Compositions and methods for increased protein production

Abstract

Disclosed herein are cell compositions with increased expression of 11S proteasomal cap proteins. The cell compositions provided herein result in increased heterologous protein levels. As such, the cell compositions may be used in methods for increasing protein production of one or more heterologous proteins, including but not limited to proteins relevant for cell and gene therapy.

Description

[0001] Compositions and Methods for Increased Protein Production

[0002] Cross-Reference to Related Applications

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 599,472, filed on November 15, 2023, the entirety of which is incorporated herein by reference.

[0004] Field of Invention

[0005] The present disclosure relates generally to cell compositions for increased protein production and methods of use thereof.

[0006] Sequence Listing

[0007] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 12, 2024, is named T120281_1010WO.XML and is 51,640 bytes in size.

[0008] Background

[0009] Therapeutic modalities that require biomanufacturing, such as monoclonal antibodies, gene therapy delivery products (e.g., AAV capsids, lentiviruses), and vaccines require heterologous expression in cells. While clinical development of these modalities has advanced significantly in recent years, manufacturing remains a frequently encountered roadblock. Biomanufacturing processes can be difficult to scale due to intrinsic limitations in cell-based heterologous protein expression and purification.

[0010] Expression of recombinant proteins can serve many purposes for biomedical research but achieving sufficient levels can be a hurdle to scientific progress. For example, structural studies of proteins can ultimately lead to the identification of new drugs but this type of research involves the production of large amounts of difficult to express recombinant proteins such as membrane proteins (e.g., GPCRs, ion channels, receptors), protein complexes, accessory proteins (e.g., G proteins), enzymes, secreted growth factors, or nanobodies in large culture volumes that are resource intensive. Summary of Invention

[0011] The present disclosure is directed to cell compositions that result in increased protein production of one or more heterologous proteins, including recombinant viruses (e.g., influenza, AAV capsids, lentiviruses), GPCRs, integral membrane proteins, enzymes, intracellular proteins, nanobodies, and monoclonal antibodies. Accordingly, in one aspect, disclosed herein is a method for increasing protein production in one or more cells or in a population of cells, the method comprising transfecting the one or more cells or cells of the population of cells with a vector, the vector comprising a promoter operably linked to a nucleic acid encoding PSME1 (PA28A). In some embodiments, the vector further comprises a promoter operably linked to a nucleic acid encoding PSME2 (PA28B). In one aspect, disclosed herein is a method for increasing protein production in one or more cells or in a population of cells, the method comprising transfecting the one or more cells or cells of the population of cells with a vector, the vector comprising a promoter operably linked to a nucleic acid encoding PSME3 (PA28G).

[0012] In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises one or more nucleic acids to produce one or more function AAV capsids. In some embodiments, the one or more nucleic acids encode an AAV capsid protein, a function rep gene, a recombinant AAV vector comprising AAV inverted terminal repeats and a transgene, and a helper protein. In some embodiments, the AAV capsid protein has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DJ / 8 and AAV2.7m8.

[0013] In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises one or more nucleic acids to produce one or more functional lentivirus particles. In some embodiments, the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and an envelope plasmid.

[0014] In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises one or more nucleic acids to produce one or more live attenuated influenza particles. In some embodiments, the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and one or more envelope plasmids.

[0015] In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises a promoter operably linked to a nucleic acid encoding a heterologous protein, such as a GPCR, an ion channel, an enzyme, a G protein, an intracellular protein, a secreted growth factor, an integral membrane protein, a nanobody, or a monoclonal antibody.

[0016] In some embodiments, the promoter is selected from the group consisting of a GUSB promoter, a SFFV promoter, a CBh promoter, a CBA promoter, a PGK promoter, a CMV promoter, a CAG promoter, a bidirectional CAG promoter, a CBA promoter, a human ubiquitin C promoter, an EFla promoter, a AcMNPV polyhedrin promoter, a AcMNPV plO promoter, a White spot syndrome virus (WSSV) iel promoter, a drosophila metallothionein (MT) promoter, a drosophila actin 5c gene (Ac5) promoter, a tetracycline-inducible (TRE) promoter, and a combination thereof.

[0017] In some embodiments, the cell is a HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a Sf9 cell, a S2 cell, MDCK cell, or a derivative thereof.

[0018] In some embodiments, the PSME1 / PA28A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 1-3, 11, or 17. In some embodiments, the PSME2 / PA28B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 7, 12, or 18. In some embodiments, the PSME3 / PA28G comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 9, 13, or 19.

[0019] In some embodiments, the endogenous PSME1 / PA28A, PSME2 / PA28B, and / or PSME3 / PA28G genes are activated via CRISPR-based or TALE-based activation methods. These methods may comprise catalytically dead Cas proteins fused to one or more transcriptional activators (e.g., VP- 16 or VP-64 activation domains) and guide RNAs directed toward the endogenous PSME1 / PA28A, PSME2 / PA28B, and / or PSME3 / PA28G genomic loci to stimulate their expression or overexpression. These methods may also comprise TALE-fused to one or more transcriptional activators (e.g, VP- 16 or VP-64 activation domains) directed toward the endogenous PSME1 / PA28A, PSME2 / PA28B, and / or PSME3 / PA28G genomic loci to stimulate their expression or overexpression.

[0020] In some embodiments, the vector further comprises a polyadenylation (polyA) site, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an optimized woodchuck hepatitis virus post-transcriptional regulatory element (oPRE), an intron, a microRNA binding motif, or a combination thereof. In some embodiments, the polyA site is selected from the group consisting of a beta-globin polyA, a bovine growth hormone polyA, a human growth hormone polyA, and a SV40 intermediate early polyA. In some embodiments, the vector further comprises a promoter operably linked to one or more selectable markers. In some embodiments, the one or more selectable markers is a resistance gene selected from the group consisting of neomycin / kanamycin, puromycin, hygromycin, and blasticidin.

[0021] In some embodiments of the above methods, the one or more cells or the population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or population of cells that does not comprise the vector. In some embodiments, the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25- fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or population of cells that does not comprise the vector.

[0022] In some embodiments of the above methods, the one or more cells or population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the heterologous protein levels as compared to a cell or population of cells that does not comprise the vector. In some embodiments, the one or more cells or population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the heterologous protein levels as compared to cells or a population of cells that does not comprise the vector.

[0023] In one aspect, disclosed herein is a stable cell line for increased protein production, wherein the stable cell line overexpresses PSME1, for example wherein the stable cell line comprises a promoter operably linked to a heterologous nucleic acid encoding PSME1 (PA28A). In some embodiments, the stable cell line overexpresses PSME2, for example wherein the stable cell line comprises a promoter operably linked to a heterologous nucleic acid sequence encoding PSME2 (PA28B).

[0024] In one aspect, disclosed herein is a stable cell line for increased protein production, wherein the stable cell line overexpresses PSME3, for example wherein the stable cell line comprises a promoter operably linked to a heterologous nucleic acid sequence encoding PSME3 (PA28G).

[0025] In some embodiments, the promoter of the stable cell line is selected from the group consisting of a GUSB promoter, a SFFV promoter, a CBh promoter, a CBA promoter, a PGK promoter, a CMV promoter, a CAG promoter, a bidirectional CAG promoter, a CBA promoter, a human ubiquitin C promoter, an EFla promoter, a AcMNPV polyhedrin promoter, a AcMNPV plO promoter, a White spot syndrome virus (WSSV) iel promoter, a drosophila metallothionein (MT) promoter, a drosophila actin 5c gene (Ac5) promoter, a tetracycline-inducible (TRE) promoter, and a combination thereof. In some embodiments, the cell is a HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a SP9 cell, a S2 cell, or a derivative thereof.

[0026] In some embodiments, the PSME1 / PA28A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 1-3, 11, or 17. In some embodiments, the PSME2 / PA28B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 7, 12, or 18. In some embodiments, the PSME3 / PA28G comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 9, 13, or 19.

[0027] In some embodiments, the polynucleotide further comprises a polyadenylation (poly A) site, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an optimized woodchuck hepatitis virus post-transcriptional regulatory element (oPRE), an intron, a microRNA binding motif, or a combination thereof. In some embodiments, the polyA site is selected from the group consisting of a beta-globin polyA, a bovine growth hormone polyA, a human growth hormone polyA, and a SV40 intermediate early polyA. In some embodiments, the polynucleotide further comprises a promoter operably linked to one or more selectable markers. In some embodiments, the one or more selectable markers is one or more resistance genes selected from the group consisting of neomycin / kanamycin, puromycin, hygromycin, and blasticidin.

