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WO2025056970A1 - Additifs de culture de cellules de mammifère - Google Patents

Additifs de culture de cellules de mammifère Download PDF

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WO2025056970A1
WO2025056970A1 PCT/IB2024/000498 IB2024000498W WO2025056970A1 WO 2025056970 A1 WO2025056970 A1 WO 2025056970A1 IB 2024000498 W IB2024000498 W IB 2024000498W WO 2025056970 A1 WO2025056970 A1 WO 2025056970A1
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composition
agent
modulator
cell culture
enhancer
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Farzin Farzaneh
David Darling
Boban STANOJEVIC
Zakeria ABDI
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Virocell Biologics Ltd
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Virocell Biologics Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/99—Serum-free medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/065—Modulators of histone acetylation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • compositions described herein enhance the viral titer by increasing the production of more virus, more infectious virus particles; by increasing the efficiency of viral infection of target cells; or a combination of such factors resulting in more virus and/or more efficient infection.
  • the disclosure provides a composition comprising a first enhancer agent that is an endosomal release promoting agent, a nuclear localization promoting agent, a chromatin modifier, an epigenetic modulator, a DNA repair modulator, or a cell cycle modulator; a second enhancer agent that is an endosomal release promoting agent, a nuclear localization promoting agent, an epigenetic modulator, or a cell cycle modulator; and an aqueous mammalian cell culture media.
  • each of the first enhancer agent and second enhancer agent is a small molecule.
  • a mammalian cell culture media is a complete cell culture media. In some embodiments, a mammalian cell culture media is chemically defined.
  • a composition lacks serum (e.g., is a serum-free media).
  • the serum comprises human serum albumin (HSA).
  • a nuclear localization promoting agent comprises a polyamine.
  • a polyamine comprises putrescine, spermine, and/or spermidine.
  • a chromatin modifier comprises a histone deacetylase (HD AC) inhibitor.
  • HD AC histone deacetylase
  • a HD AC inhibitor is selected from sodium butyrate, bufexamac, sodium propionate, vorinostat, romidepsin (FK228), belinostat (PXD101), givinostat (ITF2357), panobinostat (LBH589), valproic acid, tubacin, or trichostatin A (TSA).
  • an epigenetic modulator comprises 5 '-Azacytidine or 5'-aza-2'- deoxycytidine (decitabine).
  • a DNA repair modulator comprises caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist.
  • an adenosine receptor agonist comprises wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, or a PARP inhibitor (e.g., Olaparib).
  • a cell cycle modulator comprises a cyclin dependent kinase modulator (e.g., a cyclin dependent kinase inhibitor), hydroxyurea, or nocodazole.
  • a cyclin dependent kinase modulator is a cyclin dependent kinase inhibitor selected from Dinaciclib, NU6140, NU2058, and RO-3306.
  • a composition further comprises one or more of the following agents: teniposide, rosuvastatin, rapamycin, prostaglandin E2, 16, 16-Dimethyl Prostaglandin, MG132, etoposide, eyarestatin, dexamethasone, daunorubicin, Cyclosporin A (CsA), Cyclosporin H (CsH), BX 795, dasatinib, Akti-1, Akti-2, dimethyl sulfoxide (DMSO), and/or lithium acetate.
  • agents teniposide, rosuvastatin, rapamycin, prostaglandin E2, 16, 16-Dimethyl Prostaglandin, MG132, etoposide, eyarestatin, dexamethasone, daunorubicin, Cyclosporin A (CsA), Cyclosporin H (CsH), BX 795, dasatinib, Akti-1, Akti-2, dimethyl
  • each of a first enhancer agent and a second enhancer agent is an endosomal release promoting agent.
  • a first enhancer agent is pseudouridine, or N1 -methylpseudouridine (ml'P), and the second enhancer agent is 5-methyl- cytidine.
  • a first enhancer agent is an endosomal release promoting agent and a second enhancer agent is a nuclear localization promoting agent.
  • an endosomal release promoting agent is pseudouridine, ml'P, or 5-methyl-cytidine, and a nuclear localization promoting agent is putrescine, spermine, or spermidine.
  • a first enhancer agent is an endosomal release promoting agent and a second enhancer agent is a cell cycle modulator.
  • an endosomal release promoting agent is pseudouridine, ml'P, or 5-methyl-cytidine
  • a cell cycle modulator is a cyclin dependent kinase modulator (e.g., inhibitor), hydroxyurea, or nocodazole.
  • an endosomal release promoting agent comprises hydroxyurea and nocodazole.
  • an endosomal release promoting agent is 5-methyl-cytidine and a cell cycle modulator is hydroxyurea.
  • an endosomal release promoting agent is 5- methyl-cytidine and a cell cycle modulator is nocodazole.
  • a nuclear localization promoting agent is spermidine and an epigenetic modifier is 5 -Azacytidine. In some embodiments, a nuclear localization promoting agent is spermidine and an epigenetic modifier is 5'-aza-2'- deoxycytidine (decitabine).
  • a first enhancer agent is a nuclear localization promoting agent and a second enhancer agent is a cell cycle modulator.
  • a nuclear localization promoting agent is putrescine, spermine, or spermidine
  • a cell cycle modulator is a cyclin dependent kinase modulator (e.g., inhibitor), hydroxyurea, or nocodazole.
  • a nuclear localization promoting agent is putrescine
  • a cell cycle modulator is a cyclin dependent kinase inhibitor.
  • the cyclin dependent kinase inhibitor is Dinaciclib.
  • a first enhancer agent is a chromatin modifier
  • a second enhancer agent is an endosomal release promoting agent.
  • a chromatin modifier is an HD AC inhibitor selected from sodium butyrate, bufexamac, sodium propionate, vorinostat, romidepsin (FK228), belinostat (PXD101), givinostat (ITF2357), panobinostat (LBH589), valproic acid, tubacin, or trichostatin A (TSA) and an endosomal release promoting agent is pseudouridine, N1 -methylpseudouridine (m I T) or 5-methyl-cytidine.
  • an HD AC inhibitor is sodium butyrate and an endosomal release promoting agent is pseudouridine.
  • an HD AC inhibitor is vorinostat and an endosomal release promoting agent is pseudouridine.
  • a first enhancer agent is a chromatin modifier and a second enhancer agent is a nuclear localization promoting agent.
  • a chromatin modifier is an HD AC inhibitor selected from sodium butyrate, bufexamac, sodium propionate, vorinostat, romidepsin (FK228), belinostat (PXD101), givinostat (ITF2357), Panobinostat (LBH589), valproic acid, tubacin, or trichostatin A (TSA) and a nuclear localization promoting agent is putrescine, spermine, or spermidine.
  • an HD AC inhibitor is sodium butyrate and a nuclear localization promoting agent is putrescine.
  • a first enhancer agent is a chromatin modifier and a second enhancer agent is an epigenetic modulator.
  • a chromatin modifier is an HD AC inhibitor selected from sodium butyrate, bufexamac, sodium propionate, vorinostat, romidepsin (FK228), belinostat (PXD101), givinostat (ITF2357), Panobinostat (LBH589), valproic acid, tubacin, or trichostatin A (TSA) and an epigenetic modulator is 5 '-Azacytidine or 5'-aza-2'-deoxycytidine (decitabine).
  • an HDAC inhibitor is sodium butyrate and an epigenetic modulator is 5-Azacytidine. In some embodiments, an HDAC inhibitor is sodium butyrate and an epigenetic modulator is 5'-aza-2'-deoxycytidine (decitabine).
  • a first enhancer agent is a chromatin modifier and a second enhancer agent is cell cycle modulator.
  • a chromatin modifier is an HDAC inhibitor selected from sodium butyrate, bufexamac, sodium propionate, vorinostat, romidepsin (FK228), belinostat (PXD101), givinostat (ITF2357), Panobinostat (LBH589), valproic acid, tubacin, or trichostatin A (TSA) and a cell cycle modulator is a cyclin dependent kinase inhibitor, hydroxyurea, or nocodazole.
  • an HDAC inhibitor is sodium butyrate and a cell cycle modulator is a cyclin dependent kinase inhibitor.
  • a cyclin dependent kinase inhibitor is Dinaciclib.
  • a first enhancer agent is an epigenetic modulator and a second enhancer agent is cell cycle modulator.
  • an epigenetic modulator is 5'- Azacytidine or 5'-aza-2'-deoxycytidine (decitabine) and a cell cycle modulator is a cyclin dependent kinase inhibitor, hydroxyurea, or nocodazole.
