity of GBA in HEK293T cell lysate (FIG. 30A and FIG. 30C, respectively) and supernatant (FIG. 30B and FIG. 30D, respectively) upon transfection with pGBA-NanoLuc_STD plasmid and enhancer protein pGBA-NanoLuc_EG plasmid are shown. The total expression of both NanoLuc and GBA activity is higher in the absence of enhancer protein L. [Figure 30C] The expression of reporter protein and enzyme activity of GBA in HEK293T cell lysate (FIG. 30A and FIG. 30C, respectively) and supernatant (FIG. 30B and FIG. 30D, respectively) upon transfection with pGBA-NanoLuc_STD plasmid and enhancer protein pGBA-NanoLuc_EG plasmid are shown. The total expression of both NanoLuc and GBA activity is higher in the absence of enhancer protein L. [Figure 30D] The expression of reporter protein and enzyme activity of GBA in HEK293T cell lysate (FIG. 30A and FIG. 30C, respectively) and supernatant (FIG. 30B and FIG. 30D, respectively) upon transfection with pGBA-NanoLuc_STD plasmid and enhancer protein pGBA-NanoLuc_EG plasmid are shown. The total expression of both NanoLuc and GBA activity is higher in the absence of enhancer protein L. [Diagram 31]Figure 1 shows the relative GBA activity in GBA-NanoLuc chimeric protein normalized to NanoLuc activity. Figure 2 shows the activity in HEK293T cell lysates and supernatants upon transfection with pGBA-NanoLuc_STD plasmid and enhancer protein pGBA-NanoLuc_EG plasmid. In cell culture supernatants, the relative GBA activity was significantly higher with enhancer protein co-expression, demonstrating that enhancer protein increases the quality of expressed GBA protein. [Figure 32A] Figure 32 shows the bioluminescence imaging results of Balb / c mice treated with pGBA-NanoLuc_STD plasmid and enhancer protein GBA-NanoLuc_EG plasmid formulated in lipid nanoparticles (LNP). In Figure 32A and Figure 32B, images were taken from prone and supine positions, respectively. Figure 32C and Table 6 demonstrate that the mean coefficient of variation (CV%) of luciferase activity was higher without co-expression of L enhancer protein. [Figure 32B] Figure 32 shows the bioluminescence imaging results of Balb / c mice treated with pGBA-NanoLuc_STD plasmid and enhancer protein GBA-NanoLuc_EG plasmid formulated in lipid nanoparticles (LNP). In Figure 32A and Figure 32B, images were taken from prone and supine positions, respectively. Figure 32C and Table 6 demonstrate that the mean coefficient of variation (CV%) of luciferase activity was higher without co-expression of L enhancer protein. [Figure 32C] Figure 32 shows the bioluminescence imaging results of Balb / c mice treated with pGBA-NanoLuc_STD plasmid and enhancer protein GBA-NanoLuc_EG plasmid formulated in lipid nanoparticles (LNP). In Figure 32A and Figure 32B, images were taken from prone and supine positions, respectively. Figure 32C and Table 6 demonstrate that the mean coefficient of variation (CV%) of luciferase activity was higher without co-expression of L enhancer protein. Summary of the Invention [Means for solving the problem]

[0007] The inventors have recognized that co-expression of certain enhancer proteins with target proteins improves the quality and / or quantity of expression of recombinantly produced proteins and / or extends the duration of expression, extending the expression of a gene of interest in vitro, ex vivo and in vivo. In various embodiments, the disclosed compositions and methods exhibit one or more of the following advantages over the prior art: (1) they increase the protein expression (yield) of a target protein in a eukaryotic cell line or a living subject; (2) they control the regulation of expression of a target protein; (3) they express target proteins that exhibit reduced undesirable properties (e.g., misfolding, altered activity, incorrect post-translational modifications, and / or toxicity); (4) they increase correct folding and / or high yield of recombinant proteins; (5) they improve the performance of downstream activation pathways (e.g., GPCR signaling in cells, or in the case of in vivo expression, immune system response to an expressed antigen); (6) co-expression of enhancer proteins does not affect the functionality of the target protein and / or downstream metabolism of the cell. The present invention is not limited by these listed advantages, and some embodiments may exhibit none, some, or all of these advantages.

[0008] In one aspect, the present disclosure provides a system for recombinant expression of a target protein in eukaryotic cells, and a method for expression of a target protein in vivo, comprising one or more vectors.The vectors (or one vector) have a first polynucleotide encoding the target protein and a second polynucleotide encoding an enhancer protein.The enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or the enhancer protein is 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.The first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0009] In another aspect, the present disclosure provides a eukaryotic cell for expression of a target protein, the eukaryotic cell comprising an exogenous polynucleotide encoding an enhancer protein. The enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or the enhancer 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. The exogenous polynucleotide is operably linked to a promoter (optionally a native promoter or an exogenous promoter). In yet another aspect, the present disclosure provides a method for recombinant expression of a target protein, comprising introducing a polynucleotide encoding the target protein operably linked to a promoter into the eukaryotic cell. In yet another aspect, the present disclosure provides a method for recombinant expression of a target protein, comprising introducing the vector system of the present disclosure into a eukaryotic cell. In yet another aspect, the present disclosure provides a cell produced by introducing the vector system (or vector) of the present disclosure into a eukaryotic cell. In yet another aspect, the present disclosure provides a protein expressed by introducing the vector system (or vector) of the present disclosure into a eukaryotic cell. In yet another aspect, the present disclosure provides a method for expressing a target protein in a eukaryotic cell, comprising introducing a polynucleotide (operably linked to a promoter) encoding the target protein into a eukaryotic cell. The method utilizes co-expression of an enhancer protein to enhance the expression level, solubility and / or activity of the target protein.The enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT) and / or the enhancer 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.

[0010] In another aspect, the disclosure provides a method for generating antibodies against a target protein, comprising immunizing a subject with a cell or target protein produced using a system or method of the disclosure. In yet another aspect, the disclosure provides a method for antibody discovery by cell sorting, comprising providing a solution comprising labeled cells or labeled target proteins produced using a system or method of the disclosure and a population of recombinant cells expressing a library of polypeptides, each of which comprises an antibody or an antigen-binding fragment thereof, and sorting one or more recombinant cells from the solution by detecting the labeled cells or recombinant cells bound to the labeled target proteins. In a further aspect, the disclosure provides a method for panning a phage display library, comprising mixing the phage display library with a cell or target protein produced using a system or method of the disclosure, and purifying and / or enriching members of the phage display library that bind to the cells or target proteins.

[0011] Further aspects and embodiments are provided in the detailed disclosure that follows, but the invention is not limited by this summary. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In some embodiments, a system for recombinant expression of a target protein is provided, comprising one or more vectors. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the expression is in situ, in vivo, or ex vivo. In some embodiments, the vectors (or a vector) have a first polynucleotide encoding the target protein and a second polynucleotide encoding an enhancer protein. The enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or the enhancer protein is 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. The first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0013] Without being bound by theory, it is believed that the disclosed compositions and methods prevent cellular regulatory mechanisms from activating in response to the expression of recombinant target proteins, which improves the yield and / or functionality of the target proteins. The disclosed methods and systems can inhibit or interfere with one or more cellular mechanisms, including, but not limited to, (1) inhibition of transcription initiation, (2) inhibition of transcription termination and polyadenylation; (3) inhibition of mRNA processing and splicing, (4) inhibition of mRNA export; (5) inhibition of translation initiation; and (6) stress response (Figure 1).

[0014] In various embodiments, the compositions and methods of the present disclosure may improve target protein expression through co-expression of an enhancer protein, such as the L protein. Improved target protein expression associated with the compositions and methods of the present disclosure may include, for example, increased activity of the target protein, decreased expression levels, increased duration of expression, increased stability, increased duration in a cell or subject, increased uniformity of delivery, decreased degradation, and / or improved EC. 50 may be decreased.

[0015] Various embodiments are shown in Figures 2A-2Y and Table 1. In some embodiments, a first vector includes a polynucleotide encoding a target protein and a second vector includes a polynucleotide encoding an enhancer protein. In other embodiments, a single vector includes one or more polynucleotides encoding a target protein and an enhancer protein. A vector may include a single polynucleotide encoding both a target protein and an enhancer protein. Alternatively, two or more enhancer proteins and / or two or more target proteins are encoded by one or more vectors.

[0016] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a protein" can refer to one protein or a mixture of such proteins, reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0017] As used herein, the terms "about" or "approximately" when preceding a numerical value are intended to indicate that value plus or minus a range of 10%. For example, "about 100" includes 90 and 110.

[0018] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0019] As used herein, unless otherwise indicated, nucleotide sequences are listed in a 5' to 3' orientation and amino acid sequences are listed in an N-terminal to C-terminal orientation.

[0020] The terms "nucleic acid sequence", "nucleic acid", "nucleotide", "nucleotide sequence", and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0021] "Regulatory elements" include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may direct expression primarily in a desired tissue of interest (e.g., muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes)). Regulatory elements may also direct expression in a time-dependent manner (e.g., cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue-specific or cell type-specific. In some embodiments, the regulatory element may be a pol I promoter, a pol II promoter, a pol III promoter, or a combination thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. The term "regulatory element" also encompasses enhancer elements such as the WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I; the SV40 enhancer; and the intron sequence between exon 2 and exon 3 of rabbit β-globin.

[0022] A "vector" is used to transfer genetic material into a target cell. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that include one or more free ends, nucleic acid molecules that do not include free ends (e.g., circular); nucleic acid molecules that include DNA, RNA, or both; and various other polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which DNA or RNA sequences from a virus are present in the vector for packaging into the virus (e.g., retrovirus, adenovirus, lentivirus, and adeno-associated virus). In embodiments, viral vectors may be replication-incompetent. Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0023] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0024] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, a teenager, a child (2-14 years old), an infant (1-24 months old), or a newborn (up to 1 month old). In some embodiments, the adult is about 65 years old or older, or about 60 years old or older. In some embodiments, the subject is a pregnant woman or a woman who is about to become pregnant. In other embodiments, the subject is not a human; for example, a non-human primate; for example, a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject may be a pet, such as a dog or cat.

[0025] As used herein, "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms refer to an approach to obtain beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms during treatment. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who may not yet be affected by the disease, condition, or symptom, but who reports one or more of the physiological symptoms of the disease.

[0026] As used herein, "adalimumab" refers to the active pharmaceutical ingredient (API) in HUMIRA™, MABURA™, or EXEMPTIA™, or a functional variant thereof. Thus, adalimumab may refer to adalimumab-adaz, adalimumab-adbm, adalimumab-afzb, adalimumab-atto, adalimumab-bwwd, or adalimumab-fkjp. In some embodiments, adalimumab comprises any of the CDRs of SEQ ID NOs: 137-142 according to WO2011153477, which is incorporated herein in its entirety.

[0027] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances such as proteins, including glycoproteins, and peptides that are capable of eliciting an immune response.

[0028] As used herein, an "immunogenic response" in a subject results in the development in a subject of a humoral and / or cellular immune response to an antigen.

[0029] Polynucleotides The present disclosure relates to recombinant polynucleotides for expression of one or more target proteins and one or more enhancer proteins. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the expression is in situ, in vivo, or ex vivo. In some embodiments, the polynucleotide (or nucleic acid or nucleic acid molecule) may include one or more genes of interest and is delivered to a cell (e.g., a eukaryotic cell) using the compositions and methods of the present disclosure. The polynucleotides of the present disclosure may include DNA, RNA, and DNA-RNA hybrid molecules. In some embodiments, the polynucleotide is isolated from a natural source; prepared in vitro using techniques such as PCR amplification, in vitro transcription, or chemical synthesis; prepared in vivo, for example, via recombinant DNA technology; or prepared or obtained by any suitable method. In some embodiments, the polynucleotide is of any shape (linear, circular, etc.) or topology (single-stranded, double-stranded, linear, circular, supercoiled, kinked, nicked, etc.). Polynucleotides may also include nucleic acid derivatives, such as peptide nucleic acids (PNAS) and polypeptide-nucleic acid conjugates; nucleic acids having at least one chemically modified sugar residue, backbone, internucleotide linkage, base, nucleotide, nucleoside, or nucleotide analog or derivative, or basic site; and nucleic acids having chemically modified 5' or 3' termini; and nucleic acids having two or more such modifications. Not all linkages in a polynucleotide need be identical.

[0030] Examples of polynucleotides include, but are not limited to, oligonucleotides (including, but not limited to, antisense oligonucleotides, ribozymes, and oligonucleotides useful for RNA interference (RNAi)), aptamers, nucleic acids, artificial chromosomes, cloning vectors and constructs, expression vectors and constructs, gene therapy vectors and constructs, rRNA, tRNA, mRNA, mtRNA, and tmRNA. In some embodiments, the polynucleotide is an in vitro transcribed (IVT) mRNA. In some embodiments, the polynucleotide is a plasmid.

[0031] A polynucleotide is said to "encode" a protein if it comprises a nucleic acid sequence that can be transcribed and translated (e.g., DNA → RNA → protein) or translated (RNA → protein) to produce an amino acid sequence that corresponds to the amino acid sequence of the protein. In vivo (e.g., within a eukaryotic cell) transcription and / or translation is performed by endogenous or exogenous enzymes. In some embodiments, transcription of the polynucleotides of the disclosure is performed by the endogenous polymerase II (pol II) of the eukaryotic cell. In some embodiments, an exogenous RNA polymerase is provided on the same or a different vector. In some embodiments, the RNA polymerase is selected from T3 RNA polymerase, T5 RNA polymerase, T7 RNA polymerase, and H8 RNA polymerase.

[0032] Exemplary polynucleotides according to the present disclosure include a "first polynucleotide" that encodes a target protein; a "second polynucleotide" that encodes an enhancer protein; and a "coding polynucleotide" that encodes one or more target proteins, one or more enhancer proteins, and / or one or more separation elements.

[0033] Target Protein A polynucleotide according to the present disclosure may comprise a nucleic acid sequence that encodes one or more target proteins. The nucleic acid sequence that encodes a target protein is referred to as a gene of interest ("GOI").

[0034] In some embodiments, expression of the protein may cause cytotoxicity when expressed in a traditional expression system. In some embodiments, the protein is a protein that is expressed in a low yield in a traditional expression system. In some embodiments, expression or quality of the protein is significantly improved by expression by the disclosed methods compared to traditional expression systems. In some embodiments, expression of the target protein by the disclosed methods causes less toxicity to host cells compared to traditional expression systems. In some embodiments, expression of the target protein by the disclosed methods does not cause toxicity to host cells.

[0035] The target protein is not limited and may be any protein for which expression is desired. In some embodiments, the target protein is a viral protein. In some embodiments, the target protein is a soluble protein, a secreted protein (such as C-Inh), or a membrane protein. The target protein may be derived from any protein or polypeptide. In some embodiments, the target protein is derived from one or more animal proteins, one or more human proteins, one or more microbial proteins, one or more viral proteins, one or more fungal proteins, or a combination thereof. In some embodiments, the target protein can elicit an immunogenic response in a subject. In some embodiments, the target protein has one or more antigens.

[0036] In some embodiments, the target protein is composed of one or more proteins, one or more protein domains, one or more isoforms, or chimeric proteins. In some embodiments, the protein domain is a structural domain, a functional domain, an extracellular domain, or an intracellular domain. In some embodiments, the target protein has an altered activity and / or altered circulating half-life compared to its naturally occurring counterpart. For example, in some embodiments, the target protein is a chimeric protein composed of a functional domain of protein A and a structural domain of protein B, and the chimeric protein has a superior functional activity, circulating half-time, and / or other property compared to that of either protein A or protein B.

[0037] In some embodiments, the target protein is an antibody; an antibody-like molecule; a receptor; a monoclonal antibody; an antibody portion or fragment; a nanobody; a bispecific or multispecific antibody; or a bispecific or multispecific antibody-like molecule. In some embodiments, the antibody is adalimumab. In some embodiments, the antibody is abciximab, alemtuzumab, alirocumab, amivantamab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodalumab, brolucizumab, burosumab, canakinumab, caplacizumab, capromab, catumaxomab, semipilimab, certolizumab pegol, cetuximab, crizanlizumab, daclizumab, Daratumumab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emapalumab, emicizumab, enfortumab vedotin, eptinizumab, erenumab, ertumaxomab, etaracizumab, evolocumab, fremanezumab, galcanezumab, gemtuzumab ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, idarucizumab, imciromab, infliximab, inotuzumab · Ozogamicin, ipilimumab, isatuximab, itolizumab, ixekizumab, lanadelumab, loxivetumab, mepolizumab, mogamulizumab, moxetumomab · Pasudotox, natalizumab, necitumumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab · Vedotin, racotumomab, ramucil These include, but are not limited to, rituximab, romosozumab, rovelizumab, ruplizumab, sacituzumab govitecan, sarilumab, secukinumab, siltoximab, talquetamab, teclistamab, teprotumumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, ustekinumab, and vedolizumab.Polypeptide sequences of such antibodies are publicly available, e.g., in the Thera-SAbDab database (at opig.stats.ox.ac.uk), described in Raybould et al. (2020) Thera-SAbDab: the Therapeutic Structural Antibody Database. Nucleic Acids Res. 48(D1):gkz827.

[0038] In some embodiments, the heavy chain of adalimumab has the amino acid sequence of SEQ ID NO: 132. In some embodiments, the light chain of adalimumab has the amino acid sequence of SEQ ID NO: 133. In some embodiments, the heavy chain of adalimumab is encoded by the nucleic acid sequence of SEQ ID NO: 134. In some embodiments, the light chain of adalimumab is encoded by the nucleic acid sequence of SEQ ID NO: 135.

[0039] 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, for example, blinatumomab (MT103) and solitomab. In some embodiments, the target protein is a binding ligand or binder based on a protein scaffold (e.g., adnectin, anticalin, avimer, finomer, Kunitz domain, knottin, affibody, or DARPin).

[0040] 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 (CRP), lipoproteins (chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL)), transferrin, prothrombin, mannose-binding Lectins (MBL), albumins, globulins, fibrinogen, regulatory factors, and coagulation factors, such as Factor I, II, III, IV, V, VI, VII, IX, X, XI, XII, XIII, von Willband factor, prekallikrein, Fitzgerald factor, fibronectin, antithrombin III, heparin coenzyme 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 procoagulants. 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).

[0041] 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 motility factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor family, ciliary neurotrophic factor (CNF), and the like. NTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), colony-stimulating factor, macrophage colony-stimulating factor (M-CSF), epidermal growth factor (EGF), ephrin-ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, fibroblast growth factor (FGF) 1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF 17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, fetal bovine somatotrophin (FBS), GDNF ligand family, glial cell line-derived neurotrophic factor (GDNF), neuritin, 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), also known as migration stimulating factor (MSF), macrophage stimulating protein (MSP), 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),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, and glucagon.

[0042] In some embodiments, the target protein is an enzyme, non-limiting examples of which include α-glycosidase, glucocerebrosidase, iduronate-2-sulfate, α-galactosidase, urate oxidase, N-acetylgalactosidase, 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 invention may include proteins associated with enzyme replacement, such as agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, C3 inhibitor, Hurler and Hunter correction factors. In some embodiments, the compositions and methods of the invention are used for enzyme production. Such enzymes may be useful in the manufacture of clinical test kits or other diagnostic assays.

[0043] In some embodiments, the target protein is a membrane protein. Exemplary membrane proteins include integral membrane proteins such as ion channels, gap junctions, ionotropic receptors, transporters, cell surface receptors, proteins that shuttle between the membrane and the cytosol in response to signaling, and the like. In some embodiments, the cell surface receptor is a G protein-coupled receptor (GPCR), a tyrosine kinase receptor, an integrin, and the like. In some embodiments, the cell surface receptor is a G protein-coupled receptor. In some embodiments, the target protein is a seven-pass transmembrane domain receptor, a seven-transmembrane (7-TM) receptor, a heptahelical receptor, a serpentine receptor, or a G protein-coupled 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, an IL-1Ra, a tumor necrosis factor receptor (TNFR), or a vascular endothelial growth factor receptor (VEGFR). In some embodiments, the target protein is a receptor mimic. In some embodiments, the target protein is a protein that shuttles between the membrane and the cytosol in response to signal transduction, such as Ras proteins, Rac proteins, Raf proteins, Gα subunits, arrestins, Src proteins, and other effector proteins.

[0044] In some embodiments, the target protein is a nucleosidase, an NAD+ nucleosidase, a hydrolase, a glycosylase, a glycosylase 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.

[0045] In some embodiments, the target protein is abatacept, aflibercept, agalsidase beta, albiglutide, aldesleukin, alefecept, alglucerase, alglucosidase alfa, aliskiren, alpha-1-proteinase inhibitors, alteplase, anakinra, anthesim, anistreplase, anthrax immunoglobulin human, anti-neutrophil factor, antithrombin alpha, antithrombin III human, antithymocyte globulin, antithymocyte globulin (horse), antithymocyte globulin (rabbit), aprotinin, arcitumomab, asfotase alpha, asparaginase, asparaginase erwinia chrysanthemi), becapremin, belatacept, beractant, bivalirudin, botulinum toxin type A, botulinum toxin type B, buserelin, C1 esterase inhibitor (human), C1 esterase inhibitor, choriogonadotropin alpha, 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, drotocogine alfa, dulaglutide, efalizumab, efmoroctocog alfa, elosulfase alfa, enfuvirtide, epoetin alfa, epoetin zeta, eptifibatide, etanercept, exenatide, factor IX fibrinolysin complex (human) fibrinogen concentrate (human), fibrinolysin aka plasmin, filgrastim, filgrastim-sndz, follitropin alpha, follitropin beta, galsulfase, gastric intrinsic factor, glatiramer acetate, recombinant glucagon, glucarpidase, gramicidin D, hepatitis A vaccine, hepatitis B immunoglobulin, 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 beef, insulin degludec, insulin detemir,Insulin glargine, insulin glulisine, insulin lispro, insulin pork, insulin regular, insulin regular, insulin porcine, insulin isophane, interferon alpha-2a, recombinant, interferon alpha-2b, interferon alfacon-1, interferon alpha-n1, interferon alpha-n9, interferon beta-1a, interferon beta-1b, interferon ron gamma-1b, intravenous immunoglobulin, ipilimumab, ixekizumab, laronidase, lenograstim, lepirudin, leuprolide, liraglutide, lucinactant, lutropin alpha, lutropin alpha, mecasermin, menotropin, epoetin beta, metreleptin, muromonab, alpha interferon, nesiritide, ocriplasmin, omalizumab, oprelvekin, OspA lipoprotein, oxytocin, palifermin, pancrelipase, Poractant alfa, pramlintide, preotact, protein S human, rasburicase, reteplase, rilonacept, rituximab, romiplostim, sacrosidase, salmon calcitonin, sargramostim, satumomab pendetide, sebelipase alfa, secretin, secukinumab, sermorelin, serum albumin, iodized serum albumin, simoctocog alfa, sipuleucel-T, somatotropin recombinant, somatotropin recombinant, streptotropin The agent is selected from the group consisting of leptokinase, sulodexide, succotocog alfa, taliglucerase alfa, teduglutide, teicoplanin, tenecteplase, teriparatide, tesamorelin, thrombomodulin alfa, thymalfasin, thyroglobulin, thyrotropin alfa, thyrotropin alfa, tocilizumab, tositumomab, tuberculin purified protein derivative, turoctocog alfa, urofollitropin, urokinase, vasopressin, and velaglucerase alfa.