[0028] In one aspect, provided herein are methods of making a stable cell disclosed herein. In one aspect, disclosed herein is a method of making a stable cell line, the method comprising a) obtaining a cell disclosed herein, and b) immortalizing the cell to thereby create a stable cell line. In one aspect, disclosed herein is a method for increasing protein production in one or more cells or a population of cells, the method comprising expressing a protein of interest in the stable cell line. In some embodiments, the protein of interest is an AAV capsid or a heterologous protein. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV- DJ / 8 and AAV2.7m8. In some embodiments, the cell of the stable cell line is a HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a Sf9 cell, a S2 cell, or a derivative thereof.

[0029] In some embodiments, the protein of interest has an at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40- fold, at least 45-fold or more increase in expression levels as compared to a stable cell line that does not overexpress PSME1, a stable cell line that does not express PSME1 and PSME2, and / or a stable cell line that does not overexpress PSME3; and / or as compared to one or more cells or a population of cells that does not comprise the heterologous nucleic acid of PSME1, the heterologous nucleic acid of PSME1 and PSME2, and / or the heterologous nucleic acid of PSME3. In some embodiments, the protein of interest has an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in expression levels as compared to a stable cell line that does not overexpress PSME1, a stable cell line that does not express PSME2, and / or a stable cell line that does not overexpress PSME3; and / or as compared to a cell or population of cells that does not comprise the heterologous nucleic acid of PSME1, the heterologous nucleic acid of PSME2, and / or the heterologous nucleic acid of PSME3.

[0030] In one aspect, disclosed herein is a method for increasing protein production in one or more cells or in a population of cells, the method comprising transfecting the cell with i) a vector comprising a promoter operably linked to a nucleic acid encoding PSME1 (PA28A), and ii) a vector comprising a promoter operably linked to a nucleic acid encoding PSME2 (PA28B).

[0031] In some embodiments, the one or more cells or cells of the population of cells further comprises one or more nucleic acids to produce one or more functional AAV capsids. In some embodiments, the one or more nucleic acids encode an AAV capsid protein, a function rep gene, a recombinant AAV vector comprising AAV inverted terminal repeats and a transgene, and a helper protein. In some embodiments, the AAV capsid protein has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DI / 8 and AAV2.7m8.

[0032] In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises one or more nucleic acids to produce one or more functional lentivirus particles. In some embodiments, the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and an envelope plasmid.

[0033] In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises one or more nucleic acids to produce one or more live attenuated influenza particles. In some embodiments, the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and one or more envelope plasmids.

[0034] In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises a promoter operably linked to a nucleic acid encoding a heterologous protein, such as a GPCR, an ion channel, an enzyme, a G protein, an intracellular protein, a secreted growth factor, an integral membrane protein, a nanobody, or a monoclonal antibody, wherein practice of the method results in elevated expression of the heterologous protein.

[0035] In some embodiments, the promoter is selected from the group consisting of a GUSB promoter, a SFFV promoter, a CBh promoter, a CBA promoter, a PGK promoter, a CMV promoter, a CAG promoter, a bidirectional CAG promoter, a CBA promoter, a human ubiquitin C promoter, an EFla promoter, a AcMNPV polyhedrin promoter, a AcMNPV plO promoter, a White spot syndrome virus (WSSV) iel promoter, a drosophila metallothionein (MT) promoter, a drosophila actin 5c gene (Ac5) promoter, a tetracycline-inducible (TRE) promoter, and a combination thereof.

[0036] In some embodiments, the cell is a HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a Sf9 cell, a S2 cell, a MDCK cell, or a derivative thereof.

[0037] In some embodiments, the PSME1 / PA28A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 1-3, 11, or 17. In some embodiments, the PSME2 / PA28B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 7, 12, or 18. In some embodiments, the PSME3 / PA28G comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 9, 13, or 19.

[0038] In some embodiments, the vector further comprises a polyadenylation (poly A) site, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an optimized woodchuck hepatitis virus post-transcriptional regulatory element (oPRE), an intron, a microRNA binding motif, or a combination thereof. In some embodiments, the polyA site is selected from the group consisting of a beta-globin polyA, a bovine growth hormone polyA, a human growth hormone polyA, and a SV40 intermediate early polyA. In some embodiments, the vector further comprises a promoter operably linked to one or more selectable markers. In some embodiments, the one or more selectable markers is a resistance gene selected from the group consisting of neomycin / kanamycin, puromycin, hygromycin, and blasticidin.

[0039] In some embodiments of the above methods, the one or more cells or the population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or population of cells that does not comprise the vector. In some embodiments, the one or more cells or population of cells exhibits an at least 1.5- fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or population of cells that does not comprise the vector.

[0040] In some embodiments of the above methods, the one or more cells or population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the heterologous protein levels as compared to a cell or population of cells that does not comprise the vector. In some embodiments, the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the heterologous protein levels as compared to a cell or population of cells that does not comprise the vector.

[0041] In one aspect, disclosed herein is a method for increasing protein production in one or more cells or a population of cells, the method comprising contacting the one or more cells or cells of the population of cells with a site-directed modifying polypeptide complexed with a guide RNA (gRNA) that targets the PSME1, PSME2, and / or PSME3 genes. In some embodiments, the site-directed modifying polypeptide comprises a nuclease, for example a DNA endonuclease such as Cas9, Casl2, or a TALE nuclease. In some embodiments, the site-directed modifying polypeptide is a base editor, for example a cytosine base editor or an adenine base editor. In some embodiments, the gRNA targets a region of the PSME1, PSME2, and / or PSME3 genes that results in an upregulation of PSME1, PSME2, and / or PSME3 levels.

[0042] In some embodiments, the one or more cells or cells of the population of cells further comprises one or more nucleic acids to produce one or more functional AAV capsids. In some embodiments, the one or more nucleic acids encode an AAV capsid protein, a function rep gene, a recombinant AAV vector comprising AAV inverted terminal repeats and a transgene, and a helper protein. In some embodiments, the AAV capsid protein has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DJ / 8 and AAV2.7m8.

[0043] In some embodiments of the above methods, the one or more cells or cells of the cells of the population of cells further comprises one or more nucleic acids to produce one or more functional lentivirus particles. In some embodiments, the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and an envelope plasmid.

[0044] In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises one or more nucleic acids to produce one or more live attenuated influenza particles. In some embodiments, the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and one or more envelope plasmids, wherein performance of the methods results in increased production of the one or more live attenuated influenza particles. In some embodiments of the above methods, the one or more cells or cells of the population of cells further comprises a promoter operably linked to a nucleic acid encoding a heterologous protein, such as a GPCR, an ion channel, an enzyme, a G protein, an intracellular protein, a secreted growth factor, an integral membrane protein, a nanobody, or a monoclonal antibody, wherein performance of the methods results in increased production of the heterologous proteins.

[0045] In some embodiments of the above methods, the one or more cells or population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or population of cells that was not contacted with the site-directed modifying polypeptide. In some embodiments, the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or a population of cells that was not contacted with the site-directed modifying polypeptide.

[0046] In some embodiments of the above methods, the one or more cells or population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the heterologous protein levels as compared to a cell or a population of cells that was not contacted with the site-directed modifying polypeptide. In some embodiments, the one or more cells or population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the heterologous protein levels as compared to a cell or population of cells that was not contacted with the site-directed modifying polypeptide.

[0047] Brief Description of the Drawings

[0048] FIG. 1A-1F shows the effect of expression of PSME1 or PSME1 / PSME2 in HEK293T cells on fluorescent protein levels. FIG. 1A provides images of cells expressing ZsGreen and mKate with CMV promoters. FIG. IB provides images of cells expressing Zs-Green with PSME1 and mKate with CMV promoters. FIG. 1C provides images of cells expressing PSME1 / PSME2 and mKate with CMV promoters. FIG. ID provides images of cells expressing PSME1 / PSME2 with an EFlatandem promoter and mKate with a CMV promoter. FIG. 1E-F show the quantification of the cells from FIGS. 1A-D for ZsGreen (FIG. IE) and mKate (FIG. IF)

[0049] FIG. 2A-2F shows the effect of expression of PSME1 or PSME1 / PSME2 in HEK293T cells on fluorescent protein levels. FIG. 2A provides images of cells expressing ZsGreen and mKate with CMV promoters. FIG. 2B provides images of cells expressing Zs-Green with PSME1 and mKate with CMV promoters. FIG. 2C provides images of cells expressing PSME1 / PSME2 and mKate with CMV promoters. FIG. 2D provides images of cells expressing PSME1 / PSME2 with an EFlatandem promoter and mKate with a CMV promoter. FIG. 2E-F show the quantification of the cells from FIGS. 2A-D for ZsGreen (FIG. 2E) and mKate (FIG. 2F).

[0050] FIG. 3A-3C show the effects of expression of PSME1 or PSME1 / PSME2 on proteasome activity. FIG. 3A provides the proteasome activity as a measure of relative fluorescence units over 30 minutes. FIG. 3B is a bar graph depicting the normalized slope of the curves as shown in FIG. 3A. FIG. 3C is a Western Blot demonstrating that PSME1 and PSME2 are expressed in the HEK293T cells.