  • an epigenetic modulator is 5 '-Azacytidine and a cell cycle modulator is a cyclin dependent kinase inhibitor.
  • an epigenetic modulator is 5'-aza-2'-deoxycytidine (decitabine) and a cell cycle modulator is a cyclin dependent kinase inhibitor.
  • a cyclin dependent kinase inhibitor is Dinaciclib.
  • a first enhancer agent is a DNA repair modulator and a second enhancer agent is an endosomal release promoting agent.
  • a DNA repair modulator is caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist and an endosomal release promoting agent is pseudouridine, N1 -methylpseudouridine (m I T) or 5-methyl-cytidine.
  • a DNA repair modulator is caffeine and an endosomal release promoting agent is pseudouridine.
  • a DNA repair modulator is theophylline and an endosomal release promoting agent is pseudouridine. In some embodiments, a DNA repair modulator is theobromine and an endosomal release promoting agent is pseudouridine. In some embodiments, a DNA repair modulator is hydroxychloroquine and an endosomal release promoting agent is pseudouridine. In some embodiments, a first enhancer agent is a DNA repair modulator and a second enhancer agent is a nuclear localization promoting agent.
  • a DNA repair modulator is caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist and a nuclear localization promoting agent is putrescine, spermine, and/or spermidine.
  • a DNA repair modulator is caffeine and a nuclear localization promoting agent is putrescine.
  • a DNA repair modulator is theophylline and a nuclear localization promoting agent is putrescine.
  • a DNA repair modulator is theobromine and a nuclear localization promoting agent is putrescine.
  • a DNA repair modulator is hydroxychloroquine and a nuclear localization promoting agent is putrescine.
  • a first enhancer agent is a DNA repair modulator and a second enhancer agent is an epigenetic modulator.
  • a DNA repair modulator is caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist and an epigenetic modulator is 5'- Azacytidine or 5'-aza-2'-deoxycytidine (decitabine).
  • a DNA repair modulator is caffeine and an epigenetic modulator is 5-Azacytidine.
  • a DNA repair modulator is theophylline and an epigenetic modulator is 5-Azacytidine. In some embodiments, a DNA repair modulator is theobromine and an epigenetic modulator is 5- Azacytidine. In some embodiments, a DNA repair modulator is hydroxychloroquine and an epigenetic modulator is 5-Azacytidine.
  • a first enhancer agent is a DNA repair modulator and a second enhancer agent is a cell cycle modulator.
  • a DNA repair modulator is caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist and a cell cycle modulator is a cyclin dependent kinase modulator (e.g., inhibitor), hydroxyurea, or nocodazole.
  • a DNA repair modulator is caffeine and a cell cycle modulator is a cyclin dependent kinase inhibitor.
  • a DNA repair modulator is theophylline and a cell cycle modulator is a cyclin dependent kinase inhibitor. In some embodiments, a DNA repair modulator is theobromine and a cell cycle modulator is a cyclin dependent kinase inhibitor. In some embodiments, a DNA repair modulator is hydroxychloroquine and a cell cycle modulator is a cyclin dependent kinase inhibitor. In some embodiments, a cyclin dependent kinase inhibitor is Dinaciclib or NU 6140.
  • a population of mammalian host cells comprises HEK293 cells or derivatives thereof.
  • 3B shows GFP and CD 19 GFP expression in Jurkat cells 24 hours after transduction with filtered viral supernatant harvested from adherent 293T cells treated with lOpM panobinostat or lOpM vorinostat 24 hours after transfection.
  • FIGs. 4A-4C show comparisons of the effects of sodium butyrate and differing concentrations of panobinostat on lentiviral vector titer from suspension HEK293 cells and adherent 293T cells.
  • Transfected cells were treated with no enhancing agents, (“Control”), 5mM sodium butyrate, lOpM, 20pM, or 40pM panobinostat were added at 24 hours post-transduction.
  • Virus titer (TU/mL) was calculated from GFP expression in Jurkat cells (cell number/well/200ul in 96 well plate- flat bottom) measured via FACS sorting, 5 days after transduction with filtered supernatant.
  • FIG. 4A shows results from two representative experiments in which viral titer was calculated from GFP expression in Jurkat cells 24 hours after transduction with filtered viral supernatant harvested from suspension HEK293 cells treated with 5mM sodium butyrate or lOpM panobinostat 24 hrs after transfection.
  • FIG. 4B shows results from two representative experiments in which viral titer was calculated from GFP expression in Jurkat cells 24 hours after transduction with filtered viral supernatant harvested from suspension HEK293 cells treated with 5mM sodium butyrate, lOpM panobinostat, 20pM panobinostat, or 40pM panobinostat 24 hrs after transfection.
  • FIG. 4A top, bottom shows results from two representative experiments in which viral titer was calculated from GFP expression in Jurkat cells 24 hours after transduction with filtered viral supernatant harvested from suspension HEK293 cells treated with 5mM sodium butyrate, lOpM panobinostat, 20pM panobin
  • 4C shows results from a representative experiment in which viral titer was calculated from GFP expression in Jurkat cells 24 hours after transduction with filtered viral supernatant harvested from adherent 293T cells treated with 5mM sodium butyrate, lOpM panobinostat or 20pM panobinostat 24 hrs after transfection.
  • FIG. 5 shows the effects of sodium butyrate, panobinostat, and mocetinostat on lentiviral vector titer from HEK293 suspension cells.
  • Transfected cells were treated with no enhancing agents, (“Control”), 5mM sodium butyrate, lOpM mocetinostat, lOpM panobinostat, or lOpM mocetinostat, and lOpM panobinostat were added at 24 hours post-transduction.
  • Virus titer (TU/mL) was calculated from CD 19 GFP expression in Jurkat cells (cell number/well/200ul in 96 well plate- flat bottom) measured via FACS sorting, 5 days after transduction with filtered supernatant.
  • compositions and methods for increasing viral titer by modulating the activity, availability, efficiency, and output of one or more stages of viral replication and packaging are based, in part, on combinations of enhancer agents that function in an additive or synergistic manner to modulate certain mammalian host cell processes to result in an increase of viral particle production by the cells.
  • Retroviruses e.g., lentiviruses, y-retroviruses
  • dsDNA double-stranded DNA
  • Integrated retroviral genes are transcribed by host cell machinery into retroviral RNA, which is exported from the nucleus.
  • retroviral RNA is translated into retroviral peptides; the resulting RNA and peptides are then packaged into retroviral particles (e.g., capsids) and, optionally enveloped.
  • retroviral particles e.g., capsids
  • Production of retroviral particles thus requires several rate-limiting steps; similar bottlenecks exist for the replication and packaging of other viruses (e.g., adeno-associated viruses).
  • Non-limiting examples of rate-limiting steps include entry of viral nucleic acids into a host cell, release of viral nucleic acids from endosomes, transport of viral nucleic acids into the nucleus, integration of viral nucleic acids into the host cell genome, repair of the host cell genome after integration, transcription of viral nucleic acids, translation of viral nucleic acids, packaging of viral nucleic acids and peptides, and general cell cycle factors.
  • Titer enhancing compositions and methods which increase the activity, availability, efficiency, or output of one or more stages of rate limiting steps of viral production such that viral production is increased.
  • Titer enhancing compositions may be provided to viral packaging cells at one or more timepoints to promote the production of high viral titers, relative to titers produced by viral packaging cells in their absence. Such factors may enhance the viral titer by increasing the production of more virus, more infectious virus particles, or by increasing the efficiency of viral infection of target cells, or indeed a combination of such factors resulting in more virus and/or more efficient infection.
  • compositions comprising two or more enhancer agents (e.g., small molecules, which do not include nucleic acids or peptides) which modulate one or more of the following: entry of viral nucleic acids into a host cell, release of viral nucleic acids from endosomes or other such vesicles, transport of viral nucleic acids into the nucleus, integration of viral nucleic acids into the host cell genome, repair of the host cell genome after integration, transcription of viral nucleic acids, translation of viral nucleic acids (including the transcription and translation of helper factors encoded by the packaging/helper plasmids, packaging of viral nucleic acids and peptides, and/or cell cycle progression).
  • enhancer agents e.g., small molecules, which do not include nucleic acids or peptides
  • an “enhancer agent” is a small molecule which elicits, causes, or otherwise produces higher titers of viral particles produced by viral packaging cells (e.g., host cells, such as transient or stable vector producing cells) in the presence of the enhancer agent, relative to titers produced by viral packaging cells in their absence.