[0046] In some embodiments, the target protein is a biosimilar. In some embodiments, the target protein is a therapeutic polypeptide, such as a biological drug, also known as a biologic; a polypeptide enabling a biomarker, such as a diagnostic, prognostic, or predictive biomarker; a prophylactic polypeptide, such as an adjuvant, a soluble antigen, a subviral particle, or a virus-like particle; an auxiliary polypeptide, such as a polypeptide that supports the activity or binding of another molecule or inhibits another protein-protein interaction; 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 effect of drugs on these processes in animal models. In some embodiments, the target protein is a protein with applications in microscopy and imaging, such as a fluorescent protein. In some embodiments, the target protein is not a reporter protein, such as luciferase. In some embodiments, the target protein is a human protein.

[0047] 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 a protein complex assembly. 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 pharmacokinetic regulatory protein, a small molecule binding protein, an RNA binding protein, or a protein binding protein.

[0048] 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.

[0049] Enhancer Proteins The present disclosure relates to the co-expression of target protein and enhancer protein. In some embodiments, the enhancer protein may improve one or more aspects of target protein expression, including but not limited to yield, quality, folding, post-translational modification, activity, localization, and downstream activity, or may reduce one or more of misfolding, altered activity, incorrect post-translational modification, and / or toxicity.

[0050] In some embodiments, the enhancer protein is a nuclear pore-blocking viral protein. In some embodiments, the enhancer protein is a natural or synthetic peptide that can block a nuclear pore, thereby inhibiting nucleocytoplasmic transport ("NCT"). In some embodiments, the enhancer protein is a viral protein. In some aspects, the viral protein is an NCT inhibitor.

[0051] In some embodiments, the enhancer 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.

[0052] An enhancer protein is 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 an original protein and / or shares substantial sequence similarity with the original protein (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity) and shares one or more functional characteristics of the original protein. For example, a functional variant of an enhancer protein that is an NCT inhibitor retains the ability to inhibit NCT.

[0053] In some embodiments, the enhancer protein is a leader (L) protein from a picornavirus or a functional variant thereof. In some embodiments, the enhancer protein is a leader protein from a Cardiovirus, a Hepatovirus, or an Aphthovirus. For example, the enhancer protein may be from bovine rhinitis A virus, bovine rhinitis B virus, equine rhinitis A virus, foot and mouth disease virus, hepatovirus A, hepatovirus B, Marmota himalayana hepatovirus, Phopivirus, Cardiovirus A, Cardiovirus B, Theiler's murine encephalomyelitis virus (TMEV), Biruijsk human encephalomyelitis virus (VHEV), Theiler-like rat virus (TRV), or Saffold virus (SAF-V).

[0054] In some embodiments, the enhancer protein is the Theiler's virus L protein or a functional variant thereof. In some embodiments, the L protein shares at least 90% identity with SEQ ID NO:1. In some embodiments, the enhancer protein may comprise, consist of, or consist essentially of SEQ ID NO:1. The enhancer protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO:1.

[0055] In some embodiments, the L protein is an encephalomyocarditis virus (EMCV) L protein or a functional variant thereof. In some embodiments, the L protein may share at least 90% identity with SEQ ID NO:2. In some embodiments, the enhancer protein may comprise, consist of, or consist essentially of SEQ ID NO:2. The enhancer protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO:2.

[0056] 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.

[0057] In some embodiments, the enhancer protein is a picornavirus 2A protease or a functional variant thereof, hi some embodiments, the enhancer protein is a 2A protease from an enterovirus, a rhinovirus, an aphthovirus, or a cardiovirus.

[0058] In some embodiments, the enhancer protein is a rhinovirus 3C protease or a functional variant thereof. In some embodiments, the enhancer protein is a picornain 3C protease. In some embodiments, the enhancer protein is a 3C protease from an enterovirus, a rhinovirus, an aphthovirus, or a cardiovirus. For example, in some non-limiting embodiments, the enhancer protein is a 3C protease from a poliovirus, a coxsackievirus, a rhinovirus, a foot and mouth disease virus, or a hepatovirus A.

[0059] In some embodiments, the enhancer protein is a coronavirus ORF6 protein or a functional variant thereof, hi some embodiments, the enhancer protein is a viral protein that disrupts nuclear import complex formation and / or disrupts transport of STAT1 into the nucleus.

[0060] In some embodiments, the enhancer protein is an Ebola virus VP24 protein or a functional variant thereof. In some embodiments, the enhancer protein is an Ebola virus VP40 protein or VP35 protein. In some embodiments, the enhancer protein is a viral protein that binds to the importin protein karyopherin-alpha (KPNA). In some embodiments, the enhancer protein is a viral protein that inhibits binding of STAT1 to KPNA.

[0061] In some embodiments, the enhancer protein is a Venezuelan Equine Encephalitis Virus (VEEV) capsid protein or a functional variant thereof, hi some embodiments, the enhancer protein is a viral capsid protein that interacts with the nuclear pore complex.

[0062] In some embodiments, the enhancer protein is a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof. In some embodiments, the enhancer protein is an HSV ORF57 protein.

[0063] In some embodiments, the enhancer protein is a rhabdovirus matrix (M) protein or a functional variant thereof. In some embodiments, the enhancer protein is an M protein from a Cytorhabdovirus, Dichorhavirus, Ephemerisvirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Sprivivirus, Tibrovirus, Tupavirus, Varicosavirus, or Vesiculovirus.

[0064] In some embodiments, the enhancer protein is selected from the proteins listed in Table 1 or functional variants thereof. The polynucleotide encoding the enhancer protein may encode an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to an amino acid sequence listed in Table 1. The amino acid sequence of the enhancer protein may be at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to an amino acid sequence listed in Table 1. The amino acid sequence of the enhancer protein may be at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the enhancer protein may have an amino acid sequence that comprises, consists of, or consists essentially of one of the amino acid sequences listed in Table 1. In some embodiments, the enhancer protein may have an amino acid sequence that comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0065] [Table 1-1]

[0066] [Table 1-2]

[0067] Fusion proteins In some embodiments, the target protein and the enhancer protein are comprised in a single fusion protein. In some embodiments, the fusion protein may include a linking element. In some embodiments, the linking element may include a cleavage site for enzymatic cleavage. In other embodiments, the fusion protein or linking element does not include a cleavage site, and the expressed fusion protein includes both the target protein and the enhancer protein.

[0068] Protein Modification The target protein, enhancer protein, and / or fusion protein, or the polynucleotides encoding them, may be modified to include one or more markers, labels, or tags. For example, in some embodiments, the proteins of the present disclosure may be labeled with any label that allows for its detection, such as a radioactive label, a fluorescent agent, biotin, a peptide tag, an enzyme fragment, etc. The proteins may include an affinity tag, such as a His tag, a GST tag, a Strep tag, a biotin tag, an immunoglobulin binding domain, such as an IgG binding domain, a calmodulin binding peptide, etc. In some embodiments, the polynucleotides of the present disclosure include a selection marker, such as an antibiotic resistance marker.

[0069] In some embodiments, the target protein has one or more post-translational modifications. The type of post-translational modification is not limited and may be any post-translational modification known in the art. Non-limiting examples of post-translational modifications include glycosylation, acetylation, alkylation, methylation, biotinylation, glutamylation, glycylation, isoprenylation, lipoylation, phosphopanthenylation, phosphorylation, sufation, selenylation, C-terminal amidation, sumoylation, and any combination thereof.

[0070] Polymerase Endogenous or exogenous polymerases may be used for transcription of the polynucleotides encoding the target protein(s) and enhancer protein(s). In some embodiments, transcription of the polynucleotide(s) is performed by a native polymerase contained in the cell (e.g., a eukaryotic cell). Viral polymerases may alternatively or additionally be used. In some embodiments, viral promoters are used in combination with one or more viral polymerases. In some embodiments, eukaryotic promoters are used in combination with one or more eukaryotic polymerases. Exemplary viral polymerases include, but are not limited to, T7, T5, EMCV, HIV, influenza, SP6, CMV, T3, T1, SP01, SP2, Phi15, and the like. The viral polymerase is an RNA priming or capping polymerase. In some embodiments, an IRES element is used in conjunction with a viral polymerase.

[0071] One or more vectors according to the present disclosure may include a polynucleotide sequence encoding a polymerase. In some embodiments, the polymerase is a viral polymerase. The polynucleotide sequence encoding the polymerase may be included in a vector that includes a polynucleotide encoding a target protein and / or a polynucleotide encoding an enhancer protein. In some embodiments, the polymerase may be included in a vector that does not include a polynucleotide encoding a target protein or an enhancer protein.

[0072] In some embodiments, at least one of the one or more vectors included in the systems, methods, or cells disclosed herein may include a polynucleotide sequence encoding T7 RNA polymerase.

[0073] vector In some aspects, the present disclosure relates to vectors comprising nucleic acid sequences for expression of one or more target proteins and one or more enhancer proteins. In some embodiments, the vectors (or a vector) have a first polynucleotide encoding the target protein and a second polynucleotide encoding the enhancer protein.

[0074] The vector for use according to the present disclosure may include any vector known in the art.In certain embodiments, the vector is any recombinant vector capable of expressing a protein or polypeptide of interest or a fragment thereof, such as an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication-competent adenovirus vector, a replication-defective adenovirus vector, a herpes simplex virus, a retrovirus, a lentivirus, an alphavirus, a flavivirus, a rhabdovirus, a measles virus, a Newcastle disease virus, a poxvirus, a picornavirus, a herpes virus vector, a baculovirus vector, an adenovirus (Ad) vector, or a non-viral plasmid. In some embodiments, the vector is a viral gene delivery vector based on an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication-competent adenovirus vector, a replication-deficient adenovirus vector, a herpes simplex virus, a retrovirus, a lentivirus, an alphavirus, a flavivirus, a rhabdovirus, a measles virus, a Newcastle disease virus, a poxvirus, a picornavirus, a herpes virus vector, a baculovirus vector, an adenovirus (Ad) vector.

[0075] In some embodiments, the vector is a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, or an artificial chromosome. In some embodiments, the vector is a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC). In some embodiments, the vector is a naked or formulated plasmid DNA or a minicircle. The formulation is not limited and may be based on a non-viral DNA carrier, such as a peptide, lipid, polymer, or cation.

[0076] In some embodiments, the vector comprises a polynucleotide that is expressed constitutively, transiently, or in a regulated manner. In some embodiments, the regulation comprises a safety switch. Regulated expression of the polynucleotide from the vector may include the use of any technique known in the art, such as an inducible gene switch (e.g., synthetic receptors, protein-based switches, genetic circuits, genome editing tools, ribozymes, or aptazymes); or the use of apoptotic suicide genes and prodrugs. Protein-based switches are known in the art and may include the use of dimerization proteins or antibodies (e.g., rimiduside-induced dimerization of monomeric caspase 9).

[0077] The cells, systems, and methods disclosed herein may include one vector. In some embodiments, the cells, systems, and methods may include a single vector that includes a first polynucleotide that encodes a target protein and a second polynucleotide that encodes an enhancer protein.

[0078] The cells, systems, and methods disclosed herein may include two vectors. In some embodiments, the cells, systems, and methods may include a first vector comprising a first polynucleotide operably linked to a first promoter; and a second vector comprising a second polynucleotide operably linked to a second promoter.

[0079] The cells, systems, and methods disclosed herein may include more than two vectors, and the vectors may encode the target protein(s) and enhancer protein(s) in various combinations or configurations.

[0080] In some embodiments, a cell is provided that comprises one or more vectors of the present disclosure. In some embodiments, a cell is provided that comprises a polynucleotide of the present disclosure. In some embodiments, a cell is provided that expresses a target protein(s) and enhancer protein(s) of the present disclosure.

[0081] promoter A vector according to the present disclosure may include one or more promoters. The term "promoter" refers to a region or sequence located upstream or downstream of the transcription start site that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A polynucleotide(s) or vector(s) according to the present disclosure may include one or more promoters. The promoter may be any promoter known in the art. The promoter may be a forward promoter or a reverse promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the one or more promoters are native promoters. In some embodiments, the one or more promoters are non-native promoters. In some embodiments, the one or more promoters are non-mammalian promoters. Non-limiting examples of RNA promoters for use in the disclosed compositions and methods include U1, human elongation factor-1 alpha (EF-1 alpha), cytomegalovirus (CMV), human ubiquitin, spleen focus forming virus (SFFV), U6, H1, tRNA Lys , tRNA Ser and tRNA Arg , CAG, PGK, TRE, UAS, UbC, SV40, T7, Sp6, lac, araBad, trp, and Ptac promoters.

[0082] The term "operably linked" as used herein refers to elements or structures in a nucleic acid sequence that are linked by their operational ability and not by their physical location. The elements or structures are characterized by being capable of or achieving a desired action. It will be recognized by those skilled in the art that elements or structures in a nucleic acid sequence need not be in tandem or contiguous order to be operably linked.

[0083] In some embodiments, the promoter included in a vector according to the present disclosure is an inducible promoter.

[0084] A vector according to the present disclosure may include one or more viral promoters that allow transcription of one or more polynucleotides by one or more viral polymerases. In some embodiments, for example, a vector may include a T7 promoter configured for transcription of either or both of a first polynucleotide (i.e., a polynucleotide encoding a target protein) or a second polynucleotide (i.e., a polynucleotide encoding an enhancer protein) by T7 RNA polymerase.

[0085] Expression cassette One or more vectors according to the present disclosure may include one or more expression cassettes. The phrase "expression cassette" as used herein refers to a defined segment of a nucleic acid molecule that includes the minimum elements required for the production of another nucleic acid or protein encoded by that nucleic acid molecule. In some embodiments, the vector may include an expression cassette, which includes a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein. In some embodiments, the expression cassette includes a first promoter operably linked to the first polynucleotide; and a second promoter operably linked to the second polynucleotide. In some embodiments, the expression cassette includes a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0086] In some embodiments, the expression cassette comprises a coding polynucleotide comprising a first polynucleotide and a second polynucleotide linked by a polynucleotide encoding a separation element (e.g., a ribosomal skipping site or a 2A element), wherein the coding polynucleotides are operably linked to a shared promoter.

[0087] In some embodiments, the expression cassette comprises a coding polynucleotide that encodes an enhancer protein and a target protein linked by a separation element (e.g., a ribosomal skipping site or a 2A element), and the coding polynucleotides are operably linked to a shared promoter.

[0088] In some embodiments, the expression cassette is configured for transcription of a single messenger RNA encoding both the target protein and an enhancer protein, linked by a separation element (e.g., a ribosomal skipping site or 2A element), such that translation of the messenger RNA results in expression of the target protein and the enhancer protein (e.g., the L protein) as separate polypeptides.

[0089] In some embodiments, the expression cassette comprises a coding polynucleotide that encodes the enhancer protein and the target protein as a fusion protein with or without a polypeptide linker, optionally wherein the polypeptide linker is a cleavable linker or an intein-based cleavage system.

[0090] Separation Element In some embodiments, the target protein(s) and enhancer protein(s) according to the present disclosure are encoded on the same vector or on separate vectors. In some embodiments, when the nucleic acid sequences of one or more target proteins and one or more enhancer proteins are included in the same vector, the vector may include a separation element for separate expression of the proteins. In various embodiments, the vector is a bicistronic vector or a polycistronic vector. The separation element may be an internal ribosome entry site (IRES) or a 2A element. In some embodiments, the vector may include a nucleic acid encoding a 2A self-cleaving peptide. Exemplary 2A self-cleaving peptides include P2A, E2A, F2A, and T2A.

[0091] In some embodiments, either the first polynucleotide or the second polynucleotide, or both, are operably linked to an internal ribosome entry site (IRES).

[0092] In some embodiments, the first polynucleotide or the second polynucleotide, or both, are operably linked to a 2A element.

[0093] In some embodiments, the vector is as shown in Figure 13A or Figure 13B. In some embodiments, the vector comprises a polynucleotide that encodes SEQ ID NO: 132 and / or a polynucleotide that encodes SEQ ID NO: 133. In some embodiments, the vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 134 and / or a polynucleotide that encodes SEQ ID NO: 135.

[0094] In some embodiments, the vector comprises a nucleic acid sequence of SEQ ID NO:100, or a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:100.

[0095] SEQ ID NO:100:

[0096] In some embodiments, the vector comprises one or more of the following genetic elements:

[0097] Genetic elements of the vector shown in Figure 13B. Misc.: SEQ ID NO: 101 CGCGATGTACGGGCCAGATATACGCGTT

[0098] CMV enhancer: SEQ ID NO: 102 GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG

[0099] CMV promoter: SEQ ID NO: 103 GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTTACGGTGGAGGTCTATATAAGCAGAGCT

[0100] Misc.: SEQ ID NO: 104 GGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACG

[0101] Albumin signal peptide (codon optimized): SEQ ID NO: 105 ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTTCTTCCGCCTACAGC

[0102] Adalimumab heavy chain (variable region): SEQ ID NO: 106 GAAGTGCAGCTGGTTGAAAGCGGAGGCGGACTGGTCCAGCCAGGCAGAAGCCTGAGACTGTCTTGTGCCGCCTCTGGCTTCACCTTTGACGACTACGCCATGCACTGGGTGCGGCAGGCCCCTGGCAAGGGACTCGAGTGGGTCAGCGCCATCACCTGGAATAGCGGCCACATCGACTACG CAGATAGCGTTGAAGGCAGATTCACCATCTCCAGGGACAACGCCAAGAATTCTCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGATACCGCTGTGTACTACTGCGCCAAAGTGTCCTACCTGAGCACCGCCAGCTCCCTGGACTACTGGGCCAGGGCACCCTGGTGACAGTGAGCTCT

[0103] Human IgG1 heavy chain (constant region): SEQ ID NO: 107 GCTAGCACAAAAGGACCTAGCGTGTTTCCCCTGGCCCCTAGCAGCAAAAGCACCAGCGGCGGAACCGCCGCTCTGGGTTGTCTGGTGAAGGACTATTTCCCTGAACCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACTAGCGGCGTGCATACCTTCCCTGCCGTGCTGCAAAGCTCTGGCCTGTATAGCCTTTCTTCTGTGGTGACCGTGCCTAGCAGCTCTCTGGGCACACAGACATACATCTGCAATGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAAAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCGTGCCCCGCTCCTGAGCTGCTGGGCGGCCCTTCTGTGTTCCTGTTCCCCCCCAAACCTAAAGACACACTGATGATCAGCCGGACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTCCACAACGCCAAGACCAAACCTAGAGAGGAACAATACAACAGCACATATAGAGTGGTGTCTGTGCTGACAGTGCTCCACCAGGACTGGCTGAACGGAAAGGAATACAAGTGCAAGGTGTCCAACAAGGCCCTCCCTGCTCCAATCGAGAAGACCATTAGCAAGGCCAAGGGCCAACCTAGAGAGCCCCAGGTCTACACCCTGCCACCAAGTAGAGATGAGCTGACCAAGAACCAGGTGAGCCTAACATGCCTGGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACAACACCTCCTGTTCTGGATTCTGATGGCAGCTTCTTCCTGTACAGCAAGCTGACAGTGGATAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTTATGCACGAGGCCCTGCATAATCACTACACCCAGAAGAGCCTGTCTCTGAGCCCTGGCAAG

[0104] Adalimumab heavy chain complete: SEQ ID NO: 134

[0105] Furin cleavage site: SEQ ID NO:108 CAAGCGAAAACGGCGC

[0106] GSG linker: SEQ ID NO: 109 GGAAGCGGA

[0107] P2A self-cleaving peptide: SEQ ID NO: 110 GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGATGTGGAGGAGAACCCTGGACCT

[0108] Adalimumab light chain (variable region): SEQ ID NO: 111 GATATCCAGATGACCCAGTCTCCATCTAGCCTGAGCGCCAGCGTGGGAGATAGAGTGACCATCACCTGTAGAGCCTCTCAAGGCATCCGGAACTACCTGGCCTGGTATCAGCAGAAACCTGGCAAGGCTCCTAAGCTGCTGATCTACGCCGCTTCCACCC TGCAGAGCGGCGTTCCTTCTAGATTCAGCGGCAGCGGCTCCGGAACAGACTTCACCCTGACAATTAGCTCCCTGCAACCTGAAGATGTGGCTACATACTACTGCCAGAGATACAATCGGGCCCCTTACACCTTTGGACAGGGCACCAAGGTGGAAATCAAG

[0109] Human Ig kappa constant (light chain constant region): SEQ ID NO: 112 CGGACCGTGGCCGCCCCATCTGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAAAGCGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTCACACTGTCTAAAGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCTCCCCTGTGACAAAGAGCTTTAACAGAGGCGAGTGCTAA

[0110] Adalimumab light chain complete: SEQ ID NO: 135 GATATCCAGATGACCCAGTCTCCATCTAGCCTGAGCGCCAGCGTGGGAGATAGAGTGACCATCACCTGTAGAGCCTCTCAAGGCATCCGGAACTACCTGGCCTGGTATCAGCAGAAACCTGGCAAGGCTCCTAAGCTGCTGATCTACGCCGCTTCCACCCTGCAGAGCGGCGTTCCTTCTAGATTCAGCGGCAGCGGCTCCGGAACAGACTTCACCCTGACAATTAGCTCCCTGCAACCTGAAGATGTGGCTACATACTACTGCCAGAGATACAATCGGGCCCCTTACACCTTTGGACAGGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCCCCATCTGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAAAGCGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTCACACTGTCTAAAGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCTCCCCTGTGACAAAGAGCTTTAACAGAGGCGAGTGCTAA

[0111] Misc.: SEQ ID NO: 113 CCCCCCCCCCTA

[0112] IRES: SEQ ID NO: 114 ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAAT

[0113] Enhancer peptide: SEQ ID NO: 115 ATGGCCACAACCATGGAACAAGAGACTTGCGCGCACTCTCTCACTTTTGAGGAATGCCCAAAATGCTCTGCTCTACAATACCGTAATGGATTTTACCTGCTAAAGTATGATGAAGAATGGTACCCAGAGGAGTTATTGACTGATGGAGAGGATGATGTCTTTGATCCCGAATTAGACATGGAAGTCGTTTTCGAGTTACAGTAA

[0114] Misc.: SEQ ID NO: 116 ATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTT

[0115] SV40 poly(A) signal: SEQ ID NO: 117 AACTTGTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

[0116] Linker: SEQ ID NO:118 AGGCGTCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGC

[0117] Neomycin / kanamycin resistance gene: SEQ ID NO: 119 ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA

[0118] Misc.: Sequence No. 120 ATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC

[0119] Replication origin: SEQ ID NO: 121 TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA

[0120] Misc.: SEQ ID NO: 122 AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTTGACTCTT

[0121] Amino acid sequences of proteins expressed from the vectors shown in Figure 13B. In some embodiments, adalimumab is expressed as a single precursor polypeptide (i.e., a single open reading frame), which is co-translationally processed into mature antibody heavy and light chains. The components of the protein are as follows:

[0122] Albumin signal peptide: SEQ ID NO: 123 MKWVTFISLLFLFSSAYS

[0123] Adalimumab heavy chain (variable region): SEQ ID NO: 124 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS

[0124] Human IgG1 heavy chain (constant): SEQ ID NO: 125 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0125] Furin cleavage site (^ indicates cleavage site): SEQ ID NO: 126 RKRR^

[0126] Linker: SEQ ID NO: 127 GSG

[0127] P2A self-cleaving peptide (^ indicates the cleavage site): SEQ ID NO: 128 ATNFSLLKQAGDVEENPG^P

[0128] Adalimumab light chain (variable region): SEQ ID NO: 129 DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK

[0129] Human Ig kappa constant ( * indicates the stop codon of the precursor polypeptide): SEQ ID NO: 130 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

[0130] The enhancer peptide is expressed from an internal ribosome entry site as follows.