[0051] FIG. 4A-4B show the increased expression of mKate when co-transfected with plasmids expressing PSME1 / 2. FIG. 4A provides a representative Western Blot detecting mKate in each of the tested cells. FIG. 4B shows the relative protein levels for each of the tested promoters.

[0052] FIG. 5 provides a representative Western Blot demonstrating that PSME1 and PSME2 can be expressed using a tetracycline-inducible promoter system.

[0053] FIG. 6A-6D demonstrate that a stable HEK293T cell line was created and used to increase protein levels. FIG. 6A is a representative Western Blot showing that the stable cells constitutively express PSME1 and PSME2. FIG. 6B provides representative fluorescent images of the parental cells (left) and stable cells (right) for expression of ZsGreen. FIG. 6C provides a representative Western Blot demonstrating that the stable cell line results in increased protein levels of two heterologous-expressed proteins. FIG. 6D provides the quantification of the Western Blot shown in FIG. 6C.

[0054] FIG. 7A-7C show the increased expression of mKate in HEK293T cells when cotransfected with a tetracycline-inducible plasmid expressing PSME3. FIG. 7A provides a representative Western Blot detecting PSME3 in each of the tested cells. FIG. 7B graphically depicts PSME3 expression levels detected in HEK293T cell lysate relative to control cells with constitutive mKate2 expression. FIG. 7C graphically depicts mKate2 fluorescence levels detected in HEK293T cell lysate relative to control cells with constitutive mKate2 expression.

[0055] FIG. 8A-8C show the increased expression of mKate in CH0-K1 cells when cotransfected with a tetracycline-inducible plasmid expressing PSME3. FIG. 8A provides a representative Western Blot detecting PSME3 in each of the tested cells. FIG. 8B graphically depicts PSME3 expression levels detected in CH0-K1 cell lysate relative to control cells with constitutive mKate2 expression. FIG. 8C graphically depicts mKate2 fluorescence levels detected in HEK293T cell lysate relative to control cells with constitutive mKate2 expression.

[0056] Detailed Description

[0057] Disclosed herein are cell compositions for increased protein production and methods of use thereof. In order that the present disclosure can be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this disclosure. The word “about” if not otherwise defined means ± 5%. It is also to be noted that as used herein and in the claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise.

[0058] Definitions

[0059] As used herein, the term “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably. These terms refer to the primary structure of the molecule, and thus include double- and single-strained DNA, and double- and single-strained RNA. The nucleic acids of the instant disclosure can be made recombinantly, enzymatically, or synthetically using methods known in the art.

[0060] As used herein, the term “Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in the reference to a nucleic acid / polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purposes of determining percent nucleic acid or amino acid sequence can be achieving using methods known in the art, for example using publicly available software or algorithms.

[0061] As used herein, the term “promoter” refers to a DNA sequence recognized by the machinery of a cell, or introduced synthetic machinery, required to initiate the specific transcription of a coding sequence, such as a gene. The term “promoter” is also meant to encompass nucleic acid elements sufficient for promoter-dependent gene expression controllable for cell-type specific, tissue-type specific, or inducible by external signals or agents. Such elements can be located in the 5’ or 3’ regions of the native gene. In some embodiments, the promoter is constitutively active, active in specific cell types (e.g., a cell-type specific promoter), or an inducible promoter.

[0062] A “coding sequence” or a nucleic acid sequence “encoding” a particular molecule (e.g., PSME1 or PSME2) is a nucleic acid that is transcribed (DNA) or translated (mRNA) into a polypeptide when operably linked to a suitable regulatory sequence (e.g, a promoter). The boundaries of the coding sequence are a start codon at the 5’ terminus and a translation start codon at the 3’ terminus. A transcription termination sequence is typically located 3’ to the coding sequence.

[0063] As used herein, the term “enhancer” is a cis-acting element that stimulates or inhibits transcription of adjacent genes. An enhancer that inhibits transcription is also referred to as a silencer. Enhancers can function in either orientation and over distances of up to several kilobases from the coding sequence.

[0064] As used herein, a “transcriptional regulatory protein” or “transcriptional regulatory factor” or “transcriptional regulatory element” is a nuclear protein that binds a DNA response element and thereby regulates the expression of an associated gene(s).

[0065] As used herein, the term “regulatable promoter” is any promoter whose activity is affected by a cis- or trans-acting factor (e.g., an inducible promoter).

[0066] As used herein, the term “constitutive promoter” is any promoter that directs RNA production in many or all cell types at most times {e.g., the human CMV promoter).

[0067] As used herein, the term “termination signal sequence” can be any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation sequence. A polyadenylation signal sequence is a recognition region necessary for endonuclease cleavage of an RNA transcript that is followed by the polyadenylation consensus sequence AATAAA. This signal sequence provides a “poly A” site on an RNA transcript to which adenine residues will be added by post-transcriptional polyadenylation.

[0068] As used herein, the term “operably linked” refers to a DNA sequence and a regulatory sequence(s) connected in such a way as to permit gene expression when the appropriate molecules are bound to the regulatory sequence(s).

[0069] The term “expression construct” refers to a genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed (e.g., expressed).

[0070] The term “cistron” refers to a nucleic acid construct sufficient for expression of a gene product. In some aspects, a cistron is an expression construct. In some embodiments, the nucleic acid construct comprises two or more cistrons (e.g., multicistronic or bicistronic). In some embodiments, the nucleic acid construct is bicistronic.

[0071] As used herein, the term “vector” refers to any vehicle for the transfer of a nucleic acid (e.g., for cloning or transfer into a host cell). In some embodiments, the vector can be a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, amongst others. The term “vector” includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo, or in vivo. Insertion of a polynucleotide into a suitable vector can be accomplished using methods known in the art.

[0072] In some aspects of the disclosure, a vector may comprise an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. As used herein, the term “adeno-associated viral vector” or “AAV vector” refers to any vector that comprises components of an AAV vector and is suitable to infect mammalian cells, including but not limited to human cells. The term AAV vector typical designates an AAV-type particle or virion. The AAV vector can be derived from various serotypes, including combinations of serotypes, or from various genomes (e.g., single stranded or self-complementary). In addition, the AAV vector can be replication defective and / or targeted. The AAV serotypes useful for the cell compositions and methods disclosed herein include, but are not limited, to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DJ / 8 and AAV2.7m8. In some embodiments of the disclosure, a cell comprises one or more nucleic acids to produce functional AAV capsids (or AAV particles, used interchangeably).

[0073] As used herein, the term “transfection” refers to methods to introduce exogenous one or more nucleic acids into a cell. Methods of transfection are known in the art and include, but are not limited to, chemical methods or physical treatments.

[0074] As used herein, the term “level” refers to a level or activity or a protein, or mRNA encoding the protein, optionally as compared to a reference. The reference can be any useful reference as determined by one of ordinary skill in the art. An “increased level” of a protein or mRNA encoding a protein refers to an increase in the level of a protein or mRNA encoding a protein as compared to a reference. For example, the reference may be one-half of the amount of protein produced in an unmodified cell. As another example, the reference may be a predetermined typical or average level observed in unmodified cells. These differences reflect experimental constraints in transient transfections where half of the transfected DNA is dedicated to the protein of interest (often proxied herein as a fluorescent protein) and the other half of the transfected DNA dedicated to the PSME(l / 2 / 3)-containing plasmid. Hence, it is reasonable to compare protein expression when half of the DNA encodes a protein of interest and half is an empty vector to ensure similar transfections, while maintaining the ability to assess the contribution of PSME( 1 / 2 / 3 )-containing plasmids to enhance protein expression transiently. Constructs of Proteasome Regulatory Subunits

[0075] Proteasomes are multi-subunit complexes assembled upon association of two principal components: the 20S core and the regulatory caps. The 20S core is responsible for the proteolytic process, however its basal activity is very low in the absence of the regulatory caps. The 1 IS cap facilitates protein degradation in a ubiquitin-independent manner. Binding of the 11 S cap to the 20S core facilitates substrate access and confines the proteolytic activity to the 20S unit. The 1 IS cap comprises a hexameric ring. Normally, the 1 IS cap is a heterohexameric ring consisting of PA28A (also referred to as PSME1, used interchangeably herein) and PA28B (also referred to as PSME2, used interchangeably herein). However, the 1 IS cap is functional as a homohexameric ring consisting of PA28A alone. In addition, the 1 IS cap is functional as a homohexameric ring consisting of PA28G (also referred to as PSME3, used interchangeably herein).

[0076] Misfolded proteins and / or proteins with intrinsically disordered domains are preferentially degraded by the 20S proteasome comprising the 1 IS cap. The methods and compositions disclosed herein are based in part on the discovery that increased expression of the 11 S cap results in elevated levels of certain proteins. Without wishing to be bound by any particular theory, it is believed that proteins comprising stable tertiary and / or quaternary structures will have elevated expression levels in the methods and compositions of the instant disclosure. Representative proteins include therapeutic proteins, such as modified viruses, antibodies, enzymes, etc., and more generally, any protein conferring a desirable trait.