  • An enhancer agent may increase viral titer by modulating at least one of the following rate-limiting steps of viral production: entry of viral nucleic acids into a host cell (e.g., viral vector producing cells), for example transfection of vector and helper plasmids into the vector producing cells), release of viral nucleic acids from endosomes or other such structures, transport of viral nucleic acids into the nucleus, reverse transcription of the viral RNA genome into a DNA copy, integration of viral DNA into the host cell genome, repair of the host cell genome after integration, transcription of viral nucleic acids (either with or without, before or after, integration into the host cell genome) translation of viral nucleic acids for the production of the viral proteins encoded by the viral vector and/or helper or packaging plasmids, and the packaging of viral nucleic acids and peptides, and producer cell encoded factors.
  • a host cell e.g., viral vector producing cells
  • transfection of vector and helper plasmids into the vector producing cells e.g.
  • compositions for example cell culture media and/or cell culture media additives, comprising two or more enhancer agents.
  • a composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more enhancer agents.
  • each enhancer agent of a composition is independently selected from an endosomal release promoting agent, a nuclear localization promoting agent, a chromatin modifier, an epigenetic modulator, a DNA repair modulator, a cell cycle modulator.
  • a composition comprises 2 or more (e.g., 2, 3, 4, 5, or more) endosomal release promoting agents.
  • a composition comprises 2 or more (e.g., 2, 3, 4, 5, or more) nuclear localization promoting agents.
  • a composition comprises 2 or more (e.g., 2, 3, 4, 5, or more) chromatin modifiers. In some embodiments, a composition comprises 2 or more (e.g., 2, 3, 4, 5, or more) epigenetic modulators. In some embodiments, a composition comprises 2 or more (e.g., 2, 3, 4, 5, or more) cell cycle modulators. Examples of agents in each of the foregoing classes of enhancer agent is described in further detail below.
  • an enhancer agent is an endosomal release promoting agent.
  • an “endosomal release promoting agent” is a small molecule which increases the efficacy of release of cargo (e.g., viral nucleic acids, peptides) from an endosome into the cytoplasm of a host cell.
  • cargo e.g., viral nucleic acids, peptides
  • an endosomal release promoting agent promotes endosomal membrane fusion.
  • an endosomal release promoting agent promotes destabilization of an endosomal membrane.
  • an endosomal release promoting agent promotes the formation of endosomal pores.
  • an endosomal release promoting agent reduces recognition of cargo as foreign by toll-like receptors (TLRs). In some embodiments, an endosomal release promoting agent increases endosomal pH. In some embodiments, an endosomal release promoting agent is a chemically modified nucleotide. In some embodiments, an enhancer agent is a chemically modified nucleotide selected from pseudouridine, N1 -methylpseudouridine (ml'P), and 5-methyl-cytidine. In some embodiments, an endosomal release promoting agent comprises chloroquine.
  • an enhancer agent is a nuclear localization promoting agent.
  • a “nuclear localization promoting agent” is a small molecule which increases the efficacy of import of cargo (e.g., viral nucleic acids) into the nucleus of a host cell.
  • a nuclear localization promoting agent stabilizes cargo for transport into the nucleus.
  • a nuclear localization promoting agent promotes the formation of nuclear pore complexes.
  • a nuclear localization promoting agent promotes the association of cargo (e.g., viral nucleic acids) with nuclear transport factors (e.g., importins) in the cytoplasm of a host cell.
  • a nuclear localization promoting agent promotes the disassociation of cargo (e.g., viral nucleic acids) from nuclear transport factors (e.g., importins) in the nucleus of a host cell.
  • a nuclear localization promoting agent increases the availability of guanine triphosophates (GTP) in a host cell (e.g., a vector producing cell).
  • GTP guanine triphosophates
  • a nuclear localization promoting agent increases the availability of GTPase enzymes in a host cell (e.g., vector producing cell) or a target cell.
  • a nuclear localization promoting agent increases the availability of Ran in a host cell.
  • a nuclear localization promoting agent increases the availability of nuclear transport factors (e.g., importin). In some embodiments, a nuclear localization promoting agent increases the rate of Ran-GTP association in the nucleus. In some embodiments, a nuclear localization promoting agent increases the hydrolyzation of Ran-GTP to Ran-GDP in the cytoplasm. In some embodiments, a nuclear localization promoting agent increases the binding of Ran-GTP to a nuclear transport factor (e.g., importin). In some embodiments, an enhancer agent is dexamethasone. Aspects of the disclosure relate to nuclear localization promoting agents that are polyamines. As used herein “polyamine” refers to an organic compound having two or more amine groups.
  • polyamines are polycations.
  • a polyamine may be naturally occurring or synthetic.
  • a polyamine is a small molecule.
  • a polyamine interacts with negatively charged molecules, for example DNA, RNA, and/or negatively charged peptides.
  • certain polyamines have been observed to increase AMPK activation and subsequently promote nuclear importation of certain substrates.
  • examples of polyamines include but are not limited to cadaverine, putrescine, spermine, spermidine, ethylenediamine, and macrocyclic polyamines (e.g., cyclean).
  • a nuclear localization promoting agent is a polyamine.
  • an enhancer agent is a polyamine selected from putrescine, spermine, and spermidine.
  • an enhancer agent is a chromatin modifier.
  • a “chromatin modifier,” as used herein, is a small molecule which alters the assembly, organization, or construction of chromatin.
  • a chromatin modifier enables the incorporation of histone variants into the organization of chromatin.
  • a chromatin modifier remodels chromatin.
  • a chromatin modifier regulates post translational modifications to histones (e.g., phosphorylation, acetylation, methylation, ubiquity lation).
  • a chromatin modifier is a histone deacetylase (HD AC) inhibitor. Human histone deacetylases are generally grouped into several classes.
  • an enhancer agent is a chromatin modifier selected from sodium butyrate, sodium propionate, vorinostat, romidepsin (FK228), belinostat (PXD101), givinostat (ITF2357), panobinostat (EBH589), mocetinostat (MGCD0103), valproic acid, trichostatin A (TSA), bufexamac, and/or tubacin.
  • a composition comprises 2, 3, 4, 5, or more chromatin modifiers, for example vorinostat and bufexamac, sodium butyrate and tubacin; vorinostat and panobinostat; panobinostat and mocetinostat; etc.
  • a composition comprises panobinostat and another chromatin modifier.
  • a composition comprises panobinostat and vorinostat.
  • a compositions comprises panobinostat and mocetinostat.
  • a composition comprises an HDAC6 inhibitor and an HD AC 10 inhibitor. In some embodiments, a composition comprises an HDAC6 inhibitor, an HDAC10 inhibitor, and a non- selective HD AC inhibitor, for example sodium butyrate and bufexamac.
  • an enhancer agent is an epigenetic modulator.
  • An “epigenetic modulator” is a small molecule which alters the activity of methyltransferases.
  • an epigenetic modulator inhibits the activity of methyltransferases, thereby inhibiting DNA methylation.
  • an “epigenetic modulator” is not a “chromatin modifier.”
  • an enhancer agent is an epigenetic modulator selected from 5 '-azacytidine, 5 '-aza-2'-deoxy cytidine (decitabine), and dimethyl sulfoxide (DMSO).
  • a composition comprises 2, 3, 4, 5, or more epigenetic modulators, for example 5 '-azacytidine and 5'-aza-2'-deoxycytidine (decitabine), etc.
  • an enhancer agent is a DNA repair modulator.
  • a “DNA repair modulator” is a small molecule which modulates the activity of DNA repair mechanisms and/or frequency in a host cell.
  • DNA repair modulators alter the activity or availability of enzymes and proteins which mediate base excision repair mechanisms.
  • DNA repair modulators alter the activity or availability of enzymes and proteins which mediate nucleotide excision repair mechanisms.
  • DNA repair modulators alter the activity or availability of enzymes and proteins which mediate mismatch repair. In some embodiments, DNA repair modulators alter the activity of availability of enzymes and proteins which mediate homologous recombination. In some embodiments, DNA repair modulators alter the activity of availability of enzymes and proteins which mediate non-homologous end joining. In some embodiments, DNA repair modulators alter double-strand break repair. In some embodiments, DNA repair modulators alter the activity of translesion synthesis polymerases.