[0131] Enhancer peptide ( * indicates a stop codon) SEQ ID NO: 131 MATTMEQETCAHSLTFEECPKCSALQYRNGFYLLKYDEEWYPEELLTDGEDDVFDPELDMEVVFELQ

[0132] Adalimumab complete heavy chain: SEQ ID NO: 132 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0133] Adalimumab complete light chain: SEQ ID NO: 133 DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC *

[0134] In some embodiments, the vector comprises any of the complementarity determining regions (CDRs) of adalimumab, e.g., light chain CDRs SEQ ID NOs: 137, 139, 141:

[0135] SEQ ID NO:137 QRYNRAPYX

[0136] SEQ ID NO:139 AASTLQS

[0137] SEQ ID NO:141 RASQGIRNYLA

[0138] In some embodiments, the vector comprises any of the complementarity determining regions (CDRs) of adalimumab, e.g., heavy chain CDRs SEQ ID NOs: 138, 140, 142:

[0139] SEQ ID NO:138 VSYLSTASSLD

[0140] SEQ ID NO:140 AITWNSGHIDYADSVEG

[0141] SEQ ID NO:142 DYAMH

[0142] In some embodiments, the vector comprises a nucleic acid sequence having at least about 70% (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100%) identity to SEQ ID NO:136.

[0143] The nucleic acid sequence of the vector shown in Figure 13A. SEQ ID NO:136

[0144] Transfection, transduction, transformation. The terms "transfection," "transduction," and "transformation" refer to the process of introducing a nucleic acid into a cell (e.g., a eukaryotic cell). In some embodiments, a polynucleotide or vector described herein can be introduced into a cell (e.g., a eukaryotic cell) using any method known in the art. A polynucleotide or vector may be introduced into a cell by a variety of methods known in the art and selected in part based on the particular host cell. For example, a polynucleotide can be introduced into a cell using chemical, physical, biological, or viral means. Methods for introducing a polynucleotide or vector into a cell include, but are not limited to, the use of calcium phosphate, dendrimers, cationic polymers, lipofection, fugene, cell-penetrating peptides, peptide dendrimers, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, gene guns, magnetofection, microprojectile guns, nucleofection, and viral transduction.

[0145] Vectors containing targeting DNA and / or nucleic acids encoding the target protein and enhancer protein can be introduced into cells by various methods (e.g., injection, transformation, transfection, direct uptake, projectile bombardment, liposomes). The target protein and enhancer protein can be stably or transiently expressed in cells using expression vectors. Expression techniques in eukaryotic cells are well known to those of skill in the art. (See Current Protocols in Human Genetics: Chapter 12 "Vector Therapy" and Chapter 13 "Delivery Systems for Gene Therapy").

[0146] In some embodiments, the polynucleotide or vector can be introduced into the host cell by insertion into the genome using standard methods for generating stable cell lines, optionally through lentiviral transfection, baculovirus gene transfer into mammalian cells (BacMam), retroviral transfection, CRISPR / Cas9, and / or the use of transposons. In some embodiments, the polynucleotide or vector can be introduced into the host cell for transient transfection. In some embodiments, the transient transfection may be performed via the use of viral vectors, helper lipids, such as PEI, lipofectamine, and / or fectamine 293. The genetic elements can be encoded as DNA, for example, on a vector, or as RNA, for example, from PCR. The genetic elements can be separated separately or combined on the same vector.

[0147] The polynucleotide or vector may be introduced into the cell by a variety of methods well known in the art. For example, the polynucleotide can be introduced into the cell using chemical, physical, biological, or viral means.

[0148] In vivo delivery of targeted proteins In some embodiments, the polynucleotide or vector described herein can be introduced into a subject using any method known in the art. The polynucleotide or vector can be introduced into a subject by various methods well known in the art. The vector containing the targeting DNA and / or nucleic acid encoding the target protein and enhancer protein can be administered to a subject by various methods (e.g., injection, viral transfection, direct uptake, projectile bombardment).

[0149] Administration by injection may include, for example, intramuscular, intravenous, intracardiac, intraperitoneal, intravenous, intraarterial, intradermal, subcutaneous, intracranial, lumbar, intravitreal, intranasal, or other injections. The vector or polynucleotide can be introduced into the cells of the subject using chemical, physical, biological, or viral means. Methods of administering the polynucleotide or vector to the subject and / or introducing the polynucleotide or vector into the cells of the subject include, but are not limited to, direct injection with or without electroporation / sonoporation, with or without cationic or other polymers, lipids, lipid formulations, cell-penetrating peptides, nanoparticle-based delivery vehicles, nanogels, gene guns, jet gene devices, biolistics, and viral transduction. In some embodiments, administration is by injection under the skin. As described elsewhere in this application, in some embodiments, the vectors or polynucleotides disclosed herein may be introduced into cells of a subject using any viral gene delivery vector, such as an adenovirus, adeno-associated virus, herpes simplex virus, retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, picornavirus, or any other viral delivery system.

[0150] In some embodiments, a polynucleotide or vector encoding a target protein and an enhancer protein described herein may be administered to a subject to treat, prevent or manage at least one symptom of a disease. In some embodiments, the target protein is an antibody, such as a monoclonal antibody (e.g., adalimumab). In some embodiments, the subject is a subject with any condition known or to be discovered in the future to be treated, prevented or managed by expression of a target protein (e.g., adalimumab). For example, non-limiting examples of conditions that may be treated by administration of a polynucleotide or vector encoding adalimumab include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, hidradenitis suppurativa, uveitis, and juvenile idiopathic arthritis.

[0151] Thus, the present disclosure provides a method of treating or preventing disease in a subject, comprising administering to the subject a therapeutically effective amount of any one of the vectors or polynucleotides encoding the target protein and enhancer protein disclosed herein. The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve an outcome, e.g., to bring about a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the subject and disease symptoms being treated, the subject's weight and age, the severity of the disease symptoms, the mode of administration, etc., which can be easily determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system in which it is delivered.

[0152] The disclosed method of expressing a target protein in the presence of an enhancer has several advantages, as described below. In some embodiments, a target protein expressed in the presence of an enhancer protein using the compositions or methods disclosed herein is more functionally active than a target protein expressed in the absence of the enhancer protein. In some embodiments, a target protein expressed in the presence of an enhancer protein using the compositions or methods disclosed herein is at least about 1.2-fold (e.g., about 1.5-fold, about 1.7-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, or about 50-fold (including all values ​​and subranges therebetween)) more active than a target protein expressed in the absence of the enhancer protein.

[0153] In some embodiments, a target protein expressed in the presence of an enhancer protein using a composition or method disclosed herein is expressed for a longer period of time compared to a target protein expressed in the absence of the enhancer protein. In some embodiments, a target protein expressed in the presence of an enhancer protein using a composition or method disclosed herein is expressed for at least about 1 hour (e.g., about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 6 months, or about 1 year) longer compared to a target protein expressed in the absence of the enhancer protein.

[0154] In some embodiments, a smaller percentage of target proteins expressed in the presence of an enhancer protein using a composition or method disclosed herein exhibit undesirable properties (e.g., misfolding, altered activity, incorrect post-translational modification, and / or toxicity) compared to target proteins expressed in the absence of an enhancer protein. For example, in some embodiments, less than about 30% (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% (including all values ​​and subranges therebetween)) of target proteins expressed in the presence of an enhancer protein using a composition or method disclosed herein exhibit undesirable properties. In some embodiments, a higher percentage of target proteins expressed in the presence of an enhancer protein using a composition or method disclosed herein exhibit correct folding compared to target proteins expressed in the absence of an enhancer protein.

[0155] In some embodiments, the therapeutically effective amount of the vector or polynucleotide encoding the target protein and the enhancer protein administered to the subject is lower than the therapeutically effective amount of the control vector or polynucleotide encoding only the target protein. Without being bound by theory, it is believed that due to the improved expression quality and / or quantity of the target protein when expressed in the presence of the enhancer protein and / or the longer expression period, a lower dose of the vector or polynucleotide encoding the target protein and the enhancer protein (compared to the control vector or polynucleotide encoding only the target protein) is sufficient to induce a similar biological effect.

[0156] In some embodiments, subjects administered a vector or polynucleotide encoding a target protein and an enhancer protein show a reduced production of anti-target protein antibodies compared to control subjects administered a vector or polynucleotide encoding only the target protein. Without being bound by theory, it is also believed that the formation of poorly folded or unfolded target protein expressed in the absence of an enhancer promotes the production of anti-target protein antibodies. On the other hand, the improved expression quality and / or quantity of the target protein when expressed in the presence of an enhancer protein reduces the production of anti-target protein antibodies.

[0157] cells, cell lines, host cells Another aspect of the present disclosure relates to a cell comprising a polynucleotide and / or vector encoding one or more target proteins and one or more enhancer proteins. The polynucleotides, vectors, target proteins, and enhancer proteins may be any of those described herein.

[0158] In some embodiments, the cell is any eukaryotic cell or cell line. The disclosed polynucleotides, vectors, systems, and methods may be used in any eukaryotic primary cells and cell lines. Eukaryotic cell lines may include mammalian cell lines, such as human and animal cell lines. Eukaryotic cell lines may include insect, plant, or fungal cell lines. Non-limiting examples of such cells or cell lines generated from such cells include Bc HROC277, COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, 5P2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells, as well as insect cells, such as Spodoptera frugiperda. fugiperda (Sf, e.g., Sf9), or fungal cells, such as Saccharomyces, Pichia, and Schizosaccharomyces.

[0159] In some embodiments, the cells or cell lines for expressing the target protein(s) and enhancer protein(s) are human cells or cell lines. In certain aspects, the selection of a human cell line is beneficial for post-translational modifications ("PTMs"), such as, for example, glycosylation, phosphorylation, disulfide bonds, in the target protein. In some embodiments, human cells or cell lines are used for the expression of human target proteins.

[0160] In some embodiments, the disclosure provides a eukaryotic cell for expression of a target protein, the eukaryotic cell comprising an exogenous polynucleotide encoding an enhancer protein. In some embodiments, the exogenous polynucleotide encoding the enhancer protein is transiently transduced and / or not integrated into the genome of the cell. In some embodiments, the exogenous polynucleotide encoding the enhancer protein is stably integrated. In some embodiments, the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT). In some embodiments, the enhancer 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. The exogenous polynucleotide is operably linked to a promoter (optionally a native promoter or an exogenous promoter). In some embodiments, the polynucleotide is operably linked to an internal ribosome entry site (IRES). In some embodiments, the promoter is an inducible promoter.

[0161] In vitro and ex vivo methods The present disclosure provides a method for expressing a target protein in a eukaryotic cell. The method may include introducing a polynucleotide (operably linked to a promoter) encoding the target protein into a eukaryotic cell. The method utilizes co-expression of an enhancer protein to enhance the expression level, solubility and / or activity of the target protein. Furthermore, the method utilizes co-expression of an enhancer protein to extend the expression of the target protein for a longer period of time. In some embodiments, the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT). In some embodiments, the enhancer 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.

[0162] In some aspects, the disclosure relates to a method of producing a target protein through the use of a cell comprising a polynucleotide encoding one or more target proteins and one or more enhancer proteins. In some embodiments, the method is carried out in a eukaryotic cell comprising one or more vectors. In some embodiments, the method is carried out using the polynucleotides, vectors, and cells described in the preceding section. In some embodiments, a plurality of vectors (or a single vector) may have a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein. In some embodiments, the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0163] In some embodiments, the method may include introducing into a eukaryotic cell a polynucleotide encoding an enhancer protein operably linked to a promoter. In some embodiments, the method may include transfecting the eukaryotic cell with one or more DNA molecules, transducing the eukaryotic cell with a single viral vector, and / or transducing the eukaryotic cell with two or more viral vectors.

[0164] Further provided is a method for recombinant expression of a target protein, comprising introducing a polynucleotide encoding the target protein operably linked to a promoter into a eukaryotic cell. In some embodiments, the method of target protein expression comprises introducing the vector system of the present disclosure into a eukaryotic cell. In some embodiments, the target protein is a membrane protein. In some embodiments, the localization of the membrane protein to the cell membrane is increased compared to the localization observed when the membrane protein is expressed without an enhancer protein.

[0165] In vivo methods The present disclosure provides a method for expressing a target protein in vivo. In some embodiments, the method comprises introducing a polynucleotide (operably linked to a promoter) encoding the target protein into a cell of a subject. The method utilizes co-expression of an enhancer protein to enhance the expression level, solubility and / or activity of the target protein. In some embodiments, the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT). In some embodiments, the enhancer 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.

[0166] In some embodiments, the method induces an immune response in the subject. The immune response can be both immunogenic and immunosuppressive or immunomodulatory in nature. In some embodiments, the method treats a disease in the subject, the disease being caused by, correlated with, or associated with the target protein. In some embodiments, the method treats a disease in the subject, the expression level of the target protein in the subject being lower than the expression level of the target protein in a control subject without the disease.

[0167] In some embodiments, the present disclosure relates to a method of producing a target protein through the use of a polynucleotide encoding one or more target proteins and one or more enhancer proteins. In some embodiments, the method is carried out in vivo, comprising one or more vectors. In some embodiments, the method is carried out using the polynucleotides, vectors, and cells described in the preceding sections. In some embodiments, a plurality of vectors (or a vector) may have a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein. In some embodiments, the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0168] In some embodiments, the method may include introducing into the subject a polynucleotide encoding an enhancer protein operably linked to a promoter. In some embodiments, the method may include injection of one or more DNA molecules, a single viral vector, and / or two or more viral vectors.

[0169] Further provided are methods for in vivo expression of a target protein comprising introducing into a subject a polynucleotide encoding the target protein operably linked to a promoter. In some embodiments, the method of target protein expression comprises introducing into a subject a vector system of the present disclosure.

[0170] In some embodiments, the target protein and enhancer protein-DNA constructs are delivered via lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise a PEGylated lipid, cholesterol, and one or more ionizable lipids. In some embodiments, the LNPs comprise about 0.5% to about 2% PEGylated lipid, about 35% to about 45% cholesterol, and about 5% to about 65% one or more ionizable lipids. In some embodiments, the LNPs comprise DMG-PEG(2000), cholesterol, DOPC, and DLin-KC2-DMA in a ratio of about 1% DMG-PEG(2000) to about 40% cholesterol to about 10% DOPC and about 50% DLin-KC2-DMA.

[0171] Downstream Application In some embodiments, target proteins produced through the use of the compositions, systems, and methods of the invention are used as therapeutics, diagnostics, or for research and development. Exemplary applications include, but are not limited to, vaccines, enzyme replacement therapy, hormone replacement therapy, antibody therapy, antiviral treatments, antibacterial treatments, immunomodulatory agents, therapeutic cancer vaccines, immuno-oncology applications, bispecific T cell engagers, screening assays, diagnostic assays, clinical test kits, drug discovery, antibody discovery, and the like.

[0172] In some embodiments, target proteins produced through the use of the compositions, systems, and methods of the invention, and cells expressing such proteins, are isolated, purified, and / or used for downstream applications. Exemplary applications include, but are not limited to, small molecule screening, structure determination (e.g., X-ray crystallography, cryo-electron microscopy, etc.), activity assays, therapeutics, enzyme replacement therapy, screening assays, diagnostic assays, clinical test kits, drug discovery, antibody discovery, and the like. In some embodiments, the compositions and methods of the invention are used to produce antibodies for antibody screening assays or to produce antigens. In some embodiments, cells expressing target proteins can be used as assay systems, for example, to screen cell interactions, antibody binding, or the effects of small molecules in whole cell systems.

[0173] In some embodiments, the disclosure provides systems and methods for antibody discovery. In some embodiments, the disclosure provides methods for generating antibodies against a target protein, comprising immunizing a subject with a cell or target protein produced using the disclosed system or method. In various embodiments, the subject being immunized is a mouse, a rat, a rabbit, a non-human primate, a llama, a camel, or a human. Cells isolated from the subject can be subjected to further rounds of selection, either as isolated cells or, optionally, after generating hybridomas from the isolated cells. Gene cloning and / or sequencing can be used to isolate polynucleotide sequence(s) encoding heavy and light chains from the isolated cells or hybridomas. Gene cloning and / or sequencing can be applied to single cells or cell populations. In some embodiments, the compositions and methods of the disclosure are used to generate polyclonal antibodies via immunization of a subject followed by collection of serum from the subject.

[0174] The present disclosure further provides a method for antibody discovery by cell sorting, comprising providing a solution containing a population of labeled cells or target proteins produced using the disclosed system or method and recombinant cells expressing a library of polypeptides each comprising an antibody or antigen-binding fragment thereof, and sorting one or more recombinant cells from the solution by detecting the labeled cells or recombinant cells bound to the labeled target proteins. In other variations, cell sorting is performed on cells derived from an immunized subject. The subject may be immunized with cells or target proteins produced according to the disclosed method or with another suitable immunogen. In some embodiments, the recombinant cells contain a naive antibody library (optionally a human naive antibody library). A variety of antibody library production methods are known in the art and can be combined with the disclosed method. As used herein, the term "sorting" or "cell sorting" refers to fluorescence activated cell sorting, magnetic assisted cell sorting, and other means of selecting labeled cells in a population of labeled and unlabeled cells.

[0175] The present disclosure further provides a method for panning a phage display library, comprising mixing the phage display library with a cell or target protein produced using a system or method of the present disclosure, and purifying and / or enriching the phage display library members that bind to the cell or target protein. In some embodiments, the phage display library expresses a population of single chain variable fragments (scFv) or other types of antibodies / antibody fragments (such as Fabs).

[0176] In further embodiments, the present disclosure provides methods for screening any type of protein binder. The cells and target proteins of the present disclosure can be used to screen libraries of various types of molecules, including drugs and macromolecules (proteins, nucleic acids, and protein:nucleic acid complexes), to identify binding partners of the target protein. In other embodiments, the systems and methods of the present disclosure can be used to express libraries of target proteins in a single well, in pools of several sequences, or in libraries of gene sequences.

[0177] The ability to express antigens in their native or disease-associated form in high yields and / or present on the surface of cells allows for the discovery and / or production of more reliable antibodies, antibody fragments, and other molecules than prior art methods, which may be useful as therapeutic and / or research tools, or for other applications.

[0178] In some embodiments, the disclosed systems and methods are suitable for use in discovering antibodies that bind and / or are specific for a particular glycosylation pattern on a target molecule (e.g., a glycoprotein). In some embodiments, an antibody library is selected against a naturally glycosylated protein and counter-selected against an improperly glycosylated or deglycosylated cognate protein. Similarly, antibodies can be specifically selected against a glycosylation pattern by using a deglycosylation enzyme. In further embodiments, the cells and / or target proteins of the disclosure are used to confirm the binding and / or functional activity of novel antibodies or other macromolecules.