[0077] Accordingly, in one aspect disclosed herein are methods of increasing protein production in a cell by transfecting the cell with a vector that comprises a promoter operably linked to a nucleic acid sequence encoding PSME1 / PA28A. In some embodiments, the vector further comprises a promoter operably linked to a nucleic acid sequence encoding PSME2 / PA28B. In some embodiments, the nucleic acid sequences encoding PSME1 and PSME2 are on the same vector (i.e., a bicistronic vector). In some embodiments, the nucleic acid sequences encoding PSME1 and PSME2 are on separate vectors.

[0078] Throughout this disclosure, reference is made to PSME1. It is understood that additional names in the art exist for PSME1 and are encompassed in the present disclosure, including PA28A, IFI5111, REGa, PA28a, and HEL-S- 129m. Throughout this disclosure, reference is made to PSME2. It is understood that additional names in the art exist for PSME2 and are encompassed in the present disclosure, including PA28B, REGP, and PA280.

[0079] Throughout this disclosure, reference is made to PSME3. It is understood that additional names in the art exist for PSME3 and are encompassed in the present disclosure, including Ki, PA28G, HEL-S-283, PA28y, and REGy.

[0080] In humans, the PSME1 gene is located on chromosome 14 (bases 24,136,194 to 24,138,962; NCBI Reference Sequence NC_000014.9). PSME1 has three isoforms that are produced by alternative splicing (SEQ ID NOs 1-3, respectively).

[0081] In some embodiments, the PSME1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some aspects, the PSME1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encoding PSME1 comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0082] In humans, the PSME2 gene is located on chromosome 14 (bases 24,143,365 to 24,146,646; NCBI Reference Sequence NC_000014.9). In some embodiments, the PSME2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the nucleic acid sequence encoding PSME2 comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8.

[0083] In humans, the PSME3 gene is located on chromosome 17 (bases 42,833,397 to 42,843,760; NCBI Reference Sequence NC_000017.11). In some embodiments, the PSME3 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the nucleic acid sequence encoding PSME3 comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10. In some embodiments, the PSME1, PSME2, and / or PSME3 amino acid sequences may comprise one or more amino acid substitutions, additions, or deletions that are known in the art. For example, but not by way of limitation, in some embodiments, the PSME3 amino acid sequence may have a deleted nuclear localization sequence, corresponding to the deletion of amino acids 82-90 (SEQ ID NO: 29). In some embodiments, the PSME3 amino acid sequence may have a K188E substitution (SEQ ID NO: 30).

[0084] In some embodiments, the PSME1, PSME2, and / or PSME3 are derived from non-human sources. For example, the PSME1, PSME2, and / or PSME3 amino acid sequence may comprise the Chinese hamster (Cricetulus griseus) PSME1 (SEQ ID NO: 11), PSME2 (SEQ ID NO: 12), and / or PSME3 (SEQ ID NO: 13). In some embodiments, the nucleic acid sequence encoding the PSME1, PSME2, and / or PSME3 may comprise the Chinese hamster PSME1 (SEQ ID NO: 14), PSME2 (SEQ ID NO: 15), and / or PSME3 (SEQ ID NO: 16). In yet other embodiments, the PSME1, PSME2, and / or PSME3 amino acid sequence may comprise the mouse (Mus musculus) PSME1 (SEQ ID NO: 17), PSME2 (SEQ ID NO: 18), and / or PSME3 (SEQ ID NO: 19). In some embodiments, the nucleic acid sequence encoding the PSME1, PSME2, and / or PSME3 may comprise the mouse PSME1 (SEQ ID NO: 20), PSME2 (SEQ ID NO: 21), and / or PSME3 (SEQ ID NO: 22). In yet other embodiments, the PSME1, PSME2, and / or PSME3 amino acid sequence may comprise the dog (Canis lupus familiaris') PSME1 (SEQ ID NO: 23), PSME2 (SEQ ID NO: 24), and / or PSME3 (SEQ ID NO: 25). In some embodiments, the nucleic acid sequence encoding the PSME1, PSME2, and / or PSME3 may comprise the dog PSME1 (SEQ ID NO: 26), PSME2 (SEQ ID NO: 27), and / or PSME3 (SEQ ID NO: 28).

[0085] In one aspect, the constructs disclosed herein may be used to increase the expression of PSME1, PSME1 / PSME2, or PSME3 in a eukaryotic cell. As such, the constructs disclosed herein comprise a promoter to facilitate expression of the nucleic acid sequences within a certain cell type and / or tissue. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a cell type specific promoter. In some embodiments, the promoter is selected from the group consisting of a GUSB promoter, a SFFV promoter, a CBh promoter, a CBA promoter, a PGK promoter, a CMV promoter, a CAG promoter, a bidirectional CAG promoter, a CBA promoter, a human ubiquitin C promoter, an EFla promoter, a AcMNPV polyhedrin promoter, a AcMNPV plO promoter, a White spot syndrome virus (WSSV) iel promoter, a drosophila metallothionein (MT) promoter, a drosophila actin 5c gene (Ac5) promoter, and / or a combination thereof. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is an EFla promoter.

[0086] In some embodiments, the inducible promoter is a tetracycline inducible promoter that drives the expression of PSME1, PSME2, and / or PSME3. A tetracycline inducible promoter may comprise a constitutive tTS element, a fusion of a hybrid tetracycline repressor TetR with a nuclear localization signal and a KRAB silencing domain, and an rtTA element, a VP16 minimal transcriptional activation domain linked to a tetracycline-controlled transactivator. The tTS and rtTA elements may be linked by a 2A sequence to generate tetracycline or doxycycline responsive expression of PSME1 / 2 or PSME3 to enhance recombinant protein or viral production in cells.

[0087] Constitutive expression of PSME1 / 2, bidirectional or in tandem, or PSME3 may be further regulated by tetracycline-responsive aptazymes at the translational level. Said aptazymes are further described in Vogel et al., “A small, portable RNA device for the control of exon skipping in mammalian cells. Nucleic Acids Res (2018) 46(8):e48. A tetracycline-binding aptamer will skip a BGL2 intron in the presence of tetracycline to allow expression of PSME1 or PSME3.

[0088] Tetracycline-inducible PSME1 or PSME3 expression was achieved by the insertion of a K19 aptazyme into the 3’UTR. The aptazyme is the combination of a ribozyme that causes degradation of upstream mRNA and an RNA aptamer that interferes with ribozyme function in the presence of tetracycline, thereby permitting expression of the upstream coding sequence. In addition, this aptazyme mechanism can be combined with a tetracycline-inducible exon-skipping mechanism, which further increases the ON-OFF dynamic range of expression. The exonskipping mechanism involves the insertion into Cre recombinase of a synthetic exon flanked by BGL2 and Dhfr introns and the placement of an M2 aptamer that blocks an RNA splice site. In the presence of tetracycline, the M2 aptamer changes conformation, the splice site becomes functional, and the synthetic exon and flanking introns are removed, enabling the expression of Cre recombinase. The Cre DNA is in turn flanked by LoxP sites and is positioned upstream of the gene of interest, e.g., PSME1 or PSME3, thereby blocking that gene’s expression. Thus, the tetracycline-induced Cre protein can then excise the Cre DNA and enable the expression of the gene of interest. As is known in the art, induction of a tetracycline-inducible promoter may be achieved by treating a cell or a population of cells with a sufficient amount of tetracycline or doxycycline.

[0089] In some embodiments, the constructs may further comprise one or more additional elements, including but not limited to, a termination signal sequence (e.g., a polyadenylation (poly A) site), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an optimized woodchuck hepatitis virus post-transcriptional regulatory element (oPRE), an intron, a microRNA binding motif, or a combination thereof.

[0090] In some embodiments, the polyA site is a beta-globin poly A, a bovine growth hormone poly A, a human growth hormone polyA, an SV40 late polyA, or a SV40 intermediate early polyA.

[0091] In one aspect, the construct comprising one or more nucleic acids encoding a PSME is a bicistronic construct (e.g., PSME1 and PSME2). In some embodiments, the bicistronic construct comprises a bi-directional promoter. In some embodiments, the bicistronic construct comprises tandem expression constructs each with a promoter, a transgene encoding PSME1 or PSME2 and polyA. In some embodiments, the construct further comprises a cleavage element between an expression construct comprising a nucleic acid sequence encoding PSME1 and an expression construct comprising a nucleic acid sequence encoding PSME2. In some embodiments, the cleavage element comprises a nucleic acid sequence encoding a self-cleavage peptide. In some embodiments, the cleavage peptide is a 2A self-cleaving peptide (P2A, T2A, etc.).