  • a CDK inhibitor may be a direct inhibitor (e.g., an inhibitor that binds directly to a CDK protein or nucleic acid encoding a CDK protein) or an indirect inhibitor (e.g., an inhibitor that binds to a modulator of a CDK protein).
  • a CDK inhibitor is a selective CDK inhibitor.
  • a “selective CDK inhibitor” binds to and inhibits a single CDK protein (e.g., CDK2).
  • a CDK inhibitor is a non-selective CDK inhibitor.
  • a “non- selective CDK inhibitor” binds to and inhibits two or more of CDKs, for example CDK1 and CDK2, or CDK4, CDK2 and CDK6, etc.
  • a composition (e.g., a cell culture media or cell culture media additive) comprises one or more additional compounds selected from rosuvastatin, MG 132, Cyclosporin A (CsA), Cyclosporin H (CsH), BX 795, Dasatinib, lithium acetate, rapamycin, prostaglandin E2,16, 16-dimethyl prostaglandin, daunorubicin, and eyarestatin.
  • the amount and/or ratios of enhancer agents in a composition may vary.
  • the amount or ratio of enhancer agents in a composition may be measured according to any suitable technique, for example mass (w/w), volume (v/v), molarity (M/M), molality, etc.
  • a composition comprises a total of IpM, 5pM, lOpM, 15pM, 20pM, 25pM, 30pM, 35pM, 40pM, 50pM, 60pM, 70pM, 80pM, 90pM, or lOOpM of one or more (e.g., two or more, three or more, four or more, five or more, six or more) enhancer agents.
  • a composition comprises a total of 5pM of one or more (e.g., two or more, three or more, four or more, five or more, six or more) enhancer agents.
  • a composition comprises a total of lOp M of one or more (e.g., two or more, three or more, four or more, five or more, six or more) enhancer agents. In some embodiments, a composition comprises a total of 15pM of one or more (e.g., two or more, three or more, four or more, five or more, six or more) enhancer agents. In some embodiments, a composition comprises a total of 20pM of one or more (e.g., two or more, three or more, four or more, five or more, six or more) enhancer agents.
  • a composition comprises a total of 30pM of one or more (e.g., two or more, three or more, four or more, five or more, six or more) enhancer agents. In some embodiments, a composition comprises a total of 40pM of one or more (e.g., two or more, three or more, four or more, five or more, six or more) enhancer agents.
  • a composition comprises a total of 5pM to 40pM of panobinostat (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pM panobinostat).
  • a composition comprises a total of 5pM of panobinostat.
  • a composition comprises a total of lOpM of panobinostat.
  • a composition comprises a total of 15pM of panobinostat.
  • a composition comprises a total of 20pM of panobinostat.
  • a composition comprises a total of 25pM of panobinostat. In some embodiments, a composition comprises a total of 30pM of panobinostat. In some embodiments, a composition comprises a total of 40pM of panobinostat.
  • a composition comprises a total of 5pM to 40pM of vorinostat (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pM vorinostat).
  • a composition comprises a total of 5pM of vorinostat.
  • a composition comprises a total of lOpM of vorinostat.
  • a composition comprises a total of 15pM of vorinostat.
  • a composition comprises a total of 20pM of vorinostat.
  • a composition comprises a total of 25pM of vorinostat. In some embodiments, a composition comprises a total of 30pM of vorinostat. In some embodiments, a composition comprises a total of 40pM of vorinostat.
  • a composition comprises a total of 5pM to 40pM of mocetinostat (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pM mocetinostat).
  • a composition comprises a total of 5pM of mocetinostat.
  • a composition comprises a total of lOpM of mocetinostat.
  • a composition comprises a total of 15pM of mocetinostat.
  • a composition comprises a total of 20pM of mocetinostat.
  • a composition comprises a total of 25pM of mocetinostat. In some embodiments, a composition comprises a total of 30pM of mocetinostat. In some embodiments, a composition comprises a total of 40pM of mocetinostat.
  • a composition comprises a total of 5pM to 40pM of panobinostat and mocetinostat (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pM panobinostat and mocetinostat combined).
  • a composition comprises a total of 5pM of panobinostat and mocetinostat.
  • a composition comprises a total of lOpM of panobinostat and mocetinostat.
  • a composition comprises a total of 15pM of panobinostat and mocetinostat.
  • a composition comprises a total of 20pM of panobinostat and mocetinostat. In some embodiments, a composition comprises a total of 25pM of panobinostat and mocetinostat. In some embodiments, a composition comprises a total of 30pM of panobinostat and mocetinostat. In some embodiments, a composition comprises a total of 40pM of panobinostat and mocetinostat.
  • a composition comprises a first enhancer agent and a second enhancer agent in a ratio of at least about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:100, 1:200, 1:500, 1:1000, or more.
  • a titer enhancing composition comprises a first enhancer agent and a second enhancer agent in a ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 100:1, 200:1, 500:1, 1000:1, or more.
  • a titer enhancing composition comprises a first, second, and third enhancer agent in a ratio of about 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:1:20, 1:1:30, 1:1:40, 1:1:50, 1:1:100, 1:1:200, 1:1:500, 1:1:1000, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 1:9:1, 1:10:1, 1:20:1, 1:30:1, 1:40:1, 1:50:1, 1:100:1, 1:200:1, 1:500:1, 1:1000:1, 2:1:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, 9:1:1, 10:1:1, 20:1:1, 30:1:1, 40:1:1, 50: 1:1, 100: 1:1, 200: 1:1, 500: 1:1, 1000: 1 : 1 , or
  • a first enhancer agent is an endosomal release promoting agent, a nuclear localization promoting agent, a chromatin modifier, an epigenetic modulator, or a cell cycle modulator.
  • a second enhancer agent is an endosomal release promoting agent, a nuclear localization promoting agent, an epigenetic modulatory, or a cell cycle modulator.
  • a third, fourth, fifth, sixth or more enhancer agent is an endosomal release promoting agent, a nuclear localization promoting agent, a chromatin modifier, an epigenetic modulator, or a cell cycle modulator.
  • a composition comprises panobinostat and mocetinostat at a ratio (e.g., a molar ratio) of 10:1 to 1:10. In some embodiments, a composition comprises panobinostat and mocetinostat at a ratio (e.g., a molar ratio) of 10:1, 9:1, 8: 1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, a composition comprises panobinostat and mocetinostat at a ratio of 1:1.
  • a composition comprises panobinostat, mocetinostat, and a third enhancer agent at a ratio (e.g., a molar ratio) of 10:1:1 to 1:1:10.
  • a composition comprises a ratio of panobinostat:mocetinostat:a third enhancer ratio at a ratio of 10:1:1, 9:1:, 8:1:, 7:1:1, 6:1:1, 5:1:1, 4:1:1, 3:1:1, 2:1:1, 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 1:9:1, 1:10:1, 1:1:9, 1:1:8, 1:1:7, 1:1:6, 1:1:5, 1:1:4, 1:1:3, 1:1:2, 1:1:1.
  • a composition comprises a ratio (e.g., a molar ratio) of panobinostat:mocetinostat:a third enhancer ratio at a ratio of 1
  • enhancer agents in a mammalian cell culture may increase viral titer generated by host cells by, for example 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,
  • a titer enhancing composition comprises an endosomal release promoting agent selected from pseudouridine, N1 -methylpseudouridine (mlT), and 5-methyl- cytidine.
  • a titer enhancing composition comprises pseudouridine.
  • a titer enhancing composition comprises N1 -methylpseudouridine (m I T).
  • a titer enhancing composition comprises 5-methyl-cytidine.
  • a titer enhancing composition comprises pseudouridine, N1 -methylpseudouridine (mlT), 5-methyl-cytidine, or a combination thereof.
  • a titer enhancing composition comprises two or more endosomal release promoting agents.
  • a titer enhancing composition comprises pseudouridine and N1 -methylpseudouridine (mlT).
  • a titer enhancing composition comprises pseudouridine and 5-methyl-cytidine.
  • a titer enhancing composition comprises N1 -methylpseudouridine (m I T) and 5-methyl-cytidine.
  • a titer enhancing composition comprises an endosomal release promoting agent and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises pseudouridine and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises N1 -methylpseudouridine (mlT) and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises 5-methyl-cytidine and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises a nuclear localization promoting agent which is dexamethasone or a polyamine. In some embodiments, a titer enhancing composition comprises pseudouridine. In some embodiments, a titer enhancing composition comprises N1 -methylpseudouridine (mlT). In some embodiments, a titer enhancing composition comprises 5-methyl-cytidine. In some embodiments, a titer enhancing composition comprises pseudouridine, N1 -methylpseudouridine (mlT), 5-methyl-cytidine, or a combination thereof.