[0179] In some embodiments, target proteins produced through the use of the compositions, systems, and methods of the invention are used as therapeutics, diagnostics, or for research and development. Exemplary applications include, but are not limited to, vaccines, enzyme replacement therapy, hormone replacement therapy, antibody therapy, antiviral treatments, antibacterial treatments, immunomodulatory drugs, therapeutic cancer vaccines, bispecific T cell engager screening assays, diagnostic assays, clinical test kits, drug discovery, antibody discovery, and the like.

[0180] Exemplary Benefits The present compositions, systems, and methods may have many advantages. For example, as shown in Example 11, human NADase, which normally causes apoptosis and therefore produces undetectable yields when overexpressed in human cell lines, can be reliably expressed to produce yields of over 20 mg / L when an enhancer protein is co-expressed with the target protein. Furthermore, the NADase expressed via this exemplary method is functional (demonstrated by a phosphate release assay) and shows low batch-to-batch variability.

[0181] Similarly, in some embodiments, the methods, systems, and cells are used for reliable expression of difficult-to-express proteins. In some embodiments, the present disclosure relates to the production of proteins with low batch-to-batch variation. Proteins produced according to the present disclosure may exhibit one or more of the following improvements: purification without purification tag fusion; improved functional activity; reliable production; consistent activity; and suitability for therapeutic use.

[0182] The cells of the present disclosure may have one or more of the following advantages with respect to target protein expression: higher concentration of target membrane protein in membrane; slower / reduced target protein degradation; improved signal-to-noise ratio in whole cell assay; expression of target protein and / or enhancer protein without affecting downstream cell metabolism; increased stability against desensitization of membrane-bound membrane protein; and higher target protein yield. Example 1 provides an illustrative example of expression of enhancer protein without affecting downstream metabolism of cells. The GPCR illustrated in Example 1 can interact with its natural substrate and result in activation that can be measured in vitro.

[0183] The systems and methods may, in some embodiments, have one or more of the following advantages: compatibility with any eukaryotic cell type; reduced need for target protein expression optimization; and reliable expression of difficult-to-express proteins.

[0184] The method of expressing target protein in vivo disclosed herein has superior properties compared to the standard methods used for this purpose in the art.For example, as demonstrated in the examples, the method disclosed herein ensures stable expression of target protein for a longer period of time and reduces the variability of expression levels between animals.These properties allow the application of the method disclosed herein in the prevention and treatment of disease.

[0185] system The present disclosure provides a system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors. The present disclosure further provides a method for expressing a target protein in a subject in need thereof, comprising administering to the subject a vector system comprising one or more vectors comprising a) a first polynucleotide encoding the target protein and b) a second polynucleotide encoding an enhancer protein, wherein i) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii) the enhancer protein is 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, and the first polynucleotide and the second polynucleotide are operably linked to one or more promoters. The vectors (or one vector) may have a first polynucleotide encoding the target protein and a second polynucleotide encoding an enhancer protein. The enhancer protein may be an inhibitor of nucleocytoplasmic transport (NCT). In some embodiments, the enhancer protein 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. The first polynucleotide and the second polynucleotide may be operably linked to one or more promoters.

[0186] In some embodiments, the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT). In some embodiments, the NCT inhibitor is a viral protein.

[0187] In some embodiments, the enhancer protein is an NCT inhibitor 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.

[0188] The NCT inhibitor may be a picornavirus leader (L) protein or a functional variant thereof. In some embodiments, the NCT inhibitor may be a picornavirus 2A protease or a functional variant thereof. In some embodiments, the NCT inhibitor may be a rhinovirus 3C protease or a functional variant thereof. In some embodiments, the NCT inhibitor may be a coronavirus ORF6 protein or a functional variant thereof. In some embodiments, the NCT inhibitor may be an Ebola virus VP24 protein or a functional variant thereof. In some embodiments, the NCT inhibitor may be a Venezuelan equine encephalitis virus (VEEV) capsid protein or a functional variant thereof. In some embodiments, the NCT inhibitor is a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof. In some embodiments, the NCT inhibitor is a rhabdovirus matrix (M) protein or a functional variant thereof.

[0189] In some embodiments, the enhancer protein is an L protein, which is the L protein of Theiler's virus or a functional variant thereof. In some embodiments, the L protein may share at least 90% identity with SEQ ID NO:1.

[0190] In some embodiments, the L protein is an Encephalomyocarditis Virus (EMCV) L protein or a functional variant thereof. In some embodiments, the L protein may share at least 90% identity with SEQ ID NO:2.

[0191] 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.

[0192] The system may include a single vector comprising an expression cassette, the expression cassette comprising a first polynucleotide and a second polynucleotide. In some embodiments, the expression cassette comprises a first promoter operably linked to the first polynucleotide; and a second promoter operably linked to the second polynucleotide. In some embodiments, the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0193] In some embodiments, the expression cassette comprises a coding polynucleotide comprising a first polynucleotide and a second polynucleotide linked by a polynucleotide encoding a ribosomal skipping site, the coding polynucleotides being operably linked to a shared promoter.

[0194] In some embodiments, the expression cassette comprises a coding polynucleotide, the coding polynucleotide encoding an enhancer protein and a target protein linked by a ribosomal skipping site, the coding polynucleotide being operably linked to a shared promoter.

[0195] In some embodiments, the expression cassette is configured for transcription of a single messenger RNA encoding both the target protein and an enhancer protein, linked by a ribosomal skipping site, such that translation of the messenger RNA results in expression of the target protein and the enhancer protein (e.g., the L protein) as separate polypeptides.

[0196] The system may comprise one vector. In some embodiments, the system may comprise a single vector comprising a first polynucleotide encoding a target protein and a second polynucleotide encoding an enhancer protein.

[0197] The system may include two vectors: hi some embodiments, the system may include a first vector comprising a first polynucleotide operably linked to a first promoter; and a second vector comprising a second polynucleotide operably linked to a second promoter.

[0198] In some embodiments, either the first polynucleotide or the second polynucleotide, or both, are operably linked to an internal ribosome entry site (IRES).

[0199] In some embodiments, at least one of the one or more vectors included in the system may include a T7 promoter configured for transcription of either the first polynucleotide or the second polynucleotide, or both, by T7 RNA polymerase.

[0200] In some embodiments, at least one of the one or more vectors included in the system may include a polynucleotide sequence encoding T7 RNA polymerase.

[0201] The compositions and methods of the present disclosure provide improved expression of a target protein when co-expressed with an enhancer protein, such as the L protein. As used herein, "improved expression of a target protein" includes, but is not limited to, one or more of the following compared to the target protein: increased activity, reduced expression levels, increased duration of expression, increased stability, increased duration of detection in a cell or subject, increased uniformity of delivery, reduced degradation, and improved EC 50 Decrease in.

[0202] In some embodiments, co-expression of an enhancer protein increases the activity of a target protein in a cell or 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.

[0203] In some embodiments, co-expression of the enhancer 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%.

[0204] In some embodiments, co-expression of an enhancer protein increases the period during which active target protein is found in a cell or subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold.

[0205] The coefficient of variation (CV%) is provided as a measure of the uniformity of target protein expression and is defined as the standard deviation of the diagnostic moiety (e.g., fluorophore or radiolabel) signal divided by the signal mean. In some embodiments, co-expression of an enhancer protein increases the uniformity of expression of the target protein in a tissue or 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.

[0206] In some embodiments, co-expression of an enhancer protein reduces degradation of the target protein by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold.

[0207] In some embodiments, co-expression of an enhancer protein reduces the concentration of target protein effective to produce 50% of the maximum response (EC50). In some embodiments, the target protein is adalimumab and the response is neutralization of tumor necrosis factor-alpha (TNF-α) in a cell or subject. In some embodiments, the EC50 of adalimumab 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 in a cell or subject.

[0208] In some embodiments, co-expression of an enhancer protein (e.g., L protein) with an adalimumab protein improves the treatment of a disease selected from the following: rheumatoid arthritis, juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), plaque psoriasis (Ps), hidradenitis suppurativa (HS), and uveitis (UV). In some embodiments, co-expression of an enhancer protein with adalimumab provided herein improves the treatment of the aforementioned disease by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%, compared to adalimumab treatment without co-expression of an enhancer protein.

[0209] In some embodiments, co-expression of an enhancer protein (e.g., L protein) improves expression of a target protein, and the target protein is an antibody selected from the group including adalimumab, pembrolizumab, nivolumab, trastuzumab, bevacizumab, ustekinumab, ocrelizumab, secukinumab, vedolizumab, ibalizumab, nirsevimab, atortivimab, maftivimab, odesivimab, casirivimab, imdevimab, and brolucizumab (see also Table 8).

[0210] [Table 8-1]

[0211] [Table 8-2]

[0212] In some embodiments, co-expression of an enhancer protein (e.g., L protein) improves expression of a target protein, where the target protein is a blood protein or an immuno-oncology protein selected from the group including 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) (see also Table 9).

[0213] [Table 9-1]

[0214] [Table 9-2]

[0215] [Table 9-3]

[0216] [Table 9-4]

[0217] [Table 9-5]

[0218] [Table 9-6]

[0219] [Table 9-7]

[0220] In some embodiments, co-expression of an enhancer protein (e.g., L protein) and a set of polynucleotides of SEQ ID NOs: 191-216 may be used to improve expression of an antibody or target protein from any of Tables 8 or 9, and the target protein is expressed in place of adalimumab.

[0221] In some embodiments, co-expression of an enhancer protein (e.g., L protein) with a set of polynucleotides of SEQ ID NOs:243-272 (AAV vectors) may be used to improve expression of an antibody or target protein from any of Tables 8 or 9, and the target protein is expressed in place of adalimumab.

[0222] Exemplary embodiments Embodiment I Embodiment I-1. A system for recombinant expression of a target protein in a eukaryotic cell, comprising one or more vectors, the one or more vectors comprising: a) a first polynucleotide encoding a target protein; and b) a second polynucleotide encoding an enhancer protein; i) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii) the enhancer protein is 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; The system, wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0223] Embodiment I-2. The system according to embodiment I-1, wherein the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT).

[0224] Embodiment I-3. The system according to embodiment I-2, wherein the NCT inhibitor is a viral protein.

[0225] Embodiment I-4. The system of any one of embodiments I-1 to I-3, wherein the NCT inhibitor 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.

[0226] Embodiment I-5. The system according to embodiment I-4, wherein the NCT inhibitor is a picornavirus leader (L) protein or a functional variant thereof.

[0227] Embodiment I-6. The system according to embodiment I-4, wherein the NCT inhibitor is a picornavirus 2A protease or a functional variant thereof.

[0228] Embodiment I-7. The system according to embodiment I-4, wherein the NCT inhibitor is a rhinovirus 3C protease or a functional variant thereof.

[0229] Embodiment I-8. The system according to embodiment I-4, wherein the NCT inhibitor is a coronavirus ORF6 protein or a functional variant thereof.

[0230] Embodiment I-9. The system according to embodiment I-4, wherein the NCT inhibitor is an Ebola virus VP24 protein or a functional variant thereof.

[0231] Embodiment I-10. The system according to embodiment I-4, wherein the NCT inhibitor is a Venezuelan Equine Encephalitis Virus (VEEV) capsid protein or a functional variant thereof.

[0232] Embodiment I-11. The system according to embodiment I-4, wherein the NCT inhibitor is a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof.

[0233] Embodiment I-12. The system according to embodiment I-4, wherein the NCT inhibitor is a rhabdovirus matrix (M) protein or a functional variant thereof.

[0234] Embodiment I-13. The system according to embodiment I-5, wherein the L protein is the L protein of Theiler's virus or a functional variant thereof.

[0235] Embodiment I-14. The system according to embodiment I-5, wherein the L protein shares at least 90% identity with SEQ ID NO:1.

[0236] Embodiment I-15. The system according to embodiment I-5, wherein the L protein is the L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof.

[0237] Embodiment I-16. The system according to embodiment I-5, wherein the L protein shares at least 90% identity with SEQ ID NO:2.

[0238] Embodiment I-17. The system according to embodiment I-5, wherein the L protein is selected from the group consisting of the L protein of poliovirus, the L protein of HRV16, the L protein of Mengovirus and the L protein of Saffold virus 2 or a functional variant thereof.

[0239] Embodiment I-18. A system according to any one of embodiments I-1 to I-17, wherein the system comprises a single vector comprising an expression cassette, the expression cassette comprising a first polynucleotide and a second polynucleotide.

[0240] Embodiment I-19. The system of embodiment I-18, wherein the expression cassette comprises: a first promoter operably linked to the first polynucleotide; and a second promoter operably linked to the second polynucleotide.

[0241] Embodiment I-20. The system of embodiment I-18, wherein the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0242] Embodiment I-21. The system of embodiment I-20, wherein the expression cassette comprises a coding polynucleotide comprising a first polynucleotide and a second polynucleotide linked by a polynucleotide encoding a ribosome skipping site, the coding polynucleotide being operably linked to a shared promoter.

[0243] Embodiment I-22. The system described in embodiment I-20, wherein the expression cassette comprises a coding polynucleotide, the coding polynucleotide encoding an enhancer protein and a target protein linked by a ribosomal skipping site, the coding polynucleotide being operably linked to a shared promoter.

[0244] Embodiment I-23. A system described in any one of embodiments I-18 to I-22, wherein the expression cassette is configured for transcription of a single messenger RNA encoding both the target protein and an enhancer protein linked by a ribosomal skipping site, and translation of the messenger RNA results in expression of the target protein and the L protein as separate polypeptides.

[0245] Embodiment I-24. A system according to any one of embodiments I-1 to I-23, comprising one vector.

[0246] Embodiment I-25. A system according to any one of embodiments I-1 to I-17, a) a first vector comprising a first polynucleotide operably linked to a first promoter; and b) A system comprising a second vector comprising a second polynucleotide operably linked to a second promoter.

[0247] Embodiment I-26. A system according to any one of embodiments I-1 to I-17 or embodiment I-25, comprising two vectors.

[0248] Embodiment I-27. A system according to any one of embodiments I-1 to I-26, wherein either the first polynucleotide or the second polynucleotide, or both, are operably linked to an internal ribosome entry site (IRES).

[0249] Embodiment I-28. A system according to any one of embodiments I-1 to I-27, wherein at least one of the one or more vectors comprises a T7 promoter configured for transcription of either the first polynucleotide or the second polynucleotide or both by T7 RNA polymerase.

[0250] Embodiment I-29. A system according to any one of embodiments I-1 to I-28, wherein at least one of the one or more vectors comprises a polynucleotide sequence encoding T7 RNA polymerase.

[0251] Embodiment I-30. A vector for the recombinant expression of a target protein in a eukaryotic cell, comprising: a) a first polynucleotide encoding a target protein; and b) a second polynucleotide encoding an enhancer protein; i) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii) the enhancer 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; A vector, wherein the first polynucleotide and the second polynucleotide are operably linked to at least one promoter.

[0252] Embodiment I-31. The vector of embodiment I-30, wherein the expression cassette comprises: a first promoter operably linked to the first polynucleotide; and a second promoter operably linked to the second polynucleotide.

[0253] Embodiment I-32. The vector of embodiment I-30, wherein the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0254] Embodiment I-32.1 The vector according to embodiment I-30, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:100.

[0255] Embodiment I-32.2 A vector according to embodiment I-30, comprising a polynucleotide encoding SEQ ID NO: 132 and / or a polynucleotide encoding SEQ ID NO: 133.

[0256] Embodiment I-32.3 A vector according to embodiment I-30, comprising a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 134 and / or a polynucleotide encoding SEQ ID NO: 135.

[0257] Embodiment I-33. A eukaryotic cell for the expression of a target protein, comprising an exogenous polynucleotide encoding an enhancer protein. a) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or b) the enhancer 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; A eukaryotic cell, wherein the exogenous polynucleotide is operably linked to a promoter.

[0258] Embodiment I-34. The cell of embodiment I-33, wherein the polynucleotide is operably linked to an internal ribosome entry site (IRES).

[0259] Embodiment I-35. The cell of embodiment I-33 or embodiment I-34, wherein the promoter is an inducible promoter.

[0260] Embodiment I-36. A method for recombinant expression of a target protein, comprising introducing a polynucleotide encoding the target protein, operably linked to a promoter, into a cell according to any one of embodiments I-33 to I-35.

[0261] Embodiment I-37. A method for recombinant expression of a target protein, comprising introducing a system according to any one of embodiments I-1 to I-29 or a vector according to any one of embodiments I-30 to I-32 into a eukaryotic cell.

[0262] Embodiment I-38. The method of embodiment I-36 or embodiment I-37, wherein the target protein is a membrane protein.

[0263] Embodiment I-39. The method of any one of embodiments I-38, wherein localization of the membrane protein to the cell membrane is increased compared to the localization observed when the membrane protein is expressed without the enhancer protein.

[0264] Embodiment I-40. A cell produced by introducing a system according to any one of embodiments I-1 to I-29 or a vector according to any one of embodiments I-30 to I-32 into a eukaryotic cell.

[0265] Embodiment I-41. A target protein expressed by introducing a system according to any one of embodiments I-1 to I-29 or a vector according to any one of embodiments I-30 to I-32 into a eukaryotic cell.

[0266] Embodiment I-42. A method for expressing a target protein in a eukaryotic cell, comprising introducing into the eukaryotic cell a polynucleotide encoding the target protein (the polynucleotide operably linked to a promoter), The method utilizes co-expression of an enhancer protein to enhance the expression level, solubility and / or activity of the target protein; During the ceremony, a) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or b) the enhancer 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.

[0267] Embodiment I-43. The method of embodiment I-42, wherein co-expression of an enhancer protein comprises introducing into the eukaryotic cell a polynucleotide encoding the enhancer protein operably linked to a promoter.

[0268] Embodiment I-44. The method of embodiment I-42 or embodiment I-43, wherein the one or more introducing steps comprise transfecting a eukaryotic cell with one or more DNA molecules, transducing a eukaryotic cell with a single viral vector, and / or transducing a eukaryotic cell with two viral vectors.

[0269] Embodiment I-45. A vector system according to any one of embodiments I-1 to I-29, a vector according to any one of embodiments I-30 to I-32, a cell according to any one of embodiments I-33 to I-35 or a method according to any one of embodiments I-36 to I-44, wherein the target protein is a soluble protein.

[0270] Embodiment I-46. A vector system according to any one of embodiments I-1 to I-29, a vector according to any one of embodiments I-30 to I-32, a cell according to any one of embodiments I-33 to I-35 or a method according to any one of embodiments I-36 to I-44, wherein the target protein is a secreted protein.

[0271] Embodiment I-47. A vector system according to any one of embodiments I-1 to I-29, a vector according to any one of embodiments I-30 to I-32, a cell according to any one of embodiments I-33 to I-35 or a method according to any one of embodiments I-36 to I-44, wherein the target protein is a membrane protein.

[0272] Embodiment I-48. The vector system according to any one of embodiments I-1 to I-29, the vector according to any one of embodiments I-30 to I-32, the cell according to any one of embodiments I-33 to I-35 or the method according to any one of embodiments I-36 to I-44, wherein the target protein is dopamine receptor 1 (DRD1).

[0273] Embodiment I-49. The vector system according to any one of embodiments I-1 to I-29, the vector according to any one of embodiments I-30 to I-32, the cell according to any one of embodiments I-33 to I-35 or the method according to any one of embodiments I-36 to I-44, wherein the target protein is the cystic fibrosis transmembrane conductance regulator (CFTR).

[0274] Embodiment I-50. A vector system according to any one of embodiments I-1 to I-29, a vector according to any one of embodiments I-30 to I-32, a cell according to any one of embodiments I-33 to I-35 or a method according to any one of embodiments I-36 to I-44, wherein the target protein is C1 esterase inhibitor (C1-Inh).

[0275] Embodiment I-51. A vector system according to any one of embodiments I-1 to I-29, a vector according to any one of embodiments I-30 to I-32, a cell according to any one of embodiments I-33 to I-35 or a method according to any one of embodiments I-36 to I-44, wherein the target protein is IL2-inducible T cell kinase (ITK).

[0276] Embodiment I-52. A vector system according to any one of embodiments I-1 to I-29, a vector according to any one of embodiments I-30 to I-32, a cell according to any one of embodiments I-33 to I-35 or a method according to any one of embodiments I-36 to I-44, wherein the target protein is NADase.

[0277] Embodiment I-53. A method for producing an antibody against a target protein, comprising immunizing a subject with a cell according to any one of embodiments I-33 to I-35, a cell according to embodiment I-40, or a target protein according to embodiment I-41.

[0278] Embodiment I-54. The method of embodiment I-53, further comprising isolating one or more immune cells that express an immunoglobulin protein specific for the target protein.

[0279] The method of embodiment I-53 or embodiment I-54, comprising generating one or more hybridomas from one or more immune cells.

[0280] The method of any one of embodiments I-53 to I-55, comprising cloning one or more immunoglobulin genes from one or more immune cells.

[0281] Embodiment I-57. A method for antibody discovery by cell sorting, comprising: a) a cell or a target protein according to any one of embodiments I-33 to I-35, a cell according to embodiment I-40, or a target protein according to embodiment I-41, which is labeled; b) a population of recombinant cells expressing a library of polypeptides, each of which comprises an antibody or antigen-binding fragment thereof; and isolating one or more recombinant cells from the solution by selecting for labeled cells or recombinant cells that bind to the labeled target protein.

[0282] Embodiment I-58. A method for panning a phage display library, comprising: a) mixing a phage display library with a cell according to any one of embodiments I-33 to I-35, a cell according to embodiment I-40, or a target protein according to embodiment I-41; and b) purifying and / or enriching members of the phage display library that bind to the cell or target protein.

[0283] Embodiment I-59. A method for expressing a target protein in a subject in need thereof, comprising administering to the subject a vector system comprising one or more vectors, wherein the one or more vectors are a) a first polynucleotide encoding a target protein; and b) a second polynucleotide encoding an enhancer protein; i) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii) the enhancer protein is 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; The method, wherein the first polynucleotide and the second polynucleotide are operably linked to one or more promoters.

[0284] Embodiment I-60. The method of embodiment I-59, wherein the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT).

[0285] Embodiment I-61. The method of embodiment I-60, wherein the NCT inhibitor is a viral protein.