[0092] In some embodiments, the bicistronic construct comprises an expression construct comprising a promoter operably linked to a polynucleotide encoding PSME1 and an expression construct comprising a promoter operably linked to a polynucleotide encoding PSME2. In some embodiments, the expression constructs comprise the same promoter. In some embodiments, the expression constructs comprise different promoters. In some embodiments, the promoter is a CMV promoter and / or an EFla promoter. In some embodiments, the expression constructs further comprise a polyA site. In some embodiments, the expression constructs further comprise a WPRE. The expression constructs provided herein can be positioned within a vector in different ways. For example, a first expression construct e.g., encoding for PSME1) may be positioned adjacent to a second expression construct (e.g., encoding for PSME2). In some embodiments, a polynucleotide encoding PSME1 and a polynucleotide encoding PSME2 are operably linked to a bidirectional promoter. Compositions of expression constructs for PSME1 and PSME1 / PSME2, and orientations thereof, are further described in W02023 / 004365, incorporated herein by reference.

[0093] In some embodiments, the bicistronic construct comprises an expression construct comprising a CMV promoter operably linked to a polynucleotide encoding PSME1 and an expression construct comprising a CMV promoter operably linked to a polynucleotide encoding PSME2. In some embodiments, a polynucleotide encoding for K19 aptazyme is inserted into the 3’ UTR of the expression construct comprising a CMV promoter operably linked to the polynucleotide encoding PSME1. In some embodiments, the bicistronic construct further comprises a polynucleotide encoding a kanamycin resistance gene and / or a hygromycin resistance gene. In some embodiments, the bicistronic construct further comprises a SV40 poly (A) signal.

[0094] In some embodiments, the bicistronic construct comprises an expression construct comprising an inducible bi-directional TRE promoter operably linked to a polynucleotide encoding PSME1 and a polynucleotide encoding PSME2. The construct further comprises one or more polynucleotides encoding the necessary proteins for tetracycline induction. Said elements may comprise a constitutive tTS element, a fusion of a hybrid tetracycline repressor TetR with a nuclear localization signal and a KRAB silencing domain, and an rtTA element, a VP 16 minimal transcriptional activation domain linked to a tetracycline-controlled transactivator. The tTS and rtTA elements may be linked by a 2A sequence to generate tetracycline or doxycycline responsive expression of PSME1 and PSME2. Additionally or alternatively, the 3’UTR of the polynucleotide encoding PSME1 may further comprise a KI 9 aptazyme sequence.

[0095] In some embodiments, the construct comprises an expression construct comprising an inducible TRE promoter operably linked to a polynucleotide encoding PSME3. The construct further comprises one or more polynucleotides encoding the necessary proteins for tetracycline induction. Said elements may comprise a constitutive tTS element, a fusion of a hybrid tetracycline repressor TetR with a nuclear localization signal and a KRAB silencing domain, and an rtTA element, a VP16 minimal transcriptional activation domain linked to a tetracycline- controlled transactivator. The tTS and rtTA elements may be linked by a 2A sequence to generate tetracycline or doxycycline responsive expression of PSME3. Additionally or alternatively, the 3’UTR of the polynucleotide encoding PSME3 may further comprise a K19 aptazyme sequence. Any of the constructs expressing PSME1, PSME1 and PSME2, or PSME3 may be cotransfected with a vector comprising a polynucleotide encoding for a protein of interest, thereby resulting in a cell comprising a construct expressing PSME1, PSME 1 and PSME2, or PSME3, and a construct expressing a protein of interest. For example, but not by way of limitation, a vector may comprise an inducible TRE promoter operably linked to a polynucleotide encoding for a protein of interest.

[0096] In any of the above embodiments, the construct or bicistronic construct expressing PSME1, PSME1 and PSME2, or PSME3, may further comprise a polynucleotide encoding for one half of a puromycin resistance gene. Further, the vector expressing the protein of interest may further comprise for the second half of the puromycin resistance gene. When co-transfected into a cell, cells containing both the construct of bicistronic construct expressing PSME1, PSME1 and PSME2, or PSME3, and the vector expressing the protein of interest may be selected for using puromycin.

[0097] In one aspect, a first vector comprises a promoter operably linked to a polynucleotide encoding PSME1, and a second vector comprises a promoter operably linked to a polynucleotide encoding PSME2, In some embodiments, the expression constructs on the first and second vector comprise the same promoter. In some embodiments, the expression constructs on the first and second vector comprise different promoters. In some embodiments, the expression constructs further comprise a polyA site. In some embodiments, the expression constructs further comprise a WPRE. In other embodiments, a promoter is operably linked to a polynucleotide encoding PSME1 and PSME2 in a single open reading frame, wherein a high-efficiency cleavage site is positioned between the PSME1 and PSME2. As used herein, high-efficiency cleavage is defined as when more than 50%, more than 70%, more than 80%, or more than 90% of the translated protein is cleaved. Cleavage efficiency can be measured using methods known in the art, such as via Western Blot analysis.

[0098] In some embodiments, the constructs disclosed herein may include other components such as a selectable marker to aid in the selection of cells containing the construct, an origin of replication for the stable replication of the construct in a bacterial cell, a nuclear localization signal, or other elements which facilitate production of the DNA construct, the protein encoded thereby, or both. In some embodiments, the selectable marker is neomycin / kanamycin, puromycin, hygromycin, and blasticidin.

[0099] In some embodiments, the eukaryotic cell is HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a Sf9 cell, a S2 cell, a MDCK cell, or a derivative thereof.

[0100] In one aspect, the constructs disclosed herein may be integrated into the genome of a cell to produce a stable cell line. In one aspect, a cell comprising one or more constructs disclosed herein may be immortalized to produce a stable cell line. Methods for producing an immortalized cell are known in the art and described in, for example, US Patent No. 8,071,377.

[0101] An exemplary bicistronic construct of the instant disclosure comprises an EFla promoter operably linked to a polynucleotide encoding PSME1 and a BGH polyA site and an EFla promoter operably linked to a polynucleotide encoding PSME2 and a SV40 late polyA site. The construct further comprises a CMV promoter operably linked to a neomycin selectable marker and a BGH polyA site.

[0102] Constructs of the instant disclosure can be transfected into a cell using methods known in the art. A cell comprising said construct(s) can be used in the methods provided herein.

[0103] Methods for Increasing Protein Production

[0104] The disclosed cell compositions afford increased levels of proteins, including heterologous proteins. In one aspect, disclosed herein are methods for increasing protein production using a cell comprising a construct of the instant disclosure. In some embodiments, the construct results in the overexpression of PSME1. In some embodiments, the construct results in the overexpression of PSME1 and PSME2. In some embodiments, the construct results in the overexpression of PSME3.

[0105] As shown in Example 1 and FIGS. 1A-1F and FIGS. 2A-2F, cells that overexpress PSME1 or PSME1 / 2 result in increased levels of a heterologous protein e.g., a fluorescent protein). As such, cells comprising the constructs of the instant disclosure can be used to increase protein levels of a heterologous protein introduced into the cell (or a native protein of interest).

[0106] As shown in Example 2 and FIGS. 3A-3C, cells that overexpress PSME1 and PSME2 exhibit increased proteasome activity. Example 3 and FIGS. 4A-4B further demonstrate that cells that overexpress PSME1 and PSME2 result in increased levels of a co-expressed heterologous protein. Example 4 and FIG. 5 demonstrates that expression of PSME1 and PSME2 can be achieved using an inducible promoter, such as a tetracycline inducible promoter.

[0107] Example 5 and FIGS. 6A-6D show that a stable cell line expressing PSME1 and PSME2 can be generated. Said stable cell line can be used to increase levels of a heterologous or native protein.

[0108] Example 6 and FIGS. 7A-7C and FIGS. 8A-8C demonstrate that expression of PSME3 can be achieved using an inducible promoter, such as a tetracycline inducible promoter. Induced expression of PSME3 resulted in a 6-10X increase in levels of a co-expressed heterologous protein.

[0109] In some embodiments, a cell further comprises, in addition to a construct that results in overexpression of PSME1, PSME1 / 2, or PSME3, one or more nucleic acids encoding an AAV capsid protein, a function rep gene, a recombinant AAV vector comprising AAV inverted terminal repeats and a transgene, and a helper protein, such that a functional AAV capsid is produced. Said cells would result in increased levels of functional AAV capsids. Methods for preparing cells capable of producing AAV capsids (i.e., AAV particles) are well known in the art, and described in US Patent Publication No. 2003 / 0138772 and W02023 / 004365, the entireties of which are incorporated herein by reference.

[0110] In some embodiments, a cell further comprises, in addition to a construct that results in overexpression of PSME1, PSME1 / 2, or PSME3, one or more nucleic acids encoding a transfer plasmid, one or more packaging plasmids, and an envelope plasmid, such that a functional lentivirus particle is produced. Said cells would result in increased levels of functional AAV capsids. Methods for preparing cells capable of producing lentivirus are described in US Patent No. 10,954,530 and US Patent No. 10,993,999, incorporated herein by reference.