  • a titer enhancing composition comprises two or more endosomal release promoting agents. In some embodiments, a titer enhancing composition comprises an endosomal release promoting agent and another (e.g., one or more) enhancer agent.
  • an enhancer agent is a poly amine selected from putrescine, spermine, and spermidine.
  • a titer enhancing composition comprises putrescine.
  • a titer enhancing composition comprises spermine.
  • a titer enhancing composition comprises spermidine.
  • a titer enhancing composition comprises putrescine, spermine, spermidine, or a combination thereof.
  • a titer enhancing composition comprises two or more polyamines. In some embodiments, a titer enhancing composition comprises putrescine and spermine. In some embodiments, a titer enhancing composition comprises spermine and spermidine. In some embodiments, a titer enhancing composition comprises putrescine and spermidine.
  • a titer enhancing composition comprises a polyamine and another (e.g., one or more) enhancer agent. In some embodiments, a titer enhancing composition comprises putrescine and another (e.g., one or more) enhancer agent. In some embodiments, a titer enhancing composition comprises spermine and another (e.g., one or more) enhancer agent. In some embodiments, a titer enhancing composition comprises spermidine and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises a chromatin modifier selected from sodium butyrate, sodium propionate, vorinostat, romidepsin (FK228), belinostat (PXD101), givinostat (ITF2357), panobinostat (LBH589), mocetinostat, valproic acid, trichostatin A (TSA), bufexamac, and tubacin.
  • a titer-enhancing composition comprises sodium butyrate.
  • a titer-enhancing composition comprises sodium propionate.
  • a titer-enhancing composition comprises vorinostat.
  • a titer-enhancing composition comprises panobinostat. In some embodiments, a titer-enhancing composition comprises valproic acid. In some embodiments, a titer-enhancing composition comprises trichostatin A. In some embodiments, a titer-enhancing composition comprises tubacin. In some embodiments, a titer-enhancing composition comprises mocetinostat. In preferred embodiments, a titer-enhancing composition comprises sodium butyrate, panobinostat, mocetinostat, vorinostat, or a combination thereof. In some embodiments, a titer-enhancing composition comprising a chromatin modifier does not solely comprise sodium butyrate.
  • a titer-enhancing composition comprises two or more chromatin modifiers.
  • a titer-enhancing composition comprises sodium butyrate and another (e.g., one or more) chromatin modifier.
  • a titer-enhancing composition comprises panobinostat and another (e.g., one or more) chromatin modifier.
  • a titer-enhancing composition comprises vorinostat and another (e.g., one or more) chromatin modifier.
  • a titer-enhancing composition comprises mocetinostat and another (e.g., one or more) chromatin modifier.
  • a titerenhancing composition comprises panobinostat and mocetinostat.
  • a titer-enhancing composition comprises sodium butyrate and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises sodium butyrate and bufexamac. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, and caffeine. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, and sodium pentanoate.
  • a titerenhancing composition comprises sodium butyrate, bufexamac, caffeine, and 2’ aminopurine. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, and chloroquine. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, sodium pentanoate, and chloroquine. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, 2’ aminopurine, and chloroquine. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, and nicotinamide.
  • a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, and nicotinamide. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, sodium pentanoate, and nicotinamide. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, sodium pentanoate, chloroquine, and nicotinamide. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, 2’ aminopurine, chloroquine, and nicotinamide. In some embodiments, a titer-enhancing composition comprises sodium butyrate, bufexamac, caffeine, chloroquine, and nicotinamide.
  • a titer-enhancing composition comprises panobinostat and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises mocetinostat and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises vorinostat and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises an epigenetic modulator selected from 5-azacytidine, 5'-aza-2'-deoxycytidine (decitabine), and dimethyl sulfoxide (DMSO).
  • a titer-enhancing composition comprises 5-azacytidine.
  • a titer-enhancing composition comprises 5'-aza-2'-deoxycytidine (decitabine).
  • a titer-enhancing composition comprises DMSO.
  • a titer-enhancing composition comprises 5-azacytidine, 5'-aza-2'-deoxycytidine (decitabine), dimethyl sulfoxide (DMSO), or a combination thereof.
  • a titer enhancing composition comprises two or more epigenetic modulators.
  • a titer-enhancing composition comprises 5-azacytidine and 5'-aza-2'-deoxycytidine (decitabine).
  • a titer-enhancing composition comprises DMSO and 5-azacytidine.
  • a titer-enhancing composition comprises 5'-aza-2'-deoxycytidine (decitabine) and DMSO.
  • a titer enhancing composition comprises an epigenetic modulator and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises 5-azacytidine and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises 5'-aza-2'-deoxycytidine (decitabine) and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises DMSO and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises a DNA repair modulator selected from caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, theophylline, theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU- 55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide.
  • a titer-enhancing composition comprises caffeine.
  • a titerenhancing composition comprises nicotinamide.
  • a titer-enhancing composition comprises chloroquine diphosphate.
  • a titer-enhancing composition comprises hydroxychloroquine. In some embodiments, a titer-enhancing composition comprises NU7026. In some embodiments, a titer-enhancing composition comprises JQ1. In some embodiments, a titer-enhancing composition comprises theophylline. In some embodiments, a titer-enhancing composition comprises theobromine. In some embodiments, a titer-enhancing composition comprises an adenosine receptor agonist. In some embodiments, a titer-enhancing composition comprises wortmannin. In some embodiments, a titer-enhancing composition comprises CP-466722. In some embodiments, a titer-enhancing composition comprises KU-55933.
  • a titer-enhancing composition comprises KU-60019. In some embodiments, a titer-enhancing composition comprises KU- 559403. In some embodiments, a titer-enhancing composition comprises a PARP inhibitor. In some embodiments, a titer-enhancing composition comprises olaparib. In some embodiments, a titer-enhancing composition comprises etoposide.
  • a titer-enhancing composition comprises caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, theophylline, theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, etoposide, or a combination thereof.
  • a titer enhancing composition comprises two or more DNA repair modulators.
  • a titer-enhancing composition comprises caffeine and one or more of nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, theophylline, theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide.
  • a titer-enhancing composition comprises nicotinamide and one or more of chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, theophylline, theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide.
  • a titer-enhancing composition comprises chloroquine diphosphate and one or more of hydroxychloroquine, NU7026, JQ1, theophylline, theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU-60019, KU- 559403, PARP inhibitors, olaparib, and etoposide.
  • a titer-enhancing composition comprises CP-466722 and one or more of KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, a titer-enhancing composition comprises KU-55933 and one or more of KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, a titer-enhancing composition comprises KU-60019 and one or more of KU-559403, PARP inhibitors, olaparib, and etoposide.
  • a titer enhancing composition comprises a DNA repair modulator and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises caffeine and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises nicotinamide and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises chloroquine diphosphate and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises hydroxychloroquine and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises NU7026 and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises JQ1 and another (e.g., one or more) enhancer agent. In some embodiments, a titerenhancing composition comprises theophylline and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises theobromine and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises an adenosine receptor agonist and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises wortmannin and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises CP- 466722 and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises KU-55933 and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises KU-60019 and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises KU- 559403 and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises a PARP inhibitor and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises olaparib and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises etoposide and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises a cell cycle modulator selected from a cyclin dependent kinase modulator, hydroxyurea, nocodazole, Akti-1 and Akti- 2.
  • a titer-enhancing composition comprises a cyclin dependent kinase modulator.
  • a titer-enhancing composition comprises hydroxyurea.
  • a titer-enhancing composition comprises nocodazole.
  • a titer-enhancing composition comprises Akti-1.
  • a titerenhancing composition comprises Akti-2.
  • a titer-enhancing composition comprises cyclin dependent kinase modulator, hydroxyurea, nocodazole, Akti-1, Akti-2, or a combination thereof.
  • a titer enhancing composition comprises two or more a cell cycle modulator. In some embodiments, a titer-enhancing composition comprises a cyclin dependent kinase modulator and hydroxyurea. In some embodiments, a titer-enhancing composition comprises a cyclin dependent kinase modulator and nocodazole. In some embodiments, a titerenhancing composition comprises a cyclin dependent kinase modulator and Akti-1. In some embodiments, a titer-enhancing composition comprises a cyclin dependent kinase modulator and Akti-2. In some embodiments, a titer-enhancing composition comprises hydroxyurea and nocodazole.