[0286] Embodiment I-62. The method of any one of embodiments I-59 to I-61, wherein the NCT inhibitor 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.

[0287] Embodiment I-63. The method of embodiment I-62, wherein the NCT inhibitor is a picornavirus leader (L) protein or a functional variant thereof.

[0288] Embodiment I-64. The method of embodiment I-62, wherein the NCT inhibitor is a picornavirus 2A protease or a functional variant thereof.

[0289] Embodiment I-65. The method of embodiment I-62, wherein the NCT inhibitor is a rhinovirus 3C protease or a functional variant thereof.

[0290] Embodiment I-66. The method of embodiment I-62, wherein the NCT inhibitor is a coronavirus ORF6 protein or a functional variant thereof.

[0291] Embodiment I-67. The method of embodiment I-62, wherein the NCT inhibitor is an Ebola virus VP24 protein or a functional variant thereof.

[0292] Embodiment I-68. The method of embodiment I-62, wherein the NCT inhibitor is a Venezuelan Equine Encephalitis Virus (VEEV) capsid protein or a functional variant thereof.

[0293] Embodiment I-69. The method of embodiment I-62, wherein the NCT inhibitor is a herpes simplex virus (HSV) ICP27 protein or a functional variant thereof.

[0294] Embodiment I-70. The method of embodiment I-62, wherein the NCT inhibitor is a rhabdovirus matrix (M) protein or a functional variant thereof.

[0295] Embodiment I-71. The method of embodiment I-63, wherein the L protein is the L protein of Theiler's virus or a functional variant thereof.

[0296] The method of embodiment I-63, wherein the L protein shares at least 90% identity with SEQ ID NO:1.

[0297] Embodiment I-73. The method of embodiment I-63, wherein the L protein is an L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof.

[0298] The method of embodiment I-63, wherein the L protein shares at least 90% identity with SEQ ID NO:2.

[0299] Embodiment I-75. The method of embodiment I-63, wherein the L protein is selected from the group consisting of the L protein of poliovirus, the L protein of HRV16, the L protein of Mengovirus, and the L protein of Saffold virus 2 or a functional variant thereof.

[0300] Embodiment I-76. The method of any one of embodiments I-59 to I-75, wherein the system comprises a single vector comprising an expression cassette, the expression cassette comprising a first polynucleotide and a second polynucleotide.

[0301] Embodiment I-77. The method of embodiment I-76, wherein the expression cassette comprises a first promoter operably linked to the first polynucleotide; and a second promoter operably linked to the second polynucleotide.

[0302] Embodiment I-78. The method of embodiment I-76, wherein the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0303] Embodiment I-79. The method of embodiment I-78, wherein the expression cassette comprises a coding polynucleotide comprising a first polynucleotide and a second polynucleotide linked by a polynucleotide encoding a ribosomal skipping site, wherein the coding polynucleotide is operably linked to a shared promoter.

[0304] Embodiment I-80. The method of embodiment I-78, wherein the expression cassette comprises a coding polynucleotide, the coding polynucleotide encoding an enhancer protein and a target protein linked by a ribosomal skipping site, the coding polynucleotide being operably linked to a shared promoter.

[0305] Embodiment I-81. A method according to any one of embodiments I-76 to I-80, wherein the expression cassette is configured for transcription of a single messenger RNA encoding both the target protein and an enhancer protein linked by a ribosomal skipping site, and translation of the messenger RNA results in expression of the target protein and the L protein as separate polypeptides.

[0306] Embodiment I-82. The method of any one of embodiments I-59 to I-75, wherein the system comprises one vector.

[0307] Embodiment I-83. The method according to any one of embodiments I-59 to I-75, wherein the system comprises: a) a first vector comprising a first polynucleotide operably linked to a first promoter; and b) a second vector comprising a second polynucleotide operably linked to a second promoter.

[0308] Embodiment I-84. The method of any one of embodiments I-59 to I-75, wherein the system comprises two vectors.

[0309] Embodiment I-85. The method of any one of embodiments I-59 to I-84, wherein either the first polynucleotide or the second polynucleotide, or both, are operably linked to an internal ribosome entry site (IRES).

[0310] Embodiment I-86. The method of any one of embodiments I-59 to I-85, wherein at least one of the one or more vectors comprises a T7 promoter configured for transcription of either the first polynucleotide or the second polynucleotide, or both, by T7 RNA polymerase.

[0311] Embodiment I-87. The method of any one of embodiments I-59 to I-86, wherein at least one of the one or more vectors comprises a polynucleotide sequence encoding T7 RNA polymerase.

[0312] Embodiment I-88. A method for expressing a target protein in a subject in need thereof, comprising administering to the subject a vector, wherein the vector comprises: a) a first polynucleotide encoding a target protein; and b) a second polynucleotide encoding an enhancer protein; i) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT), and / or ii) the enhancer 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; The method, wherein the first polynucleotide and the second polynucleotide are operably linked to at least one promoter.

[0313] Embodiment I-89. The method of embodiment I-88, wherein the expression cassette comprises: a first promoter operably linked to the first polynucleotide; and a second promoter operably linked to the second polynucleotide.

[0314] Embodiment I-90. The method of embodiment I-88, wherein the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0315] Embodiment I-91. The method of any one of embodiments I-59 to I-90, wherein the target protein is a therapeutic protein.

[0316] Embodiment I-92. The method of any one of embodiments I-59 to I-91, wherein the target protein is an immunogenic protein.

[0317] Embodiment I-93. The method of any one of embodiments I-59 to I-92, wherein the target protein is an antibody, a nanobody, a receptor, a bispecific T cell engager (BiTE), a growth factor, a hormone, an enzyme, an immunomodulatory protein, an antigen, a structural protein, a blood protein, an antimicrobial polypeptide, an antiviral polypeptide, a tumor suppressor, a transcription factor, or a translation factor.

[0318] Embodiment I-94. The method of embodiment I-93, wherein the target protein is an antibody.

[0319] Embodiment I-95. The method of embodiment I-93, wherein the target protein is a blood protein.

[0320] Embodiment I-96. The method of any one of embodiments I-59 to 95, for inducing an immune response in a subject.

[0321] Embodiment I-97. A method of treating a disease in a subject, wherein the disease is caused by, correlated with, or associated with a target protein, according to any one of embodiments I-59 to I-96.

[0322] Embodiment I-98. A method of treating a disease in a subject, the method of embodiment I-97, wherein the expression level of the target protein in the subject is lower than the expression level of the target protein in a control subject without the disease.

[0323] Embodiment I-99. The target protein is selected from the group consisting of abciximab, alemtuzumab, alirocumab, amivantamab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodalumab, brolucizumab, burosumab, canakinumab, caplacizumab, capromab, catumaxomab, semipilimab, certolizumab pegol, cetuximab, crizanlizumab, daclizumab, daratumumab, denosumab, dinutuximab, dupilumab, durval. Mab, eculizumab, elotuzumab, emapalumab, emicizumab, enfortumab vedotin, eptinizumab, erenumab, ertumaxomab, etaracizumab, evolocumab, fremanezumab, galcanezumab, gemtuzumab ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, idarucizumab, imciromab, infliximab, inotuzumab ozogamicin, ipilimumab, isatuximab, itolizumab, ixekizumab, lanadelumab, loxivetumab, mepolizumab, mogamulizumab, moxekizumab Tumor vedotin, natalizumab, necitumumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab vedotin, racotumomab, ramucirumab, ranibizumab, raxibacumab, ravulizumab, reslizumab, risankizumab, rituximab, Rmab, romosozumab, lovelizumab, ruplizumab, sacituzumab govitecan, sarilumab, secukinumab, siltoximab, ta Luketamab, teclistamab, teprotumumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, ustekinumab, and vedolizumab, blinatumomab, emicizumab, solitomab, adnectins, anticalins, avimers, finomers, kunitz domains, knottins, affibodies, DARPins, thrombolytics, transferrin, t-PA, hirudin, C1 esterase inhibitors, antithrombins, plasma kallikrein inhibitors, 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 (CRP), lipoproteins (chylomicrons, VLDL, LDL, HDL), transferrin, prothrombin, mannose-binding lectin (MBL), albumin, globulins, fibrinogen, regulatory factors, and coagulation factors, such as Factor I, Factor II, and Factor III. factor IV, factor V, factor VI, factor VII, factor IX, factor X, factor XI, factor XII, factor XIII, von Willband factor, prekallikrein, Fitzgerald factor, fibronectin, antithrombin III, heparin coenzyme II, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, α2-antiplasmin, tissue plasminogen activator, urokinase, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2, cancer procoagulant, EPO, IGF-1, G-CSF, GM-GCF, BMP-2, BMP-7, KGF, PDGF-BB, TMP, adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor family, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), colony-stimulating factors, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), Ephrins - ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin (EPO), fibroblast growth factors (FGF) 1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, fetal bovine somatotrophin (FBS), GDNF ligand family,Glial cell line-derived neurotrophic factor (GDNF), neurulin, 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), hepatocyte growth factor-like protein Also known as hepatocyte growth factor receptor alpha (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), 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), Wnt signaling pathway, 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, and glucagon, α-glycosidase, glucocerebrosidase, iduronate-2-sulfate, α-galactosidase, urate oxidase, N-acetylgalactosidase, carboxypeptidase, hyaluronan, dsDNAse, asparaginase, uricase, adenosine deaminase and other enterokines, cyclases, caspases, cathepsins, oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases, agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, C3 inhibitors, Hurler and Hunter correction factors,The method of any one of embodiments I-59 to I-98, wherein the IL-11 is selected from the group consisting of an ion channel, a gap junction, an ionizable receptor, a transporter, a cell surface receptor, a signaling protein, dopamine receptor 1 (DRD1), a cystic fibrosis transmembrane conductance regulator (CFTR), C1 esterase inhibitor (C1-Inh), an IL2-inducible T cell kinase (ITK), and an NADase.

[0324] Embodiment I-100. A system according to any one of embodiments I-1 to 29, a vector according to any one of embodiments I-30 to 32, a eukaryotic cell according to any one of embodiments I-33 to 35, a method according to any one of embodiments I-36 to 39, a cell according to embodiment I-40, a target protein according to embodiment I-41, a method according to any one of embodiments I-42 to 44, a vector system according to any one of embodiments I-45 to 52, a method according to any one of embodiments I-53 to 93 and 96 to 98, wherein the target protein is an antibody.

[0325] Embodiment I-101. A system according to any one of embodiments I-1 to 29, a vector according to any one of embodiments I-30 to 32, a eukaryotic cell according to any one of embodiments I-33 to 35, a method according to any one of embodiments I-36 to 39, a cell according to embodiment I-40, a target protein according to embodiment I-41, a method according to any one of embodiments I-42 to 44, a vector system according to any one of embodiments I-45 to 52, a method according to any one of embodiments I-53 to 93 and 96 to 98, wherein the target protein is adalimumab.

[0326] Embodiment I-102. The system, vector, vector system, eukaryotic cell, method, cell or target protein according to embodiment I-101, wherein the heavy chain of adalimumab has the amino acid sequence of SEQ ID NO: 132.

[0327] Embodiment I-103. The system, vector, vector system, eukaryotic cell, method, cell or target protein according to embodiment I-101 or 102, wherein the light chain of adalimumab has the amino acid sequence of SEQ ID NO: 133.

[0328] Embodiment I-104. The system, vector, vector system, eukaryotic cell, method, cell or target protein according to any one of embodiments I-101 to 103, wherein the heavy chain of adalimumab is encoded by the nucleic acid sequence of SEQ ID NO: 134.

[0329] Embodiment I-105. A system, vector, vector system, eukaryotic cell, method, cell or target protein according to any one of embodiments I-101 to 104, wherein the light chain of adalimumab is encoded by the nucleic acid sequence of SEQ ID NO: 135.

[0330] Embodiment I-106. The method of any one of embodiments I-88 to 99, wherein the enhancer protein increases the activity of the target protein.

[0331] Embodiment I-107. The method of any one of embodiments I-88 to 99 and 106, wherein the enhancer protein reduces the expression level of the target protein.

[0332] Embodiment I-108. The method of any one of embodiments I-88 to 99 and 106 to 107, wherein the enhancer protein increases the uniformity of expression of the target protein in vivo.

[0333] Embodiment I-109. The method of any one of embodiments I-88 to 99 and 106 to 107, wherein the enhancer protein increases the duration of active target protein in a cell or organism.

[0334] Embodiment I-110. A lipid nanoparticle (LNP) comprising a vector according to any one of embodiments I-30 to 32 and one or more lipids.

[0335] Embodiment I-111. A polynucleotide encoding a leader protein and an adalimumab protein.

[0336] Embodiment I-112. The polynucleotide of embodiment I-111, encoding a leader protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 24, 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.

[0337] Embodiment I-113. The polynucleotide sequence according to embodiment I-111 or 112, wherein the polynucleotide encodes the adalimumab variable heavy chain sequence of SEQ ID NO: 124, 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 the adalimumab variable light chain sequence of SEQ ID NO: 129, 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.

[0338] Embodiment I-114. The polynucleotide of any one of embodiments I-111 to 113, wherein co-expression of the leader protein and the adalimumab protein reduces the expression level of the adalimumab 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%.

[0339] Embodiment I-120. A polynucleotide according to embodiment I-111, comprising a sequence of the set of SEQ ID NOs: 191 to 216 or a sequence of the set of SEQ ID NOs: 217 to 242.

[0340] Embodiment I-121. A vector comprising a polynucleotide according to embodiment I-111.

[0341] Embodiment I-122. The vector of embodiment I-121, which is an adeno-associated virus (AAV) vector.

[0342] Embodiment I-123. A system comprising a transfer plasmid according to embodiment I-121 and one or more polynucleotides encoding the adenoviral genes E4, E2A, VA and the Cap protein of AAV.

[0343] Embodiment I-124. A lipid nanoparticle (LNP) comprising a vector according to embodiment I-120.

[0344] Embodiment I-125. The LNP of embodiment I-123, wherein the LNP comprises a PEGylated lipid, cholesterol, and one or more ionizable lipids.

[0345] Embodiment I-126. The LNP of embodiment I-123, wherein the LNP comprises about 0.5% to about 2% PEGylated lipid, about 35% to about 45% cholesterol, and about 5% to about 65% of one or more ionizable lipids.

[0346] Embodiment I-127. The LNP of embodiment I-123, wherein the LNP comprises DMG-PEG(2000), cholesterol, DOPC and DLin-KC2-DMA in a ratio of about 1% DMG-PEG(2000) to about 40% cholesterol to about 10% DOPC and about 50% DLin-KC2-DMA.

[0347] Embodiment I-128. A method for treating a subject in need of treatment, comprising delivering a system described in embodiment I-122 and / or an LNP described in any one of embodiments I-123 to 126.

[0348] Embodiment I-129. The method of embodiment I-127, wherein the system is delivered intramuscularly or subcutaneously.

[0349] Embodiment I-130. A polynucleotide encoding a leader protein and a glucosylceramidase (GBA) protein.

[0350] Embodiment I-131. The polynucleotide of embodiment I-130, encoding a leader protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 24, 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.

[0351] Embodiment I-132. The polynucleotide of embodiment I-130 or 131, wherein the polynucleotide encodes the GBA amino acid sequence of SEQ ID NO: 406, 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.

[0352] Embodiment I-139. A vector comprising a polynucleotide according to embodiment I-130.

[0353] Embodiment I-140. The vector of embodiment I-139, which is an adeno-associated virus (AAV) vector.

[0354] Embodiment I-141. A system comprising a transfer plasmid according to embodiment I-140 and one or more polynucleotides encoding the adenoviral genes E4, E2A, VA and the Cap protein of AAV.

[0355] Embodiment I-142. A lipid nanoparticle (LNP) comprising a vector according to embodiment I-139.

[0356] Embodiment I-143. The LNP of embodiment I-142, wherein the LNP comprises a PEGylated lipid, cholesterol, and one or more ionizable lipids.

[0357] Embodiment I-144. The LNP of embodiment I-142, wherein the LNP comprises about 0.5% to about 2% PEGylated lipid, about 35% to about 45% cholesterol, and about 5% to about 65% of one or more ionizable lipids.

[0358] Embodiment I-145. The LNP of embodiment I-142, wherein the LNP comprises DMG-PEG(2000), cholesterol, DOPC and DLin-KC2-DMA in a ratio of about 1% DMG-PEG(2000) to about 40% cholesterol to about 10% DOPC and about 50% DLin-KC2-DMA.

[0359] Embodiment I-146. A method for treating a subject in need of treatment, comprising delivering a system described in embodiment I-141 and / or an LNP described in any one of embodiments I-142 to 145.

[0360] Embodiment I-147. The method of embodiment I-146, wherein the system is delivered intramuscularly or subcutaneously.

[0361] Embodiment I-148. A polynucleotide encoding a leader protein and a target protein.

[0362] Embodiment I-149. The polynucleotide of embodiment I-130, encoding a leader protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 24, 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.

[0363] Embodiment I-150. A polynucleotide according to embodiment I-130 or 131, encoding a target protein amino acid sequence of any of SEQ ID NOs: 124, 129, 374-405, and / or any of SEQ ID NOs: 406-422, 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.

[0364] Embodiment I-157. A vector comprising a polynucleotide according to embodiment I-130.

[0365] Embodiment I-158. The vector of embodiment I-139, which is an adeno-associated virus (AAV) vector.

[0366] Embodiment I-159. A system comprising a transfer plasmid according to embodiment I-140 and one or more polynucleotides encoding the adenoviral genes E4, E2A, VA and the Cap protein of AAV.

[0367] Embodiment I-160. A lipid nanoparticle (LNP) comprising a vector according to embodiment I-139.

[0368] Embodiment I-161. The LNP of embodiment I-142, wherein the LNP comprises a PEGylated lipid, cholesterol, and one or more ionizable lipids.

[0369] Embodiment I-162. The LNP of embodiment I-142, wherein the LNP comprises about 0.5% to about 2% PEGylated lipid, about 35% to about 45% cholesterol, and about 5% to about 65% of one or more ionizable lipids.

[0370] Embodiment I-163. The LNP of embodiment I-142, wherein the LNP comprises DMG-PEG(2000), cholesterol, DOPC and DLin-KC2-DMA in a ratio of about 1% DMG-PEG(2000) to about 40% cholesterol to about 10% DOPC and about 50% DLin-KC2-DMA.

[0371] Embodiment I-164. A method for treating a subject in need of treatment, comprising delivering a system described in embodiment I-141 and / or an LNP described in any one of embodiments I-142 to 145.

[0372] Embodiment I-165. The method of embodiment I-146, wherein the system is delivered intramuscularly or subcutaneously.

[0373] Embodiment II Embodiment II-52. A method for expressing an adalimumab protein in a subject in need thereof, comprising administering to the subject a vector system comprising one or more vectors, wherein the one or more vectors are a) a first polynucleotide encoding an adalimumab protein; and b) a second polynucleotide encoding a picornavirus leader (L) protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 24, 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; A method, wherein a first polynucleotide encoding an adalimumab protein and a second polynucleotide encoding an L protein are operably linked to one or more promoters, and the adalimumab protein and the L protein are co-expressed.

[0374] Embodiment II-53. The method of any 52, wherein the first polynucleotide encodes an adalimumab variable heavy chain sequence of SEQ ID NO: 124, 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 an adalimumab variable light chain sequence of SEQ ID NO: 129, 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.

[0375] Embodiment II-54. The method of any one of embodiments II-52 to II-53, wherein co-expression of the leader protein and the adalimumab protein reduces the expression level of the adalimumab protein in the cell or subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0376] Embodiment II-55. The method of any one of embodiments II-52 to II-54, wherein co-expression of the leader protein and the adalimumab protein increases the activity of the adalimumab protein in the subject's cell or 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.

[0377] Embodiment II-56. The method of any one of embodiments II-52 to II-55, wherein co-expression of the leader protein and the adalimumab protein increases the period during which the adalimumab protein is found in the subject's cells or in the subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold.

[0378] Embodiment II-57. The method of any one of embodiments II-52 to II-56, wherein co-expression of the leader protein and the adalimumab protein increases the coefficient of variation (CV%) of the target protein in a tissue or subject 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.

[0379] Embodiment II-58. The method of any one of embodiments II-52 to II-57, wherein co-expression of the leader protein and the adalimumab protein reduces degradation of the target protein by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold.

[0380] Embodiment II-59. Co-expression of leader protein and adalimumab protein enhances EC of adalimumab 50 The method according to any one of embodiments II-52 to II-58, wherein the amount of the IgG antibody 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.

[0381] Embodiment II-60. The method according to any one of embodiments II-52 to II-59, wherein the vector system comprises a polynucleotide sequence of the set of SEQ ID NOs: 191 to 216 or a sequence of the set of SEQ ID NOs: 217 to 242.

[0382] Embodiment II-61. The method according to any one of embodiments II-52 to II-60, wherein the vector system comprises one or more polynucleotides encoding the adenoviral genes E4, E2A, VA, and Cap protein of AAV.

[0383] Embodiment II-62. The method according to any one of embodiments II-52 to II-61, wherein the vector system is administered via a lipid nanoparticle (LNP).

[0384] The method of embodiment II-62, wherein the LNP comprises a PEGylated lipid, cholesterol, and one or more ionizable lipids.

[0385] The method of embodiment II-62, wherein the LNP comprises about 0.5% to about 2% PEGylated lipid, about 35% to about 45% cholesterol, and about 5% to about 65% of one or more ionizable lipids.

[0386] The method of embodiment II-62, wherein the LNPs comprise DMG-PEG(2000), cholesterol, DOPC and DLin-KC2-DMA in a ratio of about 1% DMG-PEG(2000) to about 40% cholesterol to about 10% DOPC and about 50% DLin-KC2-DMA.

[0387] The method according to any one of embodiments II-52 to II-65, wherein the system is delivered intramuscularly or subcutaneously.