[0111] In some embodiments, the cell further comprises, in addition to a construct that results in overexpression of PSME1, PSME1 / 2, or PSME3, one or more nucleic acids encoding a heterologous protein. Said cells would result in increased levels of the heterologous protein.

[0112] In one aspect, the cell compositions can result in an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% or more increase in one or more heterologous proteins (including, for example, AAV capsids) as compared to a reference level.

[0113] In one aspect, the cell compositions can result in an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35- fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in one or more heterologous proteins (including, for example, AAV capsids) as compared to a reference level. The reference level may refer to either a transient transfection of just the protein of interest or a transient transfection of one-half DNA encoding the protein of interest plus one-half of the DNA being an empty, non-expressing plasmid. This is to ensure similar levels of transfection efficiency while not biasing the results with unequal levels of DNA encoding the protein of interest.

[0114] In some embodiments, the reference level is derived from a cell without a construct that overexpresses PSME1, PSME1 and PSME2, or PSME3. In some embodiments, the reference level is derived from a cell that does not comprise a heterologous nucleic acid of PSME1, a heterologous nucleic acid of PSME1 and PSME2, or a heterologous nucleic acid of PSME3. In some embodiments, the reference level is the amount of protein produced in a cell that does not comprise a heterologous nucleic acid of PSME1, a heterologous nucleic acid of PSME1 and PSME2, or a heterologous nucleic acid of PSME3, wherein the amount is divided in half. In other embodiments, the reference level is a predetermined typical amount for an unmodified host cell grown in analogous conditions. Methods of measuring proteins are well known in the art and include, but are not limited to, Western Blot or quantitative mass spectrometry.

[0115] While not wishing to be bound to any particular theory, the method of increasing protein production in one or more cells or in a population of cells, as described herein, may be a result of increased protein production in singular cells, and / or increased protein production in a population of cells as a result of increased growth density or other factors. Examples

[0116] Example 1: Overexpression of PSME1 and PSME1 / PSME2 Increases Levels of Fluorescent Proteins

[0117] The effect of overexpressing PSME1 or PSME1 / 2 on the levels of heterologous proteins (e.g., fluorescent proteins) was determined. 10 cm dishes of HEK293T cells (~3.5xl06cells I dish) were transfected using calcium phosphate with one of four constructs: 1) pBiCMV- ZsGreen + CMV-mKate, 2) pBiCMV-ZsGreen-PSMEl + CMV-mKate, 3) pBiCMV-PSME2- PSME1 + CMV-mKate, or 4) pAAV-EFlatandem-mPSMEl / 2 + CMV-mKate. Cells were cultured for 24 hours and imaged using fluorescence microscopy with an excitation / emission of 496 / 506 nm for ZsGreen and an excitation / emission of 588 / 635 nm for mKate. FIGS. 1A-1D provide representative images of cells transfected with each construct. 48 hours after transfection (e.g., 24 hours after imaging), cells were lysed and the lysate quantified using a plate reader. As shown in FIG. IE, expression of PSME1 resulted in a 7X increase in fluorescence (ZsGreen signal) as compared to the construct without PSME1. Similarly, as shown in FIG. IF, expression of PSME1 or PSME1 / 2 resulted in an up to 24X increase in fluorescence (mKate signal) as compared to the construct without PSME1 or PSME1 / 2.

[0118] The constructs described above were also transfected in HEK293 cells (lacking the large T antigen) using the methods described above. FIGS. 2A-D provide representative images of cells transfected with each construct. As shown in FIG. 2E, overexpression of PSME1 resulted in a 4.2X increase in fluorescence (ZsGreen signal) as compared to the construct without PSME1. Similarly, as shown in FIG. 2F, expression of PSME1 or PSME1 / 2 resulted in an up to 7.4X increase in fluorescence (mKate signal) as compared to the construct without PSME1 or PSME1 / 2.

[0119] Overexpression of PSME1 or PSME1 / 2 increases total levels of fluorescent proteins in both HEK293 and HEK293T cells.

[0120] Example 2: Overexpression of PSME1 / 2 Increases Proteasome Activity

[0121] The effect of overexpressing PSME1 or PSME1 / 2 on proteasome activity in cell lysates was assessed. HEK293T cells were transfected using calcium phosphate with one of four constructs: 1) pBiCMV-ZsGreen (Control); 2) pBiCMV-ZsGreen-PSMEl; 3) pBiCMV-PSME2- PSME1; or 4) EFla-PSMEl / 2. Cell lysates of the transformed HEK293T cells was collected. Proteasome activity was measured by monitoring Suc-LLVY-AMC cleavage (chymotrypsin-like proteasome activity) via fluorescence of free AMC (excitation / emission 350 / 440 nm). RFU slopes were normalized to background proteasome activity in HEK293T cells transfected with the BiCMV-ZsGreen. FIG. 3A shows the proteasome activity observed over 30 minutes for each of the transformed HEK293T cells. Cell lysate from HEK293T cells expressing both PSME1 and PSME2 (diamond and inverted triangle) exhibited significant enhancement of proteasome activity relative to cell lysate from HEK293T control cells or cells only expressing PSME1. FIG. 3B shows the normalized slope for each of the tested cell lysates. FIG. 3C demonstrates that the pBi-CMV-PSME2-PSMEl results in the expression of PSME1 and PSME2 protein as determined by Western Blot.

[0122] Example 3: PSME1 / 2 Overexpression Increases Levels of Co-Expressed Proteins

[0123] The effect of PSME1 / 2 overexpression on the levels of a co-expressed protein, e.g., mKate fluorescent protein, was determined. HEK293T cells were co-transfected with either pBiCMV-PSME2-PSMEl or pBi-CMV-ZsGreen and mKate operably linked to one of 5 promoters - CAG, EFla, SV40, PGK, or UBC (see FIG. 4A). Following transfection, protein levels of mKate were determined using Western Blot, with GAPDH as a loading control. As shown in FIG. 4A, mKate expression was increased when co-expressed with PSME1 / 2 regardless of promoter used. FIG. 4B provides relative mKate protein levels in cells expressing PSME1 / 2 vs cells not expressing PSME1 / 2 as measured across 4 independent experiments.

[0124] Example 4: Tetracycline-Inducible Expression of PSME1 / 2

[0125] The ability to express PSME1 / 2 using a tetracycline-inducible promoter was determined. A tetracycline inducible promoter was used to co-express PSME1 and PSME2 in HEK293T cells. FIG. 5 demonstrates that PSME1 and PSME2 are only detected following induction with 100 ng / mL doxycycline. Thus, PSME1 and PSME2 expression are regulatable by a tetracyclineinducible promoter system.

[0126] Example 5: Generation of Stable Cell Lines Expressing PSME1 / 2

[0127] Stable cell lines were generated that constitutively express PSME1 / 2. HEK293T cells were co-transfected with the pBiCMV-PSME2-PSMEl vector and a hygromycin resistance gene. Cells with stable integration of the transgene were selected for with 200 pg / mL hygromycin over two weeks. Cells exhibited normal morphology but grew more slowly relative to the parental HEK293T cells. FIG. 6A demonstrates that the stable cell line expresses both PSME1 and PSME2 as detected by Western Blot. FIG. 6B provides representative fluorescence microscopy images of parental (left) or stable (right) cells, with the stable cells having enhanced expression of ZsGreen.

[0128] The stable cell line (PSME1 / 2 Stbl Cells) was tested for the ability to increase expression levels of Parrl or Parr 2 conjugated to a FLAG tag (Parrl-FLAG or Parr2-FLAG, respectively). The stable cell line expressing PSME1 / 2 was transfected with a plasmid constitutively expressing either the Parrl-FLAG or Parr2-FLAG constructs. Lysates were measured for protein levels by Western Blot. As shown in FIG. 6C and FIG. 6D, the stable cell line resulted in a 4- 14X increase in expression of the Parrl-FLAG and Parr2-FLAG constructs, respectively, as compared to the parental cells.

[0129] Example 6: Tetracycline-Inducible Expression of PSME3

[0130] The ability to express PSME3 using a tetracycline-inducible promoter was determined. A tetracycline inducible promoter (TRE) was used to co-express PSME3 and mKate2 fluorescent protein. HEK293T serum-free (SF) suspension cells were co-transfected with either pDriver (control plasmid) and TRE-mKate2, or pDriver- TRE-PSME3 and TRE-mKate2. As a control, HEK239T cells were transfected with a plasmid that constitutively expresses mKate2 (EFla- mKate2). The resultant cells were treated with or without 1 pg / mL of doxycycline to induce expression from the TRE promoter. mKate2 levels were measured by fluorescence at 588 / 635 nm excitation / emission. FIG. 7A demonstrates that PSME3 levels are increased when induced with doxycycline as measured by Western Blot. As shown in FIG. 7B, doxycycline resulted in induction of PSME3 expression by ~6X in cells transfected with pDriver-TRE-PSME3 and treated with 1 pg / mL doxycycline. FIG. 7C demonstrates that pDriver-TRE-PSME3 can drive 3- 4x greater mKate2 expression in a doxycycline-inducible manner than pDriver (control). The TRE-mKate2 expression levels are lower than constitutive mKate expression (EFla-mKate2), due to there being half of the mKate2 expressing DNA in the pDriver + TRE-mKate2 condition relative to the constitutive EFla-mKate2 control since half of the transfected DNA is dedicated to the pDriver and the TRE-mKate2 plasmids, respectively. Furthermore, EFla is a strong constitutive promoter.