  • a titer-enhancing composition comprises hydroxyurea and Akti-1. In some embodiments, a titer-enhancing composition comprises hydroxyurea and Akti-2. In some embodiments, a titer-enhancing composition comprises nocodazole and Akti-1. In some embodiments, a titer-enhancing composition comprises nocodazole and Akti-2.
  • a titer enhancing composition comprises a cell cycle modulator and another (e.g., one or more) enhancer agent and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises a cyclin dependent kinase modulator and another (e.g., one or more) enhancer agent.
  • a titerenhancing composition comprises hydroxyurea and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises nocodazole and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises Akti-1 and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises Akti-2 and another (e.g., one or more) enhancer agent.
  • a titer enhancing composition comprises a cell cycle modulator selected from dinaciclib, NU 6140, NU 2058, and Ro 3306. In some embodiments, a titerenhancing composition comprises dinaciclib. In some embodiments, a titer-enhancing composition comprises NU 6140. In some embodiments, a titer-enhancing composition comprises NU 2058. In some embodiments, a titer-enhancing composition comprises Ro 3306. In some embodiments, a titer-enhancing composition comprises dinaciclib, NU 6140, NU 2058, Ro 3306, or a combination thereof.
  • a titer enhancing composition comprises two or more cell cycle modulators. In some embodiments, a titer-enhancing composition comprises dinaciclib and NU 6140. In some embodiments, a titer-enhancing composition comprises NU 2058. In some embodiments, a titer-enhancing composition comprises dinaciclib and NU 2058. In some embodiments, a titer-enhancing composition comprises dinaciclib and Ro 3306. In some embodiments, a titer-enhancing composition comprises NU 6140 and NU 2058. In some embodiments, a titer-enhancing composition comprises NU 6140 and Ro 3306. In some embodiments, a titer-enhancing composition comprises NU 2058 and Ro 3306.
  • a titer enhancing composition comprises a cell cycle modulator and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises dinaciclib and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises NU 6140 and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises NU 2058 and another (e.g., one or more) enhancer agent.
  • a titer-enhancing composition comprises Ro 3306 and another (e.g., one or more) enhancer agent.
  • compositions comprising an aqueous mammalian cell culture media.
  • the aqueous mammalian cell culture media is a complete media.
  • “complete media” refers to mammalian cell media that comprises basal media (e.g., amino acids, glucose, and ions such as calcium, potassium, sodium, and phosphate), serum, and additional vitamins and inorganic salts that allow for in vitro culture of mammalian cells.
  • the aqueous mammalian cell culture media is a serum-free media.
  • mammalian cell culture media examples include but are not limited to Minimum Essential Media (MEM), Dulbecco’s MEM (DMEM), RPMI 1640, F12, etc.
  • MEM Minimum Essential Media
  • DMEM Dulbecco’s MEM
  • RPMI 1640 RPMI 1640
  • F12 F12
  • a mammalian cell culture media is chemically defined (e.g., does not contain any animal, plant, or yeast-derived products).
  • compositions described herein are formulated as cell culture media additives.
  • a “cell culture media additive” refers to a composition comprising two or more (e.g., 2, 3, 4, 5, or more) enhancer agents that lacks aqueous mammalian cell culture media.
  • a cell culture media additive is formulated as a solid.
  • a cell culture media additive may be a composition comprising two or more enhancer agents in a powdered (e.g., lyophilized) form.
  • media additives are formulated in an aqueous solution.
  • two or more enhancer agents may be dissolved in a solvent, such as water, ethanol, DMSO, etc.
  • a cell culture media additive comprises sodium butyrate, and bufexamac. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, and tubacin. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, and caffeine. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, and sodium pentanoate. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, and chloroquine. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, and 2’ aminopurine.
  • a cell culture media additive comprises sodium butyrate, bufexamac, and nicotinamide. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, and caffeine. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, tubacin, and sodium pentanoate. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, tubacin, and chloroquine. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, tubacin, and 2’ aminopurine.
  • a cell culture media additive comprises sodium butyrate, bufexamac, tubacin, and nicotinamide.
  • a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, and sodium pentanoate.
  • a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, and chloroquine.
  • a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, and 2’ aminopurine.
  • a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, and nicotinamide sodium butyrate, bufexamac, tubacin, and nicotinamide.
  • a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, sodium pentanoate, and chloroquine In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, sodium pentanoate, and 2’ aminopurine. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, sodium pentanoate, and nicotinamide. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, sodium pentanoate, chloroquine, and 2’ aminopurine.
  • a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, sodium pentanoate, chloroquine, and nicotinamide.
  • a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, sodium pentanoate, chloroquine, 2’ aminopurine, and nicotinamide.
  • media additives described herein are added to a basal medium
  • compositions described herein are formulated as complete cell culture media.
  • a complete cell culture medium comprises amino acids, carbon sources, vitamins, minerals, growth factors, hormones, inorganic salts, and/or trace metals.
  • a complete cell culture medium comprises one or more amino acids in L or D form, selected from glycine, alanine, arginine, asparagine, aspartic acid, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and/or valine.
  • a complete cell culture medium comprises one or more carbon source selected from sugars or sugar alcohols, including glucose, galactose, maltose, fructose, and inositol.
  • a complete cell culture medium comprises one or more vitamins selected from B vitamins (riboflavin, folic acid, cyanocobalamin, thiamine, pyridoxine, biotin), A vitamins, D vitamins, and/or E vitamins.
  • a complete cell culture medium comprises one or more minerals selected from iron, zinc, manganese, copper, aluminum, molybdenum, vanadium, nickel, and/or tin.
  • a complete cell culture comprises growth factors, including serums (e.g., fetal bovine serum, bovine calf serum, equine serum, and/or porcine serum).
  • a complete cell culture medium comprises one or more minerals selected from calcium, zinc, iron, folate, and/or magnesium.
  • a complete cell culture medium comprises inorganic salts, including, but not limited to sodium chloride, lithium chloride, sodium selenite, and ammonium meta-vanadate.
  • a complete cell culture medium comprises one or more trace metals selected from iron, zinc, manganese, copper, aluminum, molybdenum, vanadium, nickel, and/or tin.
  • a complete cell culture comprises growth factors, including serums (e.g., fetal bovine serum, bovine calf serum, equine serum, and/or porcine serum).
  • the cell culture media formulations described herein are suitable for use with antibiotics.
  • the cell culture media formulations described herein are used for adherent cell cultures.
  • Adherent cultures comprise cells grown on a substrate surface suitable for cell growth.
  • the cell culture medium formulations described herein are used for suspension cultures.
  • Suspension cultures comprise cells grown in containers comprising large surface areas suitable for appropriate gas and nutrient exchange; suspension cultures may utilize agitation mechanisms, including stirring.
  • the complete cell culture medium formulations described herein are acceptable for pharmaceutical use.
  • compositions described herein may be used in conjunction with additional supplements which advantageously supply cells with nutrients, minerals, elements, and other components for optimal growth, expansion, and/or viral vector production.
  • additional supplements include insulin, transferrin, sodium selenium, hormones (e.g., D-aldosterone, diethylstilbestrol (DES), dexamethasone, P-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin/human growth hormone (HGH), thyrotropin, thyroxine and L-thyronine), lipids (cyclodextrin, cholesterol, linoleic acid, oleic acid, and derivatives thereof).
  • hormones e.g., D-aldosterone, diethylstilbestrol (DES), dexamethasone, P-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin/human growth hormone (HGH),
  • compositions and methods described herein can be used with any host cell suitable for production of viral vectors (e.g., a viral packaging cell).
  • a viral “packaging” or “producer” cell is a host cell which produces engineered viral particles in the absence of replication competent viruses.
  • a “host cell” is a cell capable of receiving or being transformed by a heterologous nucleic acid molecule (e.g., packaging or helper plasmids).
  • a host cell receives (e.g., is transfected, transduced, or otherwise contacted by) a heterologous nucleic acid.
  • a heterologous nucleic acid is a vector (e.g., a helper vector or helper plasmid).
  • a “vector” is a vehicle for the transfer of genetic material (e.g., nucleic acids) from a first environment into a second environment.
  • a vector is a plasmid.