[0388] Embodiment II-67. A method for expressing a glucosylceramidase (GBA) protein in a subject in need thereof, comprising administering to the subject a vector system comprising one or more vectors, wherein the one or more vectors are a) a first polynucleotide encoding a glucosylceramidase (GBA) protein; and b) a second polynucleotide encoding a picornavirus leader (L) protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 24, 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; A method in which a first polynucleotide encoding a glucosylceramidase (GBA) protein and a second polynucleotide encoding an L protein are operably linked to one or more promoters, and the GBA protein and the L protein are co-expressed.

[0389] Embodiment II-68. The method of embodiment II-67, wherein the first polynucleotide encodes the GBA amino acid sequence of SEQ ID NO: 406, 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.

[0390] Embodiment II-69. The method of any one of embodiments II-67 to II-68, wherein co-expression of the leader protein and the GBA protein reduces the expression level of the GBA protein in the subject's cells or subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0391] Embodiment II-70. The method of any one of embodiments II-67 to II-69, wherein co-expression of the leader protein and the GBA protein increases the activity of GBA in a subject cell or 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.

[0392] Embodiment II-71. Co-expression of a leader protein and a GBA protein increases the time period that GBA is found in a subject's cells or a subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, ) The method according to any one of embodiments II-67 to II-70, wherein the increase is about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold.

[0393] Embodiment II-72. The method according to any one of embodiments II-67 to II-71, wherein co-expression of an enhancer protein increases the coefficient of variation (CV%) of GBA in a subject tissue or 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.

[0394] Embodiment II-73. The method of any one of embodiments II-67 to II-72, wherein co-expression of the leader protein and the GBA protein reduces the degradation of GBA 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.

[0395] Embodiment II-74. Co-expression of a leader protein and a GBA protein results in a concentration of GBA effective to produce 50% of the maximum response (EC 50 The method of any one of embodiments II-67 to II-73, wherein the amount of the β-amino acid in the α-amino acid is reduced.

[0396] Embodiment II-75. The method according to any one of embodiments II-67 to II-74, wherein the vector system comprises one or more polynucleotides encoding the adenoviral genes E4, E2A, VA, and Cap protein of AAV.

[0397] Embodiment II-76. The method according to any one of embodiments II-67 to II-75, wherein the vector system is administered via a lipid nanoparticle (LNP).

[0398] Embodiment II-77. The method of embodiment II-76, wherein the LNP comprises a PEGylated lipid, cholesterol, and one or more ionizable lipids.

[0399] The method of embodiment II-76, wherein the LNP comprises about 0.5% to about 2% PEGylated lipid, about 35% to about 45% cholesterol, and about 5% to about 65% of one or more ionizable lipids.

[0400] Embodiment II-79. The method of embodiment II-76, wherein the LNPs comprise DMG-PEG(2000), cholesterol, DOPC and DLin-KC2-DMA in a ratio of about 1% DMG-PEG(2000) to about 40% cholesterol to about 10% DOPC and about 50% DLin-KC2-DMA.

[0401] Embodiment II-80. The method of any one of embodiments II-67 to II-79, wherein the system is delivered intramuscularly or subcutaneously.

[0402] Embodiment II-81. A method for expressing a target protein in a subject in need thereof, comprising administering to the subject a vector system comprising one or more vectors, wherein the one or more vectors are a) a first polynucleotide encoding a target protein; and b) a second polynucleotide encoding a picornavirus leader (L) protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 24, 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; A method, wherein a first polynucleotide encoding a target protein and a second polynucleotide encoding an L protein are operably linked to one or more promoters, and the target protein and the L protein are co-expressed.

[0403] Embodiment II-82. The method of embodiment II-81, wherein the first polynucleotide encodes a variable heavy chain sequence of Table 8, 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 a variable light chain sequence of Table 8, 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.

[0404] Embodiment II-83. The method of embodiment II-81, wherein the first polynucleotide encodes a protein sequence of Table 9, 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.

[0405] Embodiment II-84. The method of any one of embodiments II-81 to II-83, wherein co-expression of the leader protein and the 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%.

[0406] Embodiment II-85. The method of any one of embodiments II-81 to II-84, wherein co-expression of the leader protein and the target protein increases the activity of the target protein in a cell or subject of 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.

[0407] Embodiment II-86. The method of any one of embodiments II-81 to II-85, wherein co-expression of the leader protein and the target protein increases the period during which the target protein is found in the subject's cells or in the subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold.

[0408] Embodiment II-87. The method of any one of embodiments II-81 to II-85, wherein co-expression of the leader protein and the target protein increases the coefficient of variation (CV%) of the target protein in a tissue or subject of interest 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.

[0409] Embodiment II-88. The method of any one of embodiments II-81 to II-87, wherein co-expression of the leader protein and the target protein reduces degradation of the target protein by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, or about 300-fold.

[0410] Embodiment II-89. Co-expression of a leader protein and a target protein enhances target EC 50 The method of any one of embodiments II-81 to II-88, wherein the amount of the IgG antibody 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.

[0411] Embodiment II-90. The method according to any one of embodiments II-81 to II-89, wherein the vector system comprises one or more polynucleotides encoding the adenoviral genes E4, E2A, VA, and Cap protein of AAV.

[0412] Embodiment II-91. The method of any one of embodiments II-81 to II-90, wherein the vector system is administered via a lipid nanoparticle (LNP).

[0413] Embodiment II-92. The method of any one of embodiments II-81 to II-91, wherein the LNP comprises a PEGylated lipid, cholesterol, and one or more ionizable lipids.

[0414] The method of embodiment II-92, wherein the LNP comprises about 0.5% to about 2% PEGylated lipid, about 35% to about 45% cholesterol, and about 5% to about 65% of one or more ionizable lipids.

[0415] Embodiment II-94. The method of embodiment II-92, wherein the LNPs comprise DMG-PEG(2000), cholesterol, DOPC and DLin-KC2-DMA in a ratio of about 1% DMG-PEG(2000) to about 40% cholesterol to about 10% DOPC and about 50% DLin-KC2-DMA.

[0416] The method of any one of embodiments II-81 to II-94, wherein the system is delivered intramuscularly or subcutaneously.

[0417] Embodiment II-96. A vector system for use in the method of any one of the preceding embodiments. EXAMPLES

[0418] Materials and methods for in vitro studies Construction of DNA molecules The entire assembly was made into a plasmid backbone capable of propagation in E. coli that contains 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 that is isolated from the rest of the vector by a second terminator (the transcription terminator from phage lambda). The genes containing the backbone elements were synthesized by phosphoramidite chemistry.

[0419] The structural genes used for the construction of the plasmids were synthesized by phosphoramidite chemistry, chemically amplified and cloned into the above vectors using isothermal assembly reactions such as NEB HI-FI or Gibson Assembly using the primers listed in Table 2. Selected amino acid sequences included in the exemplary constructs used in these examples are shown in Table 3.

[0420] [Table 2-1]

[0421] [Table 2-2]

[0422] [Table 2-3]

[0423] [Table 2-4]

[0424] [Table 2-5]

[0425] [Table 3-1]

[0426] [Table 3-2]

[0427] [Table 3-3]

[0428] Cell lines - culture and transfection HEK293 cells were used to illustrate the application of this system, method, and composition in human eukaryotic cells. HEK293 adherent cells (CLS) were cultured in Dulbecco's Modified Eagle Medium High Glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000U Pen Strep (Gibco). HEK293 cells were grown to 80% confluence at 37°C and 5% CO2 and then transiently transfected using 293fectin (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were harvested 48 hours later by detaching and scraping the cells using 0.5% trypsin solution for 5 minutes at 37°C. Cells were pelleted (5,000 x g, 15 minutes, 4°C) and the supernatant was discarded. Cell pellets were stored at -80°C until further use.

[0429] CHO-K1 cells are used to illustrate the application of this system, method, and composition in eukaryotic cells. CHO-K1 adherent cells (CLS) are cultured in F-12K medium (ATCC) supplemented with 10% fetal bovine serum (Gibco). CHO-K1 cells are grown to 80% confluence at 37°C and 5% CO2 before being transiently transfected using Lipofectamine LTX (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells are harvested after 48 hours by detaching and scraping the cells using 0.5% trypsin solution for 5 minutes at 37°C. The cells are pelleted (5,000 x g, 15 minutes, 4°C) and the supernatant is discarded. The cell pellet is stored at -80°C until further use.

[0430] SF9 cells are used to illustrate the application of this system, method, and composition in eukaryotic insect cells. SF9 suspension cells (CLS) are cultured in Grace's insect medium (ThermoFisher) supplemented with 10% fetal bovine serum (Gibco). SF9 cells are grown at 26°C and 130 rpm, and then transiently transfected using Cellfectin II (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells are harvested after 48 hours (5,000 x g, 15 min, 4°C) and the supernatant is discarded. Cell pellets are stored at -80°C until further use.

[0431] Example 1: Expression of the L enhancer protein with GFP reduced overexpression of the GFP protein. CMV promoter system To demonstrate the impact of introducing viral nuclear pore-blocking proteins during expression, HEK293 cells were transfected with either EG1, EG2 or co-transfected with EG3 and EG4 constructs (see Table 2 and Figure 2 for construct details). Expression of viral pore-blocking proteins resulted in a controlled regulation of protein expression. As a result, the resulting GFP signal was reduced. The reason for the controlled regulation of the gene of interest in tandem with the pore-blocking protein is the mode of action of the viral protein. Without being bound by theory, a possible mechanism for protein regulation is that by expressing the pore-blocking protein, the nuclear export of mRNA can be inhibited, resulting in downregulation of the translation of the target protein. After stabilization, the pore-blocking protein is degraded and mRNA transport resumes. This again results in the expression of both the target protein and an enhancer protein, e.g., the pore-blocking protein. This tightly controlled feedback ensures the stabilization and permanent expression of the target protein, preventing the normal regulation of eukaryotic cells that leads to the cessation of protein expression.

[0432] 3A-3D show the effect on GFP expression in the absence and presence of the L protein from ECMV as an exemplary enhancer protein according to the present disclosure. HEK293 cells were plated in 24-well plates at 0.05×10 6 Cells / well were seeded and incubated overnight at 37° C. and 5% CO2, then transiently transfected with either EG1 or EG2 as described above. GFP expression was monitored after 24 and 48 hours using a fluorescent microscope. Images were taken with a CCD camera (Amscope) and analyzed with ISCapture (Amscope). This example demonstrates improved regulation of target protein expression in an exemplary system comprising a target protein polynucleotide and an enhancer protein polynucleotide according to the present disclosure.

[0433] T7 polymerase system EG2 uses the natural polymerase of the eukaryotic host, but can use other viral polymerases, such as T7, to initiate transcription outside the nucleus. The viral polymerase is under the control of a standard eukaryotic promoter, and the corresponding mRNA is dependent on nuclear export. In the cytosol, the viral polymerase is translated and then initiates the transcription of the target protein polynucleotide and the enhancer protein polynucleotide. In some embodiments, as a result of the expression of the enhancer protein, the nuclear export of the viral polymerase is reduced. Stabilization of the system results in the degradation of the enhancer protein, and viral polymerase mRNA export resumes. Without wishing to be bound by theory, this feedback may prevent the normal regulation of the cell while overexpressing the recombinant protein. In some situations, the use of viral polymerases gives the advantage of higher expression levels on a cell-to-cell basis compared to systems using eukaryotic polymerases.

[0434] Figures 4A-4D show successful expression of ECMV-derived L protein and tandem GFP from the T7 promoter when co-transfected with a T7-carrying vector. HEK293 cells were transfected in 24-well plates with 0.05 x 10 6Cells / well were seeded and incubated overnight at 37°C and 5% CO2 before being transiently transfected with either EG1 or EG3 and EG4 as described above. GFP expression was monitored after 24 and 48 hours using a fluorescent microscope. Images were taken with a CCD camera (Amscope) and analyzed with ISCapture (Amscope). This example demonstrates the successful use of T7 as an exemplary viral polymerase in tandem with GFP as a target protein and the L protein of ECMV as an enhancer protein. As in the previous example, introduction of the L protein resulted in tighter regulation of expression and thus an overall reduction in overexpression.

[0435] Example 2: Co-expression of the L enhancer protein with DRD1-GFP resulted in improved expression and localization of the DRD1 membrane protein. DRD1 was used to illustrate the application of the disclosed system and method to co-expression of a membrane protein as a target protein in combination with a pore-blocking protein as an enhancer protein to obtain a high density of active membrane receptors. DRD1 is a G protein-coupled receptor and is known to be difficult to express using academic standards. To visualize the correct translocation to the outer membrane of the cell, a DRD1-GFP fusion (EG8) was used in this system. To illustrate the issues of GPCRs in academic and industrial settings, an academic standard (EG10) was used as a control.

[0436] Improved membrane protein expression and membrane localization DRD1-GFP fusions were expressed in HEK293 cells. HEK293 cells were plated in 24-well plates at 0.05 × 10 6 Cells / well were seeded and incubated overnight at 37°C and 5% CO2 before being transiently transfected with either EG10 or EG8 as described above. DRD1-GFP expression was monitored after 24 and 48 hours using a fluorescent microscope. Images were captured using a CCD camera (Amscope) and analyzed with ISCapture (Amscope).

[0437] Figures 5A-5D demonstrate that EG10 is unable to accurately translocate the expressed receptor. Without wishing to be bound by theory, it is believed that as a result of overexpression of the human DRD1 receptor in human cells with the EG10 construct, the cells begin to degrade or regulate the expressed target protein. This regulation of morphology results in the formation of denatured protein as inclusion bodies (Figure 5B, red arrows). Regulating the expression of membrane proteins by cells in this way can result in inactive and misfolded proteins, resulting in low quality expressed protein that cannot be used. In contrast, co-expression of the target membrane protein with an exemplary enhancer protein resulted in accurately translocated DRD1-GFP, as can be seen by the correct insertion into the membrane and the absence of inclusion bodies (Figures 5C-5D). This example demonstrates that co-expression of an exemplary enhancer protein (L protein of ECMV) in combination with an exemplary target membrane protein (DRD1) resulted in improved expression and localization of the membrane protein. Without wishing to be bound by theory, it is believed that this system provides for tight regulation of target protein expression, thereby circumventing normal cellular regulation that results in degradation of expressed membrane proteins, and thus, is suitable for high-yield expression and purification of GPCRs.

[0438] Separate expression of the target protein and the enhancer protein. To further demonstrate that enhancer proteins can be located on separate DNA molecules, the DRD1-GFP (EG10) construct is co-expressed with the L protein from ECMV (EG11) under the control of separate promoters on separate vectors. HEK293 cells are cultured in 24-well plates at 0.05 × 10 6Cells / well are seeded and incubated overnight at 37° C. and 5% CO2 before being transiently transfected with EG10 and EG11 as described above. DRD1-GFP expression is monitored after 24 and 48 hours using a fluorescent microscope. Images are captured using a CCD camera (Amscope) and analyzed with ISCapture (Amscope).

[0439] Functional activity of membrane proteins In addition to describing a correctly translocated GPCR, activity tests were performed with the DRD1-Strep fusion. The smaller strep tag ensures that the correct interaction with the G protein located in the cytosol is intact and functional assays can be performed. Upon dopamine binding, DRD1 releases the heterotrimeric G protein to its Gα subunit and its Gβγ complex. In the resting state, Gα binds to GDP, but upon activation it exchanges GTP for GDP. The Gα-GTP complex interacts with adenylate cyclase (AC), leading to activation of AC activity and consequently increasing cAMP levels. Changes in intracellular cAMP can be measured by standard cAMP assays. Again, an academic and industrial standard (EG5) was compared to the same target protein in co-expression with the L protein of ECMV.

[0440] DRD1-Strep fusions are expressed in HEK293 cells. HEK293 cells are seeded at 5,000 cells / well in 96-well white clear-bottom plates and incubated overnight at 37°C and 5% CO2 before being transiently transfected with either EG5 or EG6 as described above. Proteins are allowed to express for 48 hours and DRD1 activity is analyzed using the cAMP(glo) assay (Promega) according to the manufacturer's instructions. In detail, after 48 hours, cells are washed with sterile PBS (pH 7.2) and cells are incubated with 20 μl of dopamine substrate at concentrations ranging from 1 mM to 1 μM for 2 hours at 37°C. As a non-induced control, cells are incubated with 20 μl of PBS (pH 7.2). After incubation, cells are washed with PBS (pH 7.2) and 20 μl of lysis buffer are added. Lysis is performed for 15 minutes at room temperature (RT) with shaking. Subsequently, 40 μl of detection solution is added and cells are incubated for 20 minutes at room temperature with shaking. The reaction is stopped with 80 μl of Kinase-Glo reagent, which is incubated at room temperature for 15 min before analysis. Luminescence is measured using a plate reader (Synergy LX (BioTek)) and data is analyzed using standard analysis programs.

[0441] Example 3: The T7 viral promoter was successfully used to initiate transcription of the DRD1-GFP and L enhancer protein constructs outside the nucleus. In this example, DRD1-GFP was chosen as an exemplary expression vector for a difficult to express target membrane protein in combination with the T7 promoter to demonstrate that a viral polymerase such as T7 can be used to initiate transcription outside the nucleus. As in Example 1, the viral polymerase was under the control of a standard eukaryotic promoter and the corresponding mRNA was dependent on nuclear export.

[0442] Figures 6A-6B demonstrate successful expression of ECMV-derived L protein and tandem DRD1-GFP from the T7 promoter when co-transfected with the T7-carrying vector. HEK293 cells were transfected with 0.05 × 10 6Cells / well were seeded and incubated overnight at 37°C and 5% CO2, then transiently transfected with either EG10 or EG12 and EG4 as described above. DRD1-GFP expression was monitored after 24 and 48 hours using a fluorescent microscope. Images were taken with a CCD camera (Amscope) and analyzed with ISCapture (Amscope). This example demonstrates the successful use of T7 as a viral polymerase in tandem with DRD1-GFP as a target protein and the L protein of ECMV as an enhancer protein.

[0443] Example 4: Expression of DRD1-GFP and the L enhancer protein is compatible with different mammalian promoters. The disclosed system, method, and composition are compatible with a wide variety of mammalian promoters. DRD1-GFP was used as an exemplary target protein to demonstrate the compatibility of co-expression of target protein and enhancer protein from different promoters. As described in Example 2, the correct expression and translocation of DRD1-GFP can be easily detected by fluorescence microscopy. In addition to the CMV promoter (EG10), EF1-α (EG22) and SV40 (EG23) are used, followed by the same DRD1-GFP IRES L assembly.

[0444] DRD1-GFP fusions under the control of different mammalian promoters are expressed in HEK293 cells. HEK293 cells were plated in 24-well plates at 0.05 × 10 6 Cells / well are seeded and incubated overnight at 37° C. and 5% CO2 before being transiently transfected with either EG8, EG22 or EG23 as described above. DRD1-GFP expression is monitored after 24 and 48 hours using a fluorescent microscope. Images are captured using a CCD camera (Amscope) and analyzed with ISCapture (Amscope).

[0445] Example 5: DRD1-GFP expression and localization can be successfully enhanced using any of the tested L enhancer proteins. Without being bound by theory, one mechanism that can be used to regulate the expression of recombinantly inserted target protein polynucleotides is the introduction of pore-blocking proteins. To demonstrate that natural or synthetic pore-blocking proteins can be exchanged for each other in one embodiment of this system while still retaining the advantage of controlling cellular regulation, the leader protein of ECMV (EG10), Theiler's virus leader protein (EG19), poliovirus 2A protease (EG21) and vesicular stomatitis virus M protein (EG20) were cloned in tandem with DRD1-GFP as an exemplary target protein. As described in Example 2, the correct expression and translocation of DRD1-GFP can be easily detected by fluorescence microscopy.

[0446] DRD1-GFP fusions in tandem with different enhancer proteins are expressed in HEK293 cells. HEK293 cells were plated in 24-well plates at 0.05 × 10 6 Cells / well are seeded and incubated overnight at 37° C. and 5% CO2 before being transiently transfected with either EG8, EG19, EG20 or EG21 as described above. DRD1-GFP expression is monitored after 24 and 48 hours using a fluorescent microscope. Images are captured using a CCD camera (Amscope) and analyzed with ISCapture (Amscope).

[0447] Example 6: Expression of the L enhancer protein improves expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR was used as a further example to demonstrate that co-expression of a membrane protein as a target protein in combination with a pore-blocking protein as an enhancer protein resulted in high density of active ion channels. CFTR is a transmembrane transporter of the ABC transporter class that conducts chloride ions across epithelial cell membranes. CFTR is known to be expressed in a heterogeneous manner when using academic standards. Heterogeneity increases the difficulty in purifying or analyzing ABC transporters. To demonstrate improved homogeneity, CFTR was cloned into the backbone of an exemplary system (EG25) or used as a PCR product. For comparison, academic standards (EG24) were used together as controls.

[0448] CFTR constructs were expressed in HEK293 cells. HEK293 cells were plated in 6-well plates at 0.3 × 10 6 Cells / well were seeded and incubated overnight at 37°C and 5% CO2 before being transiently transfected with either EG25, a PCR product of EG25, or EG24 as described above. CFTR expression was monitored after 24 and 48 h using a microscope. Cells were harvested and lysed after 48 h using RIPA buffer (CellGene). Lysates were cleared and analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher) followed by Western blot (NC membrane, ThermoFisher) using anti-CFTR (Abcam, secondary anti-mouse-hrp).

[0449] Figure 6 shows the effect of co-expressing the L protein with CFTR. While the academic standard produced a broad band on Western blot, transcription and translation based on the EG25 construct produced a distinct band indicating highly homogenous expression of the ABC transporter. Furthermore, this example demonstrates that the expression system can be delivered to cells as a vector or PCR product.

[0450] Example 7: Production of C1 esterase inhibitor (C1-Inh) protein used as an exemplary protein requiring post-translational modification. C1-Inh is used as an exemplary target protein to illustrate the application of the disclosed system, where it is difficult to express secreted proteins that produce correct post-translational modifications. C1-Inh is a protease inhibitor that belongs to the serpin superfamily. As a secreted protein, C1-Inh is highly glycosylated and therefore proves to be a challenging target for recombinant expression. To demonstrate that this system can produce correctly glycosylated C1-Inh in high yields, the presented system (EG16) is used to express C1-Inh-his fusion. As a comparison, academic and industrial standards (EG15) are used together.