[0131] The constructs were further tested in a CH0-K1 adherent cell line. CH0-K1 cells were co-transfected with either pDriver (control plasmid) and TRE-mKate2, or pDriver-TRE-PSME3 and TRE-mKate2. As a control, CH0-K1 cells were transfected with a plasmid that constitutively expresses mKate2 (EFla-mKate2). The resultant cells were treated with or without 1 pg / mL of doxycycline to induce expression of the TRE promoter. mKate2 levels were measured by fluorescence at 588 / 635 nm excitation / emission. FIG. 8A demonstrates that PSME3 levels are increased when induced with doxycycline as measured by Western Blot. As shown in FIG. 8B, doxycycline resulted in induction of PSME3 expression by ~10X on in cells transfected with pDriver-TRE-PSME3. FIG. 8C demonstrates that pDriver-TRE-PSME3 can drive ~4x greater mKate2 expression in a doxycycline-inducible manner than pDriver (control). In the pDriver- TRE-PSME3 + TRE-mKate2 + 1 pg / mL doxycycline condition, mKate2 expression levels are similar to the constitutive mKate expression (EFla-mKate2) levels, this is despite there being half of the mKate2 expressing DNA in the pDriver + TRE-mKate2 + 1 pg / mL doxycycline condition relative to the constitutive EFla-mKate2 control since half of the transfected DNA is dedicated to the pDriver and the TRE-mKate2 plasmids, respectively. Differences between the strength of the EFla promoter in HEK 293T and CH0-K1 cells may explain the differences in the results between the cell lines.

[0132] Table 1: Sequences

Claims

ClaimsWe claim:

1. A method for increasing protein production in one or more cells or in a population of cells, the method comprising transfecting the one or more cells or cells of the population of cells with a vector, the vector comprising a promoter operably linked to a nucleic acid encoding PSME1 (PA28A).

2. The method of claim 1, wherein the vector further comprises a promoter operably linked to a nucleic acid encoding PSME2 (PA28B).

3. A method for increasing protein production in one or more cells or in a population of cells, the method comprising transfecting the one or more cells or cell in the population of cells with a vector, the vector comprising a promoter operably linked to a nucleic acid encoding PSME3 (PA28G).

4. The method of any one of claims 1-3, wherein the one or more cells or the cells in the population of cells further comprises one or more nucleic acids to produce one or more functional AAV capsids.

5. The method of claim 4, wherein the one or more nucleic acids encode an AAV capsid protein, a function rep gene, a recombinant AAV vector comprising AAV inverted terminal repeats and a transgene, and a helper protein.

6. The method of claim 5, wherein the AAV capsid protein has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DJ / 8 and AAV2.7m8.

7. The method of any one of claims 1-3, wherein the one or more cells or the cells in the population of cells further comprises one or more nucleic acids to produce one or more functional lentivirus particles or live attenuated influenza particles.

8. The method of claim 7, wherein the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and an envelope plasmid.

9. The method of any one of claims 1-3, wherein the one or more cells or the cells in the population of cells further comprises a promoter operably linked to a nucleic acid encoding a heterologous protein, for example wherein the heterologous protein is a GPCR, an integral membrane protein, an enzyme, an intracellular protein, a nanobody, a monoclonal antibody.

10. The method of any one of claims 1-9, wherein the promoter is selected from the group consisting of a GUSB promoter, a SFFV promoter, a CBh promoter, a CBA promoter, a PGK promoter, a CMV promoter, a CAG promoter, a bidirectional CAG promoter, a CBA promoter, a human ubiquitin C promoter, an EFla promoter, a AcMNPV polyhedrin promoter, a AcMNPV plO promoter, a White spot syndrome virus (WSSV) iel promoter, a drosophila metallothionein (MT) promoter, a drosophila actin 5c gene (Ac5) promoter, a tetracycline inducible promoter (TRE), and a combination thereof.

11. The method of any one of claims 1-10, wherein the cell is a HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a Sf9 cell, a S2 cell, a MDCK cell, or a derivative thereof.

12. The method of any one of claims 1-2 and 4-11, wherein the PSME1 / PA28A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 1-3, 11, 17, or 23.

13. The method of any one of claims 2 and 4-11, wherein the PSME2 / PA28B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 7, 12, 18, or14. The method of any one of claims 3-11, wherein the PSME3 / PA28G comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 9, 13, 19, or 25.

15. The method of any one of claims 1-14, wherein the vector further comprises a polyadenylation (poly A) site, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an optimized woodchuck hepatitis virus post-transcriptional regulatory element (oPRE), an intron, a microRNA binding motif, or a combination thereof.

16. The method of claim 15, wherein the polyA site is selected from the group consisting of a beta-globin polyA, a bovine growth hormone polyA, a human growth hormone polyA, and a SV40 intermediate early polyA.

17. The method of any one of claims 1-16, wherein the vector further comprises a promoter operably linked to one or more selectable markers.

18. The method of claim 17, wherein the one or more selectable markers is a resistance gene selected from the group consisting of neomycin / kanamycin, puromycin, hygromycin, and blasticidin.

19. The method of claim 4 or 7, wherein the one or more cells or the population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the functional AAV capsid levels, lentivirus particle levels, or live attenuatedinfluenza particle levels as compared to a cell or population of cells that does not comprise the vector.

20. The method of claim 4 or 7, wherein the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50- fold or more increase in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or population of cells that does not comprise the vector.

21. The method of claim 9, wherein the one or more cells or the population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about2500% increase, at least about 3000% increase, at least about 3500% increase, at least about4000% increase, at least about 4500% increase, at least about 5000% increase or more in the heterologous protein levels as compared to a cell or population of cells that does not comprise the vector.

22. The method of claim 9, wherein the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the heterologous protein levels as compared to a cell or population of cells that does not comprise the vector.

23. A stable cell line for increased protein production, wherein the stable cell line overexpresses PSME1, for example wherein the stable cell line comprises a promoter operably linked to a heterologous nucleic acid encoding PSME1 (PA28A).

24. The stable cell line of claim 23, wherein the stable cell line overexpresses PSME2, for example wherein the stable cell line comprises a promoter operably linked to a heterologous nucleic acid sequence encoding PSME2 (PA28B).

25. A stable cell line for increased protein production, wherein the stable cell line overexpresses PSME3, for example wherein the stable cell line comprises a promoter operably linked to a heterologous nucleic acid sequence encoding PSME3 (PA28G).

26. The stable cell line of any one of claims 23-25, wherein the promoter is selected from the group consisting of a GUSB promoter, a SFFV promoter, a CBh promoter, a CBA promoter, a PGK promoter, a CMV promoter, a CAG promoter, a bidirectional CAG promoter, a CBA promoter, a human ubiquitin C promoter, an EFla promoter, a AcMNPV polyhedrin promoter, a AcMNPV plO promoter, a White spot syndrome virus (WSSV) iel promoter, a drosophila metallothionein (MT) promoter, a drosophila actin 5c gene (Ac5) promoter, a tetracycline inducible (TRE) promoter, and a combination thereof..

27. The stable cell line of any one of claims 23-26, wherein the cell is a HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a Sf9 cell, a S2 cell, a MDCK cell, or a derivative thereof.

28. The stable cell line of any one of claims 23-24 and 26-27, wherein the PSME1 / PA28A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 1-3, 11, 17, or 23.

29. The stable cell line of any one of claims 24 and 26-28, wherein the PSME2 / PA28B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 7, 12, 18, or 24.

30. The stable cell line of any one of claims 25-27, wherein the PSME3 / PA28G comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 9, 13, 19 or 25.

31. The stable cell line of any one of claims 23-30, wherein the polynucleotide further comprises a polyadenylation (poly A) site, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an optimized woodchuck hepatitis virus post-transcriptional regulatory element (oPRE), an intron, a microRNA binding motif, or a combination thereof.

32. The stable cell line of claim 31, wherein the polyA site is selected from the group consisting of a beta-globin polyA, a bovine growth hormone polyA, a human growth hormone polyA, and a SV40 intermediate early polyA.

33. The stable cell line of any one of claims 23-32, wherein the polynucleotide further comprises a promoter operably linked to one or more selectable markers.