  • a “plasmid” is a small, extra-chromosomal nucleic acid which comprises an origin of replication and at least one gene and is capable of autonomous replication.
  • One or more plasmids may be introduced to a host cell via transfection.
  • suitable transfection methods include electroporation, heat shock, liposomes, nanoparticles, microinjection, sonoporation, photoporation, magnetofection, hydroporation, biolistics, continuous infusion, and impalefection.
  • a vector is a virion or viral particle comprising packageable nucleic acids and structural and/or functional genetic elements derived primarily from a virus.
  • a viral vector is a retroviral vector (RVV), comprising structural and/or functional genetic elements derived primarily from a retrovirus.
  • RVVs may be any suitable retrovirus of the orthoretroviriniae sub-family (e.g., betaretrovirus, gamma-retrovirus, alpharetrovirus, deltaretrovirus, lentivirus, and epsilon retrovirus) or spumaretrovirinae subfamily (e.g., bovispumavirus, equispumavirus, felispumavirus, prosimiispumavirus, simiispumavirus).
  • a viral vector is a lentiviral vector (LVV), comprising structural and/or functional genetic elements derived primarily from a lentivirus.
  • LVVs may be any suitable lentivirus, including, but not limited to, human immunodeficiency virus (HIV; including HIV-1 and HIV-2), visna-maedi virus (VMV) virus, capine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV).
  • a viral vector is a gammaretroviral vector (GW), comprising structural and/or functional genetic elements derived primarily from gamma-retroviruses.
  • GW gammaretroviral vector
  • GVVs may be any suitable gamma-retrovirus, including, but not limited to, murine leukemia virus (MLV), bovine leukemia virus (BLV), chicken syncytial virus (CSV), feline leukemia virus (FLV), gibbon ape leukemia virus (GAL), koala retrovirus (KRV), and reticuloendotheliosis virus (RE).
  • MMV murine leukemia virus
  • BLV bovine leukemia virus
  • CSV chicken syncytial virus
  • FLV feline leukemia virus
  • GAL gibbon ape leukemia virus
  • KRV koala retrovirus
  • RE reticuloendotheliosis virus
  • the terms “retrovirus” and “retroviral vector/RVV” include lentiviruses and gamma-retroviruses, and their corresponding vectors, respectively.
  • a viral vector is an adeno-associated viral vector (AAV), comprising structural and/or functional genetic elements derived primarily from an adeno-associated virus.
  • AAVs may be of any suitable serotype, including, but not limited to, 1, 2, 4, 5, 7, 8, 9 or 10.
  • a viral vector comprises some or all of a viral genome.
  • a viral vector comprises a plasmid.
  • a viral vector comprises an envelope.
  • a viral vector comprises an envelope comprising viral surface proteins.
  • a viral vector comprises and envelope comprising viral surface proteins of a different virus.
  • viral packaging cells generate LVVs. In some embodiments, a viral packaging cell generates GRVs. In some embodiments, a viral packaging cell generates AAVs. In some embodiments, a viral packaging cell generates viral vectors comprising one or more transgenes. In some embodiments, viral packaging cells contacted by the enhancing compositions described herein produce higher titers of viral vectors relative to those not contacted by the enhancing compositions described herein.
  • a host cell is contacted with a composition described herein on the same day as the host cell is contacted with a mixture of PEI and viral packaging vectors. In some embodiments, a host cell is contacted with a composition described herein at the same time as the host cell is contacted with a mixture of PEI and viral packaging vectors.
  • a host cell is first contacted with a mixture of PEI and viral packaging or helper vectors, then contacted with a composition described herein (e.g., in a cell culture medium) within 1 minute, 1 minute to 30 minutes later, 1 minute to 1 hour later, 1 hour to 2 hours later, 2 hours to 4 hours later, 4 hours to 8 hours later, 6 hours to 12 hours later, 8 hours to 18 hours later, 12 hours to 20 hours later, 21 hours later, 22 hours later, 23 hours later, or within 24 hours.
  • a composition described herein e.g., in a cell culture medium
  • a host cell is first contacted with a mixture of PEI and viral packaging or helper vectors, then contacted with a composition described herein (e.g., in a cell culture medium) within 24 to 72 hours of the contacting with the PEI and viral packaging vectors.
  • a host cell is first contacted with a mixture of PEI and viral packaging or helper vectors, then contacted with a composition described herein (e.g., in a cell culture medium) within 24 hours, 24 hours to 36 hours later, 24 hours to 48 hours later, 36 hours to 60 hours later, 48 hours to 60 hours later, 48 hours to 72 hours later, or within 72 hours of the contacting with the PEI and viral packaging vectors.
  • a host cell is first contacted with a composition described herein (e.g., in a cell culture medium), then contacted with a mixture of PEI and viral packaging or helper vectors within 24 hours.
  • a host cell is first contacted with a composition described herein (e.g., in a cell culture medium), then contacted with a mixture of PEI and viral packaging or helper vectors within 1 minute, 1 minute to 30 minutes later, 1 minute to 1 hour later, 1 hour to 2 hours later, 2 hours to 4 hours later, 4 hours to 8 hours later, 6 hours to 12 hours later, 8 hours to 18 hours later, 12 hours to 20 hours later, 21 hours later, 22 hours later, 23 hours later, or within 24 hours.
  • a host cell is first contacted with a composition described herein (e.g., in a cell culture medium), then contacted with a mixture of PEI and viral packaging or helper vectors within 24 to 72 hours.
  • a host cell is first contacted with a composition described herein (e.g., in a cell culture medium), then contacted with a mixture of PEI and viral packaging or helper vectors within 24 hours, 24 hours to 36 hours later, 24 hours to 48 hours later, 36 hours to 60 hours later, 48 hours to 60 hours later, 48 hours to 72 hours later, or within 72 hours.
  • transduced and/or transfected host cells are contacted with a composition described herein for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 36, 48, 72, or more hours by adding the composition to the culture medium without changing the culture medium.
  • packaging cells are contacted with a first composition for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 36, 48, 72, or more hours by adding the composition to the culture medium, and then contacted with a second composition for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 36, 48, 72, or more hours by adding the composition to the culture medium.
  • the first and second compositions may comprise the same enhancer agents or different enhancer agents.
  • packaging cells are contacted with a composition 1, 2, 3, 4, 5, or more times.
  • a cell is contacted with a second, third, fourth, etc. composition by changing the culture media.
  • a cell is contacted with a second, third, fourth, etc. composition without changing the culture medium.
  • titer enhancing compositions may comprise packaging cells.
  • the packaging cells and used compositions may comprise viruses, viral particles, viral vectors, and/or components thereof (e.g., nucleic acids, proteins).
  • viruses, viral particles, viral vectors, and/or components thereof may be collected (e.g., isolated) from the cells and/or supernatant according to conventional techniques.
  • viruses, viral particles, viral vectors, and/or components thereof are collected from the cells and/or supernatant in a single harvest.
  • viruses, viral particles, viral vectors, and/or components thereof are collected from the cells and/or supernatant in multiple harvests. In some embodiments, viruses, viral particles, viral vectors, and/or components thereof are collected from the cells and/or supernatant 24, 36, 48, 72, or more hours after contact with the titer enhancing compositions described herein.
  • Collected viral particles may be further purified if desired by suitable purification techniques (including ultracentrifugation, ion exchange chromatography, tangential flow filtration) known to those of skill in the art.
  • suitable purification techniques including ultracentrifugation, ion exchange chromatography, tangential flow filtration
  • Packaging cells treated with the compositions and/or methods described herein may generate titers of millions of transducing units per milliliter (TU/mL).
  • Viral stocks obtained from packaging cells treated with the compositions and/or methods described herein may result in titers of about 10 2 TU/mL, 10 3 TU/mL , 10 4 TU/mL, 10 5 TU/mL, 10 6 TU/mL, 10 7 TU/mL IO 8 TU/mL, IO 9 TU/mL, IO 10 TU/mL, IO 11 TU/mL, 10 12 TU/mL, IO 13 TU/mL, 10 14 TU/mL, 10 15 TU/mL, 10 16 TU/mL, 10 17 TU/mL, IO 18 TU/mL, IO 19 TU/mL, or IO 20 TU/mL.
  • Viral titer may be measured using any number of conventional techniques, including, but not limited to, viral plaque assays, focus forming assays, 50% tissue culture infectious dose assays, and enzyme-linked immunosorbent assays, such as commercially available p24 titer assays.