[0451] The C1-Inh-his fusion is expressed in HEK293 cells. HEK293 cells were grown in a T175 flask at 4.9×10 6 Cells are seeded and incubated overnight at 37° C. and 5% CO2 before being transiently transfected with either EG15 or EG16 as described above. C1-Inh-his expression is monitored after 24 and 48 hours using a microscope. Protein-containing supernatants are harvested after 48 hours and the supernatants are clarified by filtration (22 μm, nitrocellulose). To purify C1-Inh, His-resin (GE Healthcare HisTrap) is equilibrated with 20 mM Tris (pH 7.5), 50 mM NaCl and then added to the supernatant. The supernatant is incubated with the resin for 2 hours at 4° C. with shaking. The resin is sedimented and washed with 5 CV of 20 mM Tris (pH 7.5), 50 mM NaCl and the protein is eluted with 3 CV of 20 mM Tris (pH 7.5), 50 mM NaCl, 500 mM imidazole. The purification is analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher) and protein-containing fractions are pooled and concentrated. The protein is further polished by size-exclusion chromatography (SEC) (Superdex 200, ThermoFisher) and fractions are analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher). Protein-containing fractions are pooled and sent for analysis of glycosylation patterns and sequence analysis.

[0452] Example 8: Production of IL2-inducible T-cell kinase (ITK), used as an example of a difficult to solubilize protein. ITK was used as an exemplary target protein to illustrate the application of the disclosed system to difficult to express soluble proteins. ITK is a member of the TEC family of kinases and is believed to play a role in T cell proliferation and differentiation in T cells. ITK was also used to demonstrate the batch-to-batch enzyme activity consistency and scalability of the methods disclosed herein. ITK was expressed in 3 x 10 mL, 100 mL, and 1000 mL of growth medium. Furthermore, an ITK-L-his protein fusion construct (EG9) was used to demonstrate that enhancer proteins can be fused to recombinantly expressed target proteins without losing the ability to control regulation. ITK-his fusions were expressed in the presented system (EG17) as well as academic and industrial standards (EG18) as a comparison.

[0453] ITK-his and ITK-L-his fusions were expressed in HEK293 cells. HEK293 cells were cultured at 2×10 in 10 mL, 100 mL or 1000 mL of Expi293 medium. 6Cells were seeded at 1000 cells / ml and incubated overnight at 37° C., 120 rpm and 5% CO2 before being transiently transfected with either EG9, EG17 or EG18 as described above. Cells were harvested 48 hours later (5,000×g, 15 min, 4° C.) and cell pellets were stored at −80° C. until further use. To purify ITK, cells were resuspended in lysis buffer (40 mM Tris, 7.5 μL; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2, protease inhibitors, DNAse), lysed by sonication (2 min, 10 s ON, 10 s OFF, 40% amplitude) and crude cell extracts were clarified (100,000×g, 45 min, 4C). His-resin (GE Healthcare HisTrap) was equilibrated with wash buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2) before being added to the clarified lysate. The lysate was incubated with the resin for 2 hours at 4° C. with shaking. The resin was pelleted, washed with 5 CV of wash buffer, and the protein was eluted with 3 CV of elution buffer (wash buffer + 300 mM imidazole).

[0454] The purification was analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher). Figure 9A shows the SDS-PAGE results of His-tag purification of ITK. From left to right, the lanes show protein ladder (Seeblue2+ prestained), 500ng GFP, 2μg ITK, 5μg ITK, and 10μg ITK. The SDS-PAGE gel was then analyzed by non-reducing Western blot. Proteins were transferred to a nitrocellulose membrane, bound with a primary mouse anti-His antibody and a secondary anti-mouse HRP antibody, and then visualized using NBT / BCIP solution. Figure 9B shows the results of the Western blot analysis, with the ITK protein monomer and dimer indicated by arrows.

[0455] After SDS-PAGE analysis, protein-containing fractions are pooled and concentrated. Proteins are further purified by size-exclusion chromatography (SEC) (Superdex 200, ThermoFisher) and fractions are analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher). Protein-containing fractions are pooled and sent for analysis of phosphorylation patterns and sequence analysis.

[0456] Analyze ITK activity using the ADP-Glo ​​Kinase Assay (Promega) according to the manufacturer's instructions. E4Y1 is used as the ITK substrate. ITK concentrations vary from 0.1 to 500 ng. To compare the quality of recombinantly expressed ITK with standard available ITK, use the ITK Kinase Enzyme System (Promega). Dilute ITK, substrate and ATP to working concentrations in wash buffer. Mix ITK with substrate and ATP and incubate for 60 min at room temperature (RT). Add ADP-Glo ​​reagent and incubate the reaction at room temperature for 40 min. Stop the reaction by adding kinase detection reagent and incubate at room temperature for 30 min. Measure luminescence using a plate reader (Synergy LX (BioTek)) and analyze the data using a standard analysis program.

[0457] Example 9: Production of IL2-inducible T cell kinase (ITK) was compatible with CHO-K1 cells. To demonstrate the compatibility of this embodiment of the system with other eukaryotic cell lines, the experiment of Example 7 is repeated using CHO cells instead of HEK293. ITK-his is expressed either in the presented system (EG17) or in the industrial and academic standard (EG18).

[0458] The ITK-his fusion is expressed in CHO-K1 cells. 2×10 CHO-K1 cells 6Cells / mL are seeded at 100 mL and incubated overnight at 37° C., 120 rpm and 5% CO2 before being transiently transfected with either EG17 or EG18 as described above. Cells are harvested 48 hours later (5,000×g, 15 min, 4° C.) and cell pellets are stored at −80° C. until further use. To purify ITK, cells are resuspended in lysis buffer (40 mM Tris, 7.5 μL; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2, protease inhibitors, DNAse), lysed by sonication (2 min, 10 s ON, 10 s OFF, 40% amplitude) and the crude cell extract clarified (100,000×g, 45 min, 4C). His-resin (GE Healthcare HisTrap) is equilibrated with wash buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2) and then added to the clarified lysate. The lysate is incubated with the resin for 2 h at 4 °C with shaking. The resin is sedimented, washed with 5 CV of wash buffer, and the protein is eluted with 3 CV of elution buffer (wash buffer + 300 mM imidazole). The purification is analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher) and protein-containing fractions are pooled and concentrated. The protein was further polished by size exclusion chromatography (SEC) (Superdex 200, ThermoFisher) and fractions were analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher). Protein-containing fractions are pooled and sent for analysis of phosphorylation patterns and sequence analysis.

[0459] Example 10: Production of IL2-inducible T cell kinase (ITK) was compatible with Sf9 cells. To demonstrate the compatibility of the proposed system with other eukaryotic cell lines, the experiment of Example 7 is repeated using Sf9 cells instead of HEK293. ITK-his is expressed either in the proposed system (EG17) or in the industrial and academic standard (EG18).

[0460] The ITK-his fusion is expressed in CHO-K1 cells. 2×10 CHO-K1 cells 6 Cells / mL are seeded at 100 mL and incubated overnight at 26° C. and 130 rpm before being transiently transfected with either EG17 or EG18 as described above. Cells are harvested 48 hours later (5,000×g, 15 min, 4° C.) and cell pellets are stored at −80° C. until further use. To purify ITK, cells are resuspended in lysis buffer (40 mM Tris, 7.5 μL; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2, protease inhibitors, DNAse), lysed by sonication (2 min, 10 s ON, 10 s OFF, 40% amplitude), and crude cell extracts are clarified (100,000×g, 45 min, 4C). His-resin (GE Healthcare HisTrap) is equilibrated with wash buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg / ml BSA; 50 μM DTT; and 2 mM MnCl2) and then added to the clarified lysate. The lysate is incubated with the resin for 2 h at 4 °C with shaking. The resin is sedimented, washed with 5 CV of wash buffer, and the protein is eluted with 3 CV of elution buffer (wash buffer + 300 mM imidazole). The purification is analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher) and protein-containing fractions are pooled and concentrated. The protein is further polished by size exclusion chromatography (SEC) (Superdex 200, ThermoFisher) and fractions are analyzed by SDS-PAGE (6-12% BOLT, ThermoFisher). Protein-containing fractions are pooled and sent for analysis of phosphorylation patterns and sequence analysis.

[0461] Example 11: Materials and Methods for In Vivo Studies Construction of DNA molecules The entire assembly was made into a plasmid backbone capable of propagation in E. coli that contains 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 that is isolated from the rest of the vector by a second terminator (the transcription terminator from phage lambda). The genes containing the backbone elements were synthesized by phosphoramidite chemistry.

[0462] The structural genes used in the construction of the plasmids were synthesized by phosphoramidite chemistry and cloned into the above vectors with assembly sites on GATG for the 3' region and TAAG for the 5' region using Golden Gate assembly via restriction digestion and Esp3I restriction, see Figure 10.

[0463] The construct shown in Figure 10A was synthesized and cloned into an adapted version of pVax1 using ESP3I with assembly sites of GATG for the 3' and TAAG for the 5' to yield the plasmid shown in Figure 10B.

[0464] The construct shown in Figure 10C was synthesized and cloned into an adapted version of pVax1 using ESP3I with assembly sites of GATG for the 3' and TAAG for the 5' to yield the plasmid shown in Figure 13D.

[0465] [Table 7-1]

[0466] [Table 7-2]

[0467] [Table 7-3]

[0468] Cell lines - culture and transfection The constructs were validated in vitro using HEK293 cells before being injected into animals. HEK293 adherent cells (CLS) were cultured in Dulbecco's Modified Eagle Medium High Glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 50,000U of Pen Strep (Gibco). HEK293 cells were grown to 80% confluence at 37°C and 5% CO2 and then transiently transfected using 293fectin (ThermoFisher) according to the manufacturer's instructions. Protein-expressing cells were harvested 48 hours later by detaching and scraping the cells using 0.5% trypsin solution for 5 min at 37°C. The cells were pelleted (5,000 x g, 15 min, 4°C) and the supernatant was discarded. The cell pellets were stored at -80°C until further use.

[0469] HEK293 suspension-adapted cells were cultured in Expi293 serum-free medium (Gibco). HEK293 cells were seeded at 3.0×10^6 cells / mL at 37°C, 5% CO2 and 120 rpm and then transiently transfected using ExpiFectamin 293 (Gibco) according to the manufacturer's instructions. Depending on the example, for protein expression, cells were harvested after 48-72 hours by pelleting (5,000×g, 15 min, 4°C) and the supernatant was discarded. The cell pellet was stored at -80°C until further use. For secreted proteins, the supernatant was harvested after 96 hours and clarified by centrifugation (5,000×g, 15 min, 4°C). The supernatant was used immediately for further analysis or purification.

[0470] animal For animal studies, BALB / c mice (Charles River Laboratories) and wild-type mice were used. Corresponding disease models can also be used. Ages are indicated in the Examples. Animals of the same sex were group-housed in polycarbonate cages containing appropriate bedding. Mice were identified either by a visible tail tattoo or by implantation of an electronic identification chip. Mice were acclimated for at least 5 days before procedures to familiarize the animals with the laboratory 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 environmental enrichment. Target environmental conditions were a temperature of 19-25°C, humidity of 30%-70%, and a photoperiod of 12 hours light and 12 hours dark. Food (Lab Diet Certified CR Rodent Diet 5CR4) was provided ad libitum in the form of pellets. Water was provided ad libitum in the form of municipal tap water treated by reverse osmosis and ultraviolet irradiation.

[0471] Example 12: In vivo expression of the L enhancer protein with luciferase increases the stability and duration of luciferase expression. To assess the expression of firefly luciferase in vivo, groups of 4 mice each of 6-8 week old female BALB / c mice were used. Groups were injected intradermally once with 50 μl of 25 μg in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 pH 7.4) of (a) a plasmid encoding luciferase as shown in FIG. 9B, or (b) a plasmid encoding luciferase contiguous with an enhancer protein as shown in FIG. 9D. For baseline measurements, 4 mice were injected intradermally with 50 μl of PBS alone. Dose volumes were administered using a syringe / needle within a defined area in the lumbar or sacral region of the back.

[0472] Animals were randomized into groups and body weights were recorded on day 1 and then every other week until the end of the study. Adverse events (RM, SD, RD) were recorded according to good laboratory standards. Any individual animal that had a single observed weight loss of >30% or three consecutive measurements of weight loss of >25% was euthanized.

[0473] Luciferase expression was measured by whole animal bioluminescence imaging on days 2 (24 hours post-injection), 3 (48 hours post-injection), 4 (72 hours post-injection), 11, 18, 25, 32, 38, 53, and 67. The study endpoint, when all animals were euthanized, was day 67. Bioluminescence imaging was performed under anesthesia. Dorsal images were taken 10 minutes after luciferin substrate injection. Substrate was administered at 150 mg / kg ip at 10 mL / kg based on recent body weight. See Figures 11 and 12.

[0474] These results show that when luciferase is expressed together with an enhancer protein such as the L1 protein of EMCV, the in vivo expression of luciferase is maintained for a longer period. As shown in Figure 10, the expression level of luciferase in control animals is initially high, then decreases by 30 days after treatment. In contrast, the expression level of luciferase is maintained at a steady level for a longer period, even up to 110 days after treatment, when it is expressed in combination with the L1 protein. That is, when luciferase is expressed together with the L1 protein, there is less fluctuation in the luciferase expression level over time.

[0475] Furthermore, the variation in luciferase expression levels between animals expressing luciferase and L1 proteins is less than between animals expressing luciferase alone. This effect is particularly evident over time, for example at day 53. See FIG. 11. Only 25% (1 out of 4) of animals injected with the plasmid of FIG. 9B show detectable luciferase expression at day 53 post-injection, whereas 100% (3 out of 3) of animals injected with the plasmid of FIG. 9D show luciferase expression at day 53. Thus, the disclosed method reduces the variability in target protein expression levels between animals and provides more reproducibility in target protein expression, which has important applications in therapy.

[0476] Finally, Figure 12 further demonstrates that expression of luciferase in the presence of enhancer protein results in more stable expression over a longer period of time. While control mice expressing luciferase alone show high levels of luciferase for several days, followed by a gradual decrease in expression to a minimum, mice expressing luciferase in the presence of L1 protein show detectable and stable luciferase expression for 123 days. This result demonstrates the superior advantages of the disclosed compositions and methods for in vivo expression of target proteins.

[0477] To demonstrate that the beneficial effect of reporter gene expression over time was not specific to either the route of administration or the site of injection, the experiment was repeated using subcutaneous administration. Briefly, groups of four female BALB / c mice aged 6-8 weeks were used. These groups were injected subcutaneously once with 200 μl of 30 μg naked plasmid in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 pH 7.4) together with either the plasmid encoding luciferase shown in Figures 9A and 9B, or the plasmid encoding luciferase in succession with an enhancer protein shown in Figures 9C and 9D. For baseline measurements, four control mice were treated with PBS only. The dose was administered using a needle as a subcutaneous injection into the interscapular space.

[0478] Mice were dosed on day 0 and luciferase expression was measured by bioluminescence imaging of the whole body of the animals on days 1, 2, 3, 7, 15, 21, 28, 35 and 42 (imaging data quantified in FIG. 19). For imaging, luciferin (i.e., a substrate for firefly luciferase) was injected into the intraperitoneal (ip) cavity of mice at a dose of 150 mg / kg in a volume of 10 mL / kg. Images were acquired 10 minutes after administration of the substrate under anesthesia. Mice were imaged in a prone position to focus on the injection site.

[0479] FIG. 19 shows the results of bioluminescence imaging of firefly luciferase (Fluc) after subcutaneous treatment of Balb / c mice with either a plasmid expressing firefly luciferase in combination with an enhancer protein (Fluc EG) or the same plasmid without the enhancer protein (Fluc Std). As described above, the addition of the enhancer protein increases the time during which the reporter gene is functionally expressed. The enhancer protein results in stable expression over the entire duration of the experiment, whereas without the enhancer protein, loss of active luciferase expression can be detected within a few days. Surprisingly, the stabilizing effect of the enhancer protein is independent of the injection site. This result demonstrates the general applicability of the compositions and methods disclosed herein for in vivo expression of target proteins.

[0480] Example 13: Expression of the L enhancer protein with adalimumab improves the quality and expression levels of adalimumab protein To test whether adalimumab was expressed in HEK293T cells from a control plasmid containing a nucleic acid sequence encoding adalimumab (EG141, FIG. 13A) and a plasmid containing an enhancer gene in combination with a nucleic acid sequence encoding adalimumab (EG140, FIG. 13B), the following experiment was performed.

[0481] On day 0, HEK293 T cells were seeded at 20,000 cells / well in 24-well plates. On day 1, the growth medium was replaced with Opti-MEM (450 μl / well). Cells were then transfected using 0.5 μg of plasmid and 1 μg of PEI per well in a 1:2 ratio according to standard transfection procedures. A total of six replicates were used. On day 3, the cell culture supernatant was collected for cell fixation for ELISA and immunofluorescence microscopy. Remaining cell debris was removed using centrifugation performed at 500×g for 5 min. The clear supernatant was then pipetted into a new 1.5 mL Eppendorf tube and stored at −20° C. until analysis by ELISA.

[0482] Cells were fixed with 10% neutral buffered formalin for 10 min at room temperature (RT) and permeabilized with 0.2% Triton® X-100 for 10 min at room temperature. Cells were stained with DyLight488-labeled anti-human antibody (IgG Fc Cross-Adsorbed Goat anti-Human, DyLight® 488, Invitrogen-PISA510134 ANTI-HUMAN IGG-FC XMIN D488) at 1:500 dilution for 1 h at room temperature and washed. Cells were imaged using a FLoid fluorescence microscope.

[0483] Immunofluorescence results are shown in Figure 14. The results show that adalimumab was expressed from both EG140 and EG141 plasmids in HEK293T cells. Expression of adalimumab from either plasmid was easily detected, but expression of adalimumab in combination with EMCV L1 protein (from EG141) was slightly lower than when adalimumab was expressed alone (from EG140). Surprisingly, expression of adalimumab in combination with L1 enhancer protein results in a more uniform intracellular distribution of the antibody (Figure 14B) compared to the absence of enhancer protein (Figure 14A). Furthermore, when adalimumab is expressed in combination with L1 enhancer, there are no intracellular foci indicative of the presence of misfolded or unfolded protein seen when adalimumab is expressed alone. These results further support that the presence of L1 enhancer protein improves the quality and / or quantity of expression of recombinant adalimumab antibody expressed in cells.

[0484] Furthermore, whether adalimumab can be detected in cell culture supernatants was tested using a direct enzyme-linked immunosorbent assay (ELISA) method. For ELISA experiments, frozen cell culture supernatants were thawed and used to coat ELISA high-binding plates. A two-fold dilution series of cell culture supernatants was used for coating at 75 μl / well. For positive controls, a dilution series of recombinant human anti-TNFα antibody (NBP2-62567, Novus Biologicals) was used instead. Coating was performed overnight at 4°C.

[0485] The next day, ELISA plates were washed once with PBS-T and blocked with EZ Block at 37°C for 2 hours at 150ul per well. Plates were washed and incubated with secondary antibody (Anti-Human HRP-Conjugated Antibody, IgG(H+L) Goat Anti-Human, HRP, Invitrogen-A18805 GTXHU IGG HRP AFFINITY) at a dilution of 1:2000 for 1 hour at room temperature. Plates were washed 5 times with PBS-T. TMB substrate was added (75ul / well) and incubated for 20 minutes, followed by addition of stop solution (75ul / well). Absorbance at 450nm was measured using a Biotek plate reader.

[0486] The results are shown in Table 15 below. Since the ELISA plate was coated with cell culture supernatant from adalimumab-expressing HEK293T cells, the results suggest that adalimumab is easily detectable in the supernatant of both EG140- and EG141-transfected cells. The level of adalimumab upon expression of EG140 (control) is >3-fold higher than upon expression of EG141 (plasmid encoding adalimumab and L1 enhancer).

[0487] Example 14: Expression of the L enhancer protein significantly improves the quality and expression levels of the adalimumab protein. To determine whether antibodies expressed and secreted from EG140- or EG141-transfected cells were capable of specifically binding to recombinant human TNF-α, the following experiment was performed.

[0488] In addition to plasmids EG140 and EG141, the same expression cassettes differing from the EG140 and EG141 backbones by different polyadenylation signals and flanked by inverted terminal repeats (ITRs) were cloned into the AAV transfer plasmid backbone for the purpose of generating recombinant AAV vectors for in vivo use downstream of AAV in animals (Figure 20). To test whether adalimumab from AAV transfer plasmids without (Figure 21) and with (Figure 22) enhancer proteins expressed in HEK293T cells could specifically bind recombinant human TNF-α, the following experiment was performed.

[0489] Cells were grown and transfected as described in Example 13. In contrast to experiment 13, however, high-binding ELISA plates were first coated with recombinant TNF-α (1ug / ml, 75μl / well) overnight at 4°C. On the second day, ELISA plates were washed with PBST and blocked with EZ-Block reagent. Cell culture supernatant samples were diluted in Opti-MEM (1:256) and added to the blocked wells (75μl per well, 1 hour at 37°C). Wells were washed three times with PBST and secondary antibody (anti-human IgG HRP) was added at 1:2000 dilution (75μl per well, 1 hour at 37°C). Wells were then washed five times with PBST. Bound antibodies were detected by adding 75μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The reaction was stopped using 75 μl of TMB stop solution and the signal was read by measuring absorbance at 450 nm using a Biotek microplate reader.

[0490] In some experiments, the total concentration of secreted adalimumab was measured using a quantitative ELISA. In these experiments, the ELISA was performed as described above with the following modifications: Cell culture supernatants were pre-diluted in EZ Block reagent and added as samples to pre-coated and blocked wells. The positive control antibody, recombinant monoclonal adalimumab (Novus Biologicals NB001486), was diluted to concentrations of 0, 0.1, 1, 10 and 100 ng / ml in EZ Block reagent and added as a standard curve to pre-coated and blocked wells. In all other respects, the ELISA was performed as described above. After reading the absorbance at 450 nm, the sample absorbance values ​​were converted to total secreted adalimumab concentrations in ng / ml using the absorbance values ​​of the standard curve.