34. The stable cell line of claim 33, wherein the one or more selectable markers is one or more resistance genes selected from the group consisting of neomycin / kanamycin, puromycin, hygromycin, and blasticidin.

35. A method of making the stable cell of any one of claims 23-34.

36. A method of making a stable cell line, the method comprising: a) obtaining the cell of any one of claims 1-22; and b) immortalizing the cell to thereby create a stable cell line.

37. A method for increasing protein production, the method comprising expressing a protein of interest in the stable cell line of any one of claims 23-34.

38. The method of claim 37, wherein the protein of interest is an AAV capsid, a lentivirus particle, a live attenuated influenza particle, or a heterologous protein, for example wherein the heterologous protein is a GPCR, an integral membrane protein, an enzyme, an intracellular protein, a nanobody, a monoclonal antibody.

39. The method of claim 38, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DJ / 8 and AAV2.7m8.

40. The method of any one of claims 35-39, wherein the cell of the stable cell line is a HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a Sf9 cell, a S2 cell, a MDCK cell, or a derivative thereof.

41. The method of any one of claims 37-40, wherein the protein of interest has an at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold or more increase in expression levels as compared to a stable cell line that does not overexpress PSME1, a stable cell line that does not express PSME1 and PSME2, and / or a stable cell line that does not overexpress PSME3; and / or as compared to a cell that does not comprise the heterologous nucleic acid of PSME1, the heterologous nucleic acid of PSME1 and PSME2, and / or the heterologous nucleic acid of PSME3.

42. The method of any one of claims 37-40, wherein the protein of interest has an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in expression levels as compared to a stable cell line that does not overexpress PSME1, a stable cell line that does not express PSME2, and / or a stable cell line that does not overexpress PSME3; and / or as compared to a cell that does not comprise the heterologous nucleic acid of PSME1, the heterologous nucleic acid of PSME2, and / or the heterologous nucleic acid of PSME3.

43. A method for increasing protein production in one or more cells or in a population of cells, the method comprising transfecting the one or more cells or cells in the population of cells with i) a vector comprising a promoter operably linked to a nucleic acid encoding PSME1 (PA28A), and ii) a vector comprising a promoter operably linked to a nucleic acid encoding PSME2 (PA28B).

44. The method of claim 43, wherein the one or more cells or the cells in the population of cells further comprises one or more nucleic acids to produce one or more functional AAV capsids.

45. The method of claim 44, wherein the one or more nucleic acids encode an AAV capsid protein, a function rep gene, a recombinant AAV vector comprising AAV inverted terminal repeats and a transgene, and a helper protein.

46. The method of claim 45, wherein the AAV capsid protein has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DJ / 8 and AAV2.7m8.

47. The method of claim 43, wherein the one or more cells or the cells in the population of cells further comprises one or more nucleic acids to produce one or more functional lentivirus particles or live attenuated influenza particles.

48. The method of claim 47, wherein the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and an envelope plasmid.

49. The method of any one of claims 43, wherein the one or more cells or the cells in the population of cells further comprises a promoter operably linked to a nucleic acid encoding a heterologous protein, for example wherein the heterologous protein is a GPCR, an integral membrane protein, an enzyme, an intracellular protein, a nanobody, a monoclonal antibody.

50. The method of any one of claims 43-49, wherein the promoter is selected from the group consisting of a GUSB promoter, a SFFV promoter, a CBh promoter, a CBA promoter, a PGK promoter, a CMV promoter, a CAG promoter, a bidirectional CAG promoter, a CBA promoter, a human ubiquitin C promoter, an EFla promoter, a AcMNPV polyhedrin promoter, a AcMNPV plO promoter, a White spot syndrome virus (WSSV) iel promoter, a drosophila metallothionein (MT) promoter, a drosophila actin 5c gene (Ac5) promoter, a tetracycline inducible (TRE) promoter, and a combination thereof.

51. The method of any one of claims 43-50, wherein the one or more cells or the cells in the population of cells is a HEK293 cell, a HEK293T cell, a CHO cell, a U2OS cell, a Sf9 cell, a S2 cell, a MDCK cell, or a derivative thereof.

52. The method of any one of claims 43-51, wherein the PSME1 / PA28A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 1-3, 11, or 17.

53. The method of any one of claims 43-51, wherein the PSME2 / PA28B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 7, 12, or 18.

54. The method of any one of claims 43-53, wherein the vector further comprises a polyadenylation (poly A) site, a woodchuck hepatitis virus post-transcriptional regulatory element(WPRE), an optimized woodchuck hepatitis virus post-transcriptional regulatory element (oPRE), an intron, a microRNA binding motif, or a combination thereof.

55. The method of claim 54, wherein the polyA site is selected from the group consisting of a beta-globin polyA, a bovine growth hormone polyA, a human growth hormone polyA, and a SV40 intermediate early polyA.

56. The method of any one of claims 43-55 wherein the vector further comprises a promoter operably linked to one or more selectable markers.

57. The method of claim 56, wherein the one or more selectable markers is a resistance gene selected from the group consisting of neomycin / kanamycin, puromycin, hygromycin, and blasticidin.

58. The method of claim 44 or 47, wherein the one or more cells or the population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels, as compared to a cell or population of cells that does not comprise the vector.

59. The method of claim 44 or 47, wherein the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the functional AAV capsid levels or lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or population of cells that does not comprise the vector.

60. The method of claim 49, wherein the one or more cells or the population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the heterologous protein levels as compared to a cell or population of cells that does not comprise the vector.

61. The method of claim 49, wherein the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the heterologous protein levels as compared to a cell or population of cells that does not comprise the vector.

62. A method for increasing protein production in one or more cells or in a population of cells, the method comprising contacting the one or more cells or cells in the population of cells with a site-directed modifying polypeptide complexed with a guide RNA (gRNA) that targets the PSME1, PSME2, and / or PSME3 genes.

63. The method of claim 62, wherein the site-directed modifying polypeptide comprises a nuclease, for example a DNA endonuclease such as Cas9, Casl2, or a TALE nuclease.

64. The method of claim 62, wherein the site-directed modifying polypeptide is a base editor, for example a cytosine base editor or an adenine base editor.

65. The method of any one of claims 62-64, wherein the gRNA targets a region of the PSME1, PSME2, and / or PSME3 genes that results in an upregulation of PSME1, PSME2, and / or PSME3 levels.

66. The method of any one of claims 62-65, wherein the one or more cells or cells in the population of cells further comprises one or more nucleic acid(s) to produce one or more functional AAV capsids.

67. The method of claim 66, wherein the one or more nucleic acids encode an AAV capsid protein, a function rep gene, a recombinant AAV vector comprising AAV inverted terminal repeats and a transgene, and a helper protein.

68. The method of claim 67, wherein the AAV capsid protein has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DJ / 8 and AAV2.7m8.

69. The method of any one of claims 62-65, wherein the one or more cells or cells in the population of cells further comprises one or more nucleic acids to produce one or more functional lentivirus particles or live attenuated influenza particles.

70. The method of claim 69, wherein the one or more nucleic acids encode a transfer plasmid, one or more packaging plasmids, and an envelope plasmid.

71. The method of any one of claims 62-65, wherein the one or more cells or cells in the population of cells further comprises a promoter operably linked to a nucleic acid encoding a heterologous protein, for example wherein the heterologous protein is a GPCR, an integral membrane protein, an enzyme, an intracellular protein, a nanobody, or a monoclonal antibody.

72. The method of claim 66-70, wherein the one or more cells or population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about 1000% increase, at least about 1500% increase, at least about 2000% increase, at least about 2500% increase, at least about 3000% increase, at least about 3500% increase, at least about 4000% increase, at least about 4500% increase, at least about 5000% increase or more in the functional AAV capsid levels, lentivirus particle levels, or live attenuated influenza particle levels, as compared to a cell or population of cells that was not contacted with the site-directed modifying polypeptide.

73. The method of claim 66-70, wherein the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50- fold or more increase in the functional AAV capsid levels or lentivirus particle levels, or live attenuated influenza particle levels as compared to a cell or population of cells that was not contacted with the site-directed modifying polypeptide.

74. The method of claim 71, wherein the one or more cells or the population of cells exhibits an at least about 20% increase, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, at least about 400% increase, at least about 500% increase, at least about 600% increase, at least about 700% increase, at least about 800% increase, at least about1000% increase, at least about 1500% increase, at least about 2000% increase, at least about2500% increase, at least about 3000% increase, at least about 3500% increase, at least about4000% increase, at least about 4500% increase, at least about 5000% increase or more in theheterologous protein levels as compared to a cell or population of cells that was not contacted with the site-directed modifying polypeptide.

75. The method of claim 71, wherein the one or more cells or the population of cells exhibits an at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more increase in the heterologous protein levels as compared to a cell or population of cells that was not contacted with the site-directed modifying polypeptide.

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