Mammalian cell culture additives

Abstract

Aspects of the disclosure relate to compositions and methods for production of recombinant viral vectors (e.g., recombinant lentiviral particles) in mammalian cells. The disclosure relates, in part, to combinations of agents that modulate one or more host cell processes such as nucleic acid transport, epigenetic modulation of gene expression, and viral transduction. In some embodiments, combinations of agents described by the disclosure are useful as mammalian cell culture media additives. In some embodiments, compositions and methods described by the disclosure result in increased viral particle production by host cells relative to host cells that have not been treated with the compositions. In some embodiments, compositions described by the disclosure enhance the vector titer by enhancing the efficiency with which viral particles produced using methods of the disclosure infect target cells.

Description

[0001] MAMMALIAN CELL CULTURE ADDITIVES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. 119(e) of U.S. provisional Application Serial Number 63 / 581,994, entitled “MAMMALIAN CELL CULTURE ADDITIVES”, filed on September 12, 2023, the entire contents of which are incorporated by reference herein.

[0004] BACKGROUND OF INVENTION

[0005] Recombinant retroviral vectors, such as lentivirus vectors (LVVs), show great promise for industrial, research, and clinical use. LVVs are used in research settings to introduce heterologous gene products into cells and animal models and are increasingly of interest for the delivery of genetic treatments of human diseases. However, the production of recombinant retroviral vectors, especially LVVs, at sufficient yield is difficult and costly. Low viral titers resulting from standard retroviral vector production methods greatly hinder the practicality of further industrial, research, and clinical use.

[0006] SUMMARY OF INVENTION

[0007] The disclosure relates, in some aspects, to compositions and methods for production of recombinant viral vectors (e.g., recombinant lentiviral particles) in mammalian cells. In some embodiments, combinations of agents are disclosed herein that modulate one or more host cell (e.g., mammalian cell) processes, for example nucleic acid transport, epigenetic modulation of gene expression, and viral transduction. In some embodiments, the agents are small molecules. In some embodiments, compositions comprising combinations of agents described by the disclosure may be added to mammalian cell culture media to increase or improve production of viral vectors by the mammalian cells. The disclosure also relates, in some aspects, to methods for producing viral vectors in a mammalian cell culture system. In some embodiments, 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. Accordingly, in some aspects, 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. In some embodiments, each of the first enhancer agent and second enhancer agent is a small molecule.

[0008] In some embodiments, a mammalian cell culture media is a complete cell culture media. In some embodiments, a mammalian cell culture media is chemically defined.

[0009] In some embodiments, a composition lacks serum (e.g., is a serum- free media). In some embodiments, the serum comprises human serum albumin (HSA).

[0010] In some embodiments, an endosomal release promoting agent comprises pseudouridine, N1 -methylpseudouridine (ml'P), or 5-methyl-cytidine.

[0011] In some embodiments, a nuclear localization promoting agent comprises a polyamine. In some embodiments, a polyamine comprises putrescine, spermine, and / or spermidine.

[0012] In some embodiments, a chromatin modifier comprises a histone deacetylase (HD AC) inhibitor.

[0013] In some embodiments, 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).

[0014] In some embodiments, an epigenetic modulator comprises 5 '-Azacytidine or 5'-aza-2'- deoxycytidine (decitabine).

[0015] In some embodiments, a DNA repair modulator comprises caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist. In some embodiments, an adenosine receptor agonist comprises wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, or a PARP inhibitor (e.g., Olaparib).

[0016] In some embodiments, a cell cycle modulator comprises a cyclin dependent kinase modulator (e.g., a cyclin dependent kinase inhibitor), hydroxyurea, or nocodazole. In some embodiments, a cyclin dependent kinase modulator is a cyclin dependent kinase inhibitor selected from Dinaciclib, NU6140, NU2058, and RO-3306. In some embodiments, 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.

[0017] In some embodiments, each of a first enhancer agent and a second enhancer agent is an endosomal release promoting agent. In some embodiments, a first enhancer agent is pseudouridine, or N1 -methylpseudouridine (ml'P), and the second enhancer agent is 5-methyl- cytidine.

[0018] In some embodiments, a first enhancer agent is an endosomal release promoting agent and a second enhancer agent is a nuclear localization promoting agent. In some embodiments, an endosomal release promoting agent is pseudouridine, ml'P, or 5-methyl-cytidine, and a nuclear localization promoting agent is putrescine, spermine, or spermidine.

[0019] In some embodiments, a first enhancer agent is an endosomal release promoting agent and a second enhancer agent is an epigenetic modulator. In some embodiments, an endosomal release promoting agent is pseudouridine, ml'P, or 5-methyl-cytidine, and an epigenetic modulator is 5 '-Azacytidine or 5'-aza-2'-deoxycytidine (decitabine).

[0020] In some embodiments, a first enhancer agent is an endosomal release promoting agent and a second enhancer agent is a cell cycle modulator. In some embodiments, an endosomal release promoting agent is pseudouridine, ml'P, or 5-methyl-cytidine, and a cell cycle modulator is a cyclin dependent kinase modulator (e.g., inhibitor), hydroxyurea, or nocodazole. In some embodiments, an endosomal release promoting agent comprises hydroxyurea and nocodazole. In some embodiments, an endosomal release promoting agent is 5-methyl-cytidine and a cell cycle modulator is hydroxyurea. In some embodiments, an endosomal release promoting agent is 5- methyl-cytidine and a cell cycle modulator is nocodazole.

[0021] In some embodiments, a first enhancer agent is a nuclear localization promoting agent and a second enhancer agent is an epigenetic modifier. In some embodiments, a nuclear localization promoting agent is putrescine, spermine, or spermidine, and an epigenetic modifier is 5 '-Azacytidine or 5 '-aza-2'-deoxy cytidine (decitabine). In some embodiments, a nuclear localization promoting agent is putrescine and an epigenetic modifier is 5-Azacytidine. In some embodiments, a nuclear localization promoting agent is putrescine and an epigenetic modifier is5'-aza-2'-deoxycytidine (decitabine). In some embodiments, 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).

[0022] In some embodiments, a first enhancer agent is a nuclear localization promoting agent and a second enhancer agent is a cell cycle modulator. In some embodiments, a nuclear localization promoting agent is putrescine, spermine, or spermidine, and a cell cycle modulator is a cyclin dependent kinase modulator (e.g., inhibitor), hydroxyurea, or nocodazole. In some embodiments, a nuclear localization promoting agent is putrescine, and a cell cycle modulator is a cyclin dependent kinase inhibitor. In some embodiments, the cyclin dependent kinase inhibitor is Dinaciclib.

[0023] In some embodiments, a first enhancer agent is a chromatin modifier, and a second enhancer agent is an endosomal release promoting agent. In some embodiments, 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. In some embodiments, an HD AC inhibitor is sodium butyrate and an endosomal release promoting agent is pseudouridine. In some embodiments, an HD AC inhibitor is vorinostat and an endosomal release promoting agent is pseudouridine.

[0024] In some embodiments, a first enhancer agent is a chromatin modifier and a second enhancer agent is a nuclear localization promoting agent. In some embodiments, 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. In some embodiments, an HD AC inhibitor is sodium butyrate and a nuclear localization promoting agent is putrescine.

[0025] In some embodiments, a first enhancer agent is a chromatin modifier and a second enhancer agent is an epigenetic modulator. In some embodiments, 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). In some embodiments, 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).

[0026] In some embodiments, a first enhancer agent is a chromatin modifier and a second enhancer agent is cell cycle