  • Example 1 Treatment with sodium butyrate, panobinostat, or vorinostat improves viral titer during transfection of suspension cells
  • Sodium butyrate an HD AC inhibitor
  • sodium butyrate has only been shown to be effective for increasing viral titer when added at specific concentrations and during specific time points.
  • HD AC inhibitors include panobinostat (having the chemical structure shown in FIG. 1A) is a hydroxamic acid HD AC inhibitor that differs significantly from sodium butyrate (chemical structure shown in FIG. IB).
  • panobinostat panobinostat
  • vorinostat i.e., SAHA
  • suspension HEK 293 suspension cells were expanded in a 10 cm culture dish in 10 ml LV Max media (3% Fetal Bovine Serum (FBS); 1% Penicillin-Streptomycin (PS)). Cells were dispensed into T125 flasks at 20mL volume with a cell density of 4xl0 6 cells/mL. On day 2 (24 hours post-expansion), cells were transfected with 20pg of 3:1 (pL:pg) ratio of polyethylenimine (PEI): lentiviral packaging DNA plasmid (DNA) mixture.
  • PEI polyethylenimine
  • DNA lentiviral packaging DNA plasmid
  • the DNA mixture consisted of four standard plasmids encoding various viral vector production or helper components, including: 14.75pg VSV-G plasmid, 24.4pg Gag-Pol plasmid, 12.06pg Rev plasmid, and 48.8pg of CARCD19 or GFP-encoding plasmids, per lOOpg of DNA mixture.
  • On day 2 (with transfection) or day 3 (24 hours post-transfection) cells were treated with: 5mM sodium butyrate (in water), lOmM sodium butyrate (in water), I Op m Panobinostat (in DMSO), or 10pm vorinostat (in DMSO).
  • Viral supernatant was collected from the cells on Day 4 (48hrs post-transfection), centrifuged at 300g for 5 mins, and filtered through a 0.45-micron filter. Filtered viral supernatant was frozen as aliquots of 1 ml or greater.
  • Jurkat cells (2.75xl0 5 cells) were plated in a flat-bottom 96 well plate (200pl/well), transduced with O.lpL-lpL of viral supernatant collected from cells transfected as described above, and incubated.
  • expression of viral vector encoded GFP was analyzed using fluorescent activated cell sorting (FACS) method. Titer was calculated using standard formulas in the art, for example as shown below.
  • suspension HEK293 suspension cells were expanded in a 10 cm culture dish in lOmL LV Max (10% FBS, 1% PS) culture media.
  • lOmL LV Max 10% FBS, 1% PS
  • cells were dispensed into T125 flasks at 20ml volume with a cell density of 4xl0 6 cells/ml.
  • cells were treated with lOpM Panobinostat (in DMSO).
  • cell suspension was harvested from the cells and centrifuged at 300g for 5 mins and filtered through a 0.45-micron filter. Filtered cell suspension was frozen as aliquots of O.lpL-lmL or larger volumes.
  • panobinostat at both Ohrs and 24hrs post- transfection was found to increase viral titer by 4.3- to 23-fold in suspension cells; similar findings were observed for vorinostat, which increased viral titer by 3.5- to 23.8-fold.
  • panobinostat and vorinostat may be useful for increasing viral titer in lentiviral producer suspension cells.
  • Example 2 Treatment with sodium butyrate, panobinostat, or vorinostat improves viral titer during transfection of adherent cells
  • HEK 293T cells On Day 1, adherent HEK 293T cells (seeded at 80,000 cells/cm 2 ) were expanded in a 10 cm culture dish in 10 ml DMEM media (10% Fetal Bovine Serum (FBS); 1% Penicillin- Streptomycin (PS)). On day 2 (24 hours post-expansion), cells (60-80% confluence) were transfected with O.lpg/cm 2 of 3:1 (pL:pg) ratio of PEEDNA.
  • FBS Fetal Bovine Serum
  • PS Penicillin- Streptomycin
  • the DNA mixture consisted of four standard plasmids encoding various viral vector production components, including: 14.75pg VSV-G plasmid, 24.4pg Gag-Pol plasmid, 12.06pg Rev plasmid, and 48.8pg of CARCD19 or GFP-encoding plasmids, per lOOpg of DNA mixture.
  • FIG. 3A for GFP expression
  • FIG. 3B for GFP and CD 19 GFP expression.
  • panobinostat treatment generally surpassed treatment other additives, increasing viral titer by 1.4- to 7.68-fold in adherent cells, though vorinostat also provided viral titer increases of 1.3 to 6.9-fold.
  • panobinostat and vorinostat may be useful for increasing viral titer in lentiviral producer suspension cells.
  • Example 3 Higher doses of panobinostat, or vorinostat improves viral titer during transfection of suspension cells and adherent cells
  • Panobinostat has previously been shown to have cytotoxic effects in Jurkat cells; however, findings in Example 1 and Example 2 indicate potential use in improving viral titer. In this Example, the effects of higher doses of panobinostat on viral titer during transfection were assessed.
  • Suspension cells HEK 293 suspension cells
  • adherent cells HEK 293T cells
  • panobinostat lOpM, 20pM, or 40pM
  • viral supernatant volumes used for transduction in 20pM and 40pM panobinostat conditions were reduced (0.05pL-0.5pL) due to potential toxicity of panobinostat.
  • 1 to O.lpl virus volume was used for transfection after producer cells were treated with 20 to 100 pM panobinostat.
  • Panobinostat transduction controls were prepared and viral titer was assessed as described in Example 1.
  • FIG. 4A shows representative experiments comparing 5mM sodium butyrate and lOpM panobinostat on viral titer in lentiviral producer suspension cells. Similar to the results found in Example 1, panobinostat was at least comparable to sodium butyrate (FIG. 4A, BOTTOM; Table 1 (Run 2)), and induced up to a 19.1-fold improvement in viral titer (FIG. 4A, TOP; Table 1 (Run 1)). Surprisingly, increased doses of panobinostat (20pM and 40pM) during transfection yielded similar or higher viral titer compared to the I Op M condition (FIG. 4B, TOP, BOTTOM; Table 1 (Runs 3-4)). Table 1. Panobinostat-induced viral titer effects in lentiviral producer HEK293 suspension cells
  • FIG. 4C shows a representative experiment comparing 5mM sodium butyrate and lOpM or 20pM panobinostat on viral titer in lentiviral producer adherent cells. Similar to the results found in Example 2, panobinostat was at least comparable to sodium butyrate (FIG. 4C; Table 2); notably, and similar to results in suspension cells, higher doses of panobinostat further improved viral titer.
  • panobinostat are useful for increasing viral titer in lentiviral producer suspension cells.
  • Suspension cells (HEK 293 suspension cells) were prepared as described in Example 1, respectively, differing only by addition of panobinostat (lOpM), mocetinostat (lOpM), or both (lOpM each). Viral titer was assessed as described in Example 1.
  • panobinostat and mocetinostat increased lentiviral titer in suspension cells by as much 4.3-fold relative to control, equivalent to a roughly 2-fold increase relative to panobinostat alone and about 27% better than sodium butyrate alone.
  • the viral titer increases induced by both panobinostat and mocetinostat is greater than additive, suggesting a synergistic effect of the combination.
  • panobinostat and mocetinostat in combination significantly improve lentiviral titer over the leading enhancer agent, sodium butyrate.

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Abstract

Des aspects de la divulgation concernent des compositions et des procédés de production de vecteurs viraux recombinants (par exemple, des particules lentivirales recombinantes) dans des cellules de mammifère. La divulgation concerne, en partie, des combinaisons d'agents qui modulent un ou plusieurs processus de cellule hôte tels que le transport d'acide nucléique, la modulation épigénétique de l'expression génique et la transduction virale. Dans certains modes de réalisation, des combinaisons d'agents décrits par la divulgation sont utiles en tant qu'additifs de milieux de culture de cellules de mammifère. Dans certains modes de réalisation, des compositions et des procédés décrits par la divulgation conduisent à une production accrue de particules virales par des cellules hôtes par rapport à des cellules hôtes qui n'ont pas été traitées avec les compositions. Dans certains modes de réalisation, les compositions décrites par la divulgation améliorent le titre vectoriel par amélioration de l'efficacité avec laquelle des particules virales produites à l'aide des procédés de l'invention infectent des cellules cibles.
PCT/IB2024/000498 2023-09-12 2024-09-11 Additifs de culture de cellules de mammifère Pending WO2025056970A1 (fr)

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