[0491] The results showed that cell culture supernatants from EG140-transfected cells contained approximately 3-fold more TNF-α-binding human antibodies compared to EG141-transfected cells (FIG. 16).

[0492] Results for AAV transfer plasmids encoding adalimumab with (SEQ ID NO: 243-272) and without (SEQ ID NO: 217-242) enhancer L protein are shown in FIG. 23, along with repeat experiments of transfected plasmids encoding adalimumab without (STD) and with (EG) enhancer protein. Secreted adalimumab concentrations were quantified in ng / ml by quantitative ELISA. In both cases, the presence of enhancer protein reduced the total amount of adalimumab in the cell supernatant. Notably, the presence of adalimumab in the standard plasmid together with enhancer protein reduced the presence of adalimumab protein in the supernatant by 39-fold, while the enhancer protein in the AAV transfer plasmid reduced the presence of adalimumab protein in the supernatant by 77-fold.

[0493] Analysis of adalimumab quality was tested by the level of TNF-α activation in cell culture supernatants. Active adalimumab blocks TNF-α. This experiment was performed using luciferase TNF-α reporter cells.

[0494] The isolated cell culture supernatants were analyzed using reporter cells designed to monitor the level of bioactive TNF-α in the samples by assessing the activation of NF-κB. The reporter HEK-Dual TNF-a cell line (Invivogen) was generated by transfecting HEK293 cells with NF-κB-inducible secreted firefly luciferase. Upon TNF-α treatment, the NF-κB pathway is activated, which leads to the expression of secreted luciferase, which can be detected using a luciferase substrate. In the presence of cell culture supernatants of EG140- and EG141-transfected cells, or TNF-α neutralizing antibodies in the same expression cassette cloned into the AAV transfer plasmid backbone, NF-κB activation was predicted to be inhibited and the luciferase signal reduced.

[0495] The experiment was performed as follows: Reporter cells were seeded in 96-well plates (5000 cells per well) in DMEM + 10% FBS. The next day, cell culture supernatants from EG140-transfected and EG141-transfected cells were diluted in cell culture medium at the indicated ratios and mixed with 1 ng / ml human recombinant TNF-α for 30 min at room temperature. The cell medium of TNF-α reporter cells pre-seeded in 96-well plates was then replaced with pre-incubated cell culture supernatant / TNF-α samples using 100 μl of the mixture per well. The reporter cells were then incubated for 5 h at 37°C. The secreted luciferase signal was then detected using the Quanti-Luc Gold assay (Invivogen) according to the manufacturer's instructions to detect TNF-α activation, and luciferase activity was measured using a microplate reader.

[0496] The results are shown in Figure 17, Figure 24 and Tables 4 and 5. Cell culture supernatants isolated from both EG140- and EG141-transfected cells were able to inhibit TNF-α-mediated activation of NF-κB-driven luciferase expression, although supernatants from EG140-transfected cells were approximately three-fold more active in suppressing TNF-α activation compared to supernatants from EG 141-transfected cells.

[0497] [Table 4]

[0498] [Table 5]

[0499] Furthermore, to estimate the relative quality of secreted adalimumab, the total amount of secreted adalimumab in the supernatant (in ng / ml as measured by quantitative ELISA) was compared with the potency of the same supernatant to inhibit TNF-α signaling (EC measured by using HEK Dual TNF-α reporter cells). 50 Units) were compared.

[0500] Interestingly, although the amount of Adalimumab produced and secreted for the standard system was higher (Figure 24 and Tables 4 and 5), the relative quality of the Adalimumab produced, as defined by biological activity, was higher in the construct with the Enhancer L protein: the activity with the Enhancer protein was 22.2-fold higher in standard construct-transfected cells and 65.3-fold higher in AAV construct-transfected cells.

[0501] In particular, the relative percentage of biologically active adalimumab was further analyzed. Figure 25 shows that the relative amount of active adalimumab with enhancer protein co-expression was higher than without it. Importantly, the percentage of active expressed adalimumab was up to 60% higher with enhancer protein co-expression.

[0502] Example 15: Expression of the L enhancer appears to reduce the number of inactive adalimumab expression products. To visualize the total amount of secreted antibodies in cell culture supernatants of HEK293T cells transiently transfected with EG140 and EG141 plasmids, the following experiment was performed. Cells were seeded in 15 cm cell culture dishes at 4E6 cells / dish and transfected with 40ug of plasmid and 80ug of PEI. Antibodies were purified from cell culture supernatants using Protein A / G agarose resin (MPbio) according to the manufacturer's instructions. Equal volumes of purified antibodies were analyzed by SDS-Page and Western blotting (GenScript) according to the manufacturer's instructions.

[0503] The results of SDS PAGE (FIG. 18A) and Western blot (FIG. 18B) show that purified adalimumab light and heavy chains were detected in the cell culture supernatants of both EG140 and EG141 transfected HEK293T cells. The results show that the amount of purified adalimumab expressed in the control EG140 transfected cells is significantly greater than that expressed in the presence of the L1 enhancer in EG141 transfected cells. Without wishing to be bound by theory, it is believed that the majority of secreted adalimumab expressed by EG140 transfected cells is potentially non-functional due to misfolding and / or inappropriate localization to intracellular foci compared to secreted adalimumab expressed by EG141 transfected cells.

[0504] Example 16: Expression of the L enhancer protein in vivo reduces injection site dependent effects. To evaluate adalimumab expression in vivo, groups of six 6- to 8-week-old female BALB / c mice were administered 2 × 10 6-well plates of recombinant AAV in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO pH 7.4). 1150 μl of genome copies were injected once via either intramuscular (im) or subcutaneous (sc) route. The recombinant AAV vectors used were made using either (a) a plasmid encoding adalimumab as shown in FIG. 21 or (b) a plasmid encoding adalimumab with enhancer protein as shown in FIG. 22. The recombinant AAV vectors were made by VectorBuilder Inc. by triple transfecting AAV transfer plasmid together with a helper plasmid (encoding adenoviral genes E4, E2A and VA) and a Rep-Cap plasmid (encoding the Cap protein of AAV serotype 8, AAV8) into HEK293T packaging cells. Similarly, any replication gene sufficient for viral genome replication and packaging and any viral capsid gene sufficient for viral encapsidation may be used in place of Rep-Cap. Two days after transfection, cells were harvested, concentrated by PEG and purified by CsCl gradient purification.

[0505] The pharmacokinetics of adalimumab in AAV8-treated mice was compared to that of mice treated with recombinantly produced adalimumab protein as a control, mimicking the current standard of care for anti-TNFα therapy. In the control group, three mice were injected subcutaneously with 50 μl of 100 μg of recombinantly produced adalimumab in PBS.

[0506] Animals were randomized into groups and body weights were recorded on day 1 and then every other week until the end of the study. Adverse events (RM, SD, RD) were recorded according to good laboratory standards. Any individual animal that showed a single observation of >20% weight loss, wounds that interfered with normal physiological function (e.g., feeding, drinking and mobility), or clinical observations of weakness, seizures and bleeding was euthanized.

[0507] Whole blood was collected via the submandibular vein and processed to collect serum for analysis. Blood was collected on days 0 (pre-dose), 3, 7, 14, 21, 28, and 42. Serum was analyzed for adalimumab concentration by quantitative ELISA.

[0508] Quantitative ELISA was performed as described in Example 14 above by using mouse serum samples (pre-diluted with EZ Block reagent) as samples. Positive control antibody, recombinant monoclonal adalimumab (Novus Biologicals NB001486), was diluted in EZ Block reagent to concentrations of 0, 0.1, 1, 10 and 100 ng / ml and used as a standard curve. After reading the absorbance at 450 nm, the absorbance values ​​of the samples were converted to total secreted adalimumab concentrations in serum in μg / ml using the absorbance values ​​of the standard curve.

[0509] The results of the ELISA experiment are shown in Figures 26A and 26B. The straight dotted line threshold indicates the 5 μg / ml adalimumab concentration above which the therapeutic effect of adalimumab is observed in the rheumatoid arthritis model. Figure 26A shows the results of intramuscular injection of AAV with (EG) and without (STD) enhancer protein. Figure 26B shows subcutaneous injection of the same material. As mentioned above with respect to other experiments presented herein, the line without enhancer protein (STD) had higher adalimumab concentrations in serum compared to the line with enhancer protein (EG).

[0510] Notably, the concentration of adalimumab without enhancer protein co-expression was highly dependent on the injection site. In total, a 10-fold difference could be observed between injection sites. This can be very challenging for certain gene therapies where the injection site cannot be varied, since relatively small deviations in treatment dosing can lead to large changes in the steady-state serum levels of the therapeutic transgene.

[0511] In contrast, the system with enhancer protein (EG) showed bloodstream administration independent of injection site. These data demonstrated that enhancer protein ensured similar protein expression levels regardless of the cell type being treated, which is an important result for achieving robust and reproducible therapeutic effects. Importantly, the same levels of adalimumab were found in the serum of treated mice both in intramuscular and subcutaneous administration, both over the required therapeutic concentration. Surprisingly, the addition of enhancer protein ensured that adalimumab was expressed at a stable and constant level, independent of injection site.

[0512] Example 17: Expression of the L enhancer protein increases the relative activity of secreted glucosylceramidase (GBA) in vitro To test glucosylceramidase (GBA) expression in HEK293T cells in the presence and absence of enhancer L protein, the following experiment was performed.

[0513] HEK293T cells were transfected with a control plasmid expressing GBA-NanoLuc fusion protein (Figure 28A) and a plasmid co-expressing enhancer protein L with GBA-NanoLuc chimera (Figure 28B). On day 0, HEK 293T cells were seeded in 24-well cell culture microplates in 500 μl of DMEM containing 10% FBS. On day 1, cells were transfected with the respective pGBA-NanoLuc_STD (SEQ ID NOs: 273-296) and pGBA-NanoLuc_EG (SEQ ID NOs: 297-324) plasmids using Mirus TransIT-Lenti transfection reagent according to the manufacturer's instructions. Each well was treated with complexes made with 0.2 μg of plasmid, 0.6 μl of Mirus TransIT-Lenti reagent and 50 μl of Opti-MEM medium. On day 3, transfected cells and cell culture supernatants were assayed for NanoLuc luciferase and GBA activity and processed for Western blotting.

[0514] For the NanoLuc assay, cell culture supernatants were collected in microcentrifuge tubes and cellular debris was removed by centrifugation. Cells that remained attached to the 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 were loaded into an opaque black 96-well microplate. Fresh 1X Nano-Glo assay reagent in 50 μl of 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. Cell lysates were further analyzed for their protein concentration using 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, absorbance at 660 nm was measured using a Biotek Synergy LX plate reader, and protein concentration was quantified based on a standard curve. Luminescence of cell lysates was normalized per μg of cell lysate protein (i.e., giving luminescence values ​​in relative light units (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 RLU / ml of supernatant).

[0515] To determine GBA activity, cells were seeded and transfected with the respective plasmids as in the NanoLuc assay above. On day 3, cell culture supernatants were harvested, centrifugally removed to remove cell debris, and saved for assaying secreted GBA activity. Adherent cells were detached from the plate using 500 μl of 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) that had been pre-equilibrated to 37°C prior to lysis. Protein concentrations of cell lysates were determined using the Pierce A660 protein assay according to the manufacturer's instructions, after which cell lysates were diluted to a final protein concentration of 125 μg / ml using 1× GBA assay buffer. 40 μl of pre-diluted cell lysates were pipetted into individual wells of a clear 96-well plate in duplicate. 20 μl of cell culture supernatant collected earlier was added in duplicate to individual wells of a clear 96-well plate, and 20 μl of 2×GBA assay buffer was added to each well containing cell culture supernatant. Then, 20 μl of assay substrate (6 mM 4-MU-β-D-glucopuranoside prepared in 1×GBA assay buffer) was added to each well containing cell lysate or cell culture supernatant. In adjacent wells, a calibration curve using 4-methyl-umbelliferone was prepared in 1×GBA assay buffer. Samples were incubated in the presence of assay substrate at 37°C for 30 min to 4 h, followed by the addition of 100 μl of stop solution (0.5 M glycine, 0.3 M NaOH, pH 10). The fluorescence of each sample and standard was then measured using a Biotek Synergy LX plate reader at excitation / emission wavelengths of 360 / 445 nm. GBA activity in cell lysate samples was calculated using the following formula: activity = [B / (T x V x P)] x D = pmol / min / mg = μU / mg, where B is converted to 4-MU amount (pmol) calculated using the standard curve, T is reaction time (min), V is sample volume (ml), P is initial protein sample concentration, and D is sample dilution factor (if applicable).GBA activity in cell culture supernatants was calculated using the following formula: activity = [B / (T × V)] × D = pmol / min / ml = μU / ml, where B is the amount of 4-MU calculated using the standard curve (pmol), T is the reaction time (min), V is the sample volume (ml), and D is the sample dilution factor (if applicable).

[0516] For Western blotting, transfected cells were harvested by scraping and pelleted by centrifugation. Cell pellets were lysed using 100 μl of RIPA buffer (Cell Signalling technologies) supplemented with 1× protease inhibitor cocktail and 10 U of universal nuclease, incubated on ice for 10 min, followed by sonication for 10 s. Lysed samples were centrifuged at 14,000 g for 10 min, and 80 μl of supernatant was collected for analysis. Protein concentration of each sample was determined using the Pierce A660 assay according to the manufacturer's instructions. 40 μg of cell lysate was loaded into each well of a 4-12% Bis-Tris MES gel, and Western blotting was continued as previously described in Example 15. To detect GBA, rabbit anti-GBA antibody (Abcam ab128879) was used at a 1:500 dilution overnight.

[0517] Western blotting revealed that the designed pGBA-NanoLuc_STD and pGBA-NanoLuc_EG constructs were detected as a single band of the exact predicted size for the pGBA-NanoLuc protein chimera of approximately 75 kDa when expressed in HEK 293T cells. The band of the pGBA-NanoLuc_STD construct was somewhat stronger than that of the pGBA-NanoLuc_EG construct ( FIG. 29 ), which corresponds to previous observations with expressed secreted proteins shown in Example 15.

[0518] By using NanoLuc-tagged GBA protein during the analysis, it was possible to distinguish between the total expression of the protein of interest (NanoLuc reporter readout) and the expression of the functionally active protein of interest (GBA enzyme activity). The total amount of both reporter protein (NanoLuc) and enzyme activity (GBA) was high in the case of the pGBA-NanoLuc_STD construct (Figure 30A-30D), consistent with the Western blot results above. However, the relative quality of the produced protein was much higher in the case of the pGBA-NanoLuc_EG construct when the enhancer protein was co-expressed (Figure 31). The relative quality of the expressed GBA protein was estimated by first normalizing the GBA enzyme activity using the NanoLuc reporter signal (i.e., to obtain the specific GBA activity GBA_S = GBA_activity / NanoLuc_signal) and then comparing the GBA_S of the two different constructs. This analysis revealed that in cell lysates, the specific GBA activity was similar between the pGBA-NanoLuc_STD and pGBA-NanoLuc_EG constructs.

[0519] However, it is noteworthy that in the cell supernatant, the specific GBA activity in the presence of enhancer protein (i.e., pGBA-NanoLuc_EG construct) was approximately 300% higher than that in the absence of enhancer L protein (i.e., pGBA-NanoLuc_STD construct) (Figure 31). Considering that endogenous GBA is a secreted protein that is fully active in the supernatant, these data support the increase in protein quality when co-expressed with enhancer protein L, as presented herein.

[0520] Example 18: Expression of the L enhancer protein improves the uniformity of glucosylceramidase (GBA) expression in vivo. To evaluate the expression of GBA in vivo, the following experiment was performed. Both pGBA-NanoLuc_STD (without enhancer protein L, SEQ ID NOs: 273-296) and pGBA-NanoLuc_EG (with enhancer protein L, SEQ ID NOs: 297-324) plasmids were formulated into lipid nanoparticles (LNPs) for in vivo delivery as follows: 300 μg of plasmid was dissolved in 2.6 mL of encapsulation buffer (EB, 25 mM sodium acetate, pH 4). The LNP lipid mixture was prepared in 2.6 mL of 100% EtOH containing 1% DMG-PEG(2000), 39% cholesterol, 10% DOPC and 50% DLin-KC2-DMA ionizable lipids, with a final total lipid concentration of 4 mM. Equal volumes (2.6 mL each) of plasmid in EB and lipid in EtOH were combined by rapid mixing. Immediately after mixing, 5.2 mL of neutrbio similar affer (NB, 300 mM NaCl + 20 mM sodium citrate, pH 6) was added to the lipid / plasmid mixture, mixed rapidly, and incubated at 37°C for 30 minutes. The mixture was diafiltered against PBS using an Amicon Ultra 15-ml 100 kDa MWCO spin filter. The encapsulation efficiency and total concentration of loaded plasmid was determined using the SYBRSafe encapsulation efficiency assay as follows: 5 μl of plasmid / LNP was mixed with 1× SYBRSafe DNA binding dye in either TE buffer (to detect the amount of plasmid not loaded into the LNP) or TE buffer containing 1% Triton X-100 (to detect the total amount of plasmid, i.e., both plasmid loaded and not loaded into the LNP). For absolute plasmid DNA calculations, a standard curve was constructed using known amounts of reference naked plasmid DNA mixed in 1x SYBRSafe DNA binding dye in either TE buffer or TE buffer containing 1% Triton X-100, respectively. Samples or standards were incubated for 5 minutes and fluorescence was read using a Biotek Synergy LX fluorescence microplate reader using a filter set for FITC.The encapsulation efficiency was calculated using the formula: loading_efficiency=(plasmid_total-plasmid_unloaded) / plasmid_total×100%.

[0521] Loading efficiency was >90%. Plasmids / LNPs were diluted with PBS to an equivalent plasmid concentration of 30 μg plasmid per 200 μl PBS. Female Balb / c mice were anesthetized and individually administered a fixed volume of 200 μl for subcutaneous injection between the shoulder blades (N=3 / group). At the indicated time points, whole-body bioluminescence imaging (BLI) was performed by intraperitoneally (ip) injecting mice with Nano-Glo® In Vivo Substrate, FFz, and imaged in prone and supine positions. Prone position focused on the injection site, supine position focused on the liver region. Luminescence values ​​were quantified, tabulated, and plotted.

[0522] Bioluminescence imaging (BLI) results showed that the detected luminescence signal was relatively comparable, if not slightly higher, in the LNP-pGBA-NanoLuc_STD group compared to the LNP-pGBA-NanoLuc_EG group (Figure 32A-C and Table 6), consistent with the in vitro experiments. Strong signals were detected in both prone BLI (i.e., at the site of injection) as well as supine (i.e., the site of expressed GBA-NanoLuc accumulation, which may be the liver, indicating that the protein produced in vivo is secreted and accumulates in target tissues).

[0523] [Table 6]

[0524] The BLI signal in the LNP-pGBA-NanoLuc_STD group was more variable over time and between individual animals when measured at the same time points compared to the LNP-pGBA-NanoLuc_EG group. This was further investigated by quantifying the coefficient of variation (CV%) of the signal at each measurement time point and then calculating the average CV% for each treatment group. CV% was defined as the standard deviation of the signal divided by the signal mean. This analysis revealed that the average CV% was higher in the absence of enhancer protein (Figure 32C and Table 6). In the prone position (i.e., the site of LNP administration), the difference in CV% was relatively small, but in the prone position (i.e., the site of secreted GBA-NanoLuc protein accumulation), the difference was approximately two-fold. This suggests that in vivo use of vectorized secreted proteins in the presence of enhancer protein L is more uniform and robust compared to the absence of enhancer protein L.

Claims

1. 1. A system for use in expressing a target protein in a subject in need thereof, said system comprising: a) a first polynucleotide encoding the target protein; and b) a second polynucleotide encoding an enhancer protein; i) the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT inhibitor); and / or ii) the enhancer protein is 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; system.

2. The system of claim 1 , wherein the enhancer protein is an inhibitor of nucleocytoplasmic transport (NCT inhibitor).

3. The system according to claim 2 , wherein the NCT inhibitor is a viral protein.

4. The system according to claim 3 , wherein the NCT inhibitor is a picornavirus leader (L) protein or a functional variant thereof.

5. The system of claim 4, wherein the L protein is the L protein of Theiler's virus or a functional variant thereof.

6. The system of claim 4, wherein the L protein shares at least 90% identity with SEQ ID NO:

1.

7. The system according to claim 4, wherein the L protein is the L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof.

8. The system of claim 4, wherein the L protein shares at least 90% identity with SEQ ID NO:

2.

9. The system of claim 4, wherein 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.

10. The system of claim 1 , wherein the target protein is a therapeutic protein.

11. The system of claim 1 , wherein the target protein is an immunogenic protein.

12. The system described in claim 1, wherein the enhancer protein increases the activity of the target protein in the subject, decreases the expression level of the target protein in the subject, increases the uniformity of expression of the target protein at an injection site of the subject, increases the duration of active target protein in the subject, or a combination thereof.

13. The system of claim 1, further comprising a delivery system.

14. The system of claim 13 , wherein the delivery system comprises a lipid nanoparticle (LNP).

15. 10. The system of claim 1, wherein the system is delivered intramuscularly, subcutaneously, intravenously, intradermally or intranasally.

16. The system described in claim 1, wherein at least one of the first polynucleotide or the second polynucleotide is RNA.

17. The system described in claim 16, wherein the RNA is messenger RNA (mRNA).

18. The system described in claim 17, wherein the mRNA is in vitro transcribed (IVT) mRNA.

19. The system described in claim 1, wherein the target protein is derived from one or more animal proteins, one or more human proteins, one or more microbial proteins, one or more viral proteins, one or more fungal proteins, or a combination thereof.

20. The system of claim 1, wherein the target protein is a vaccine antigen.

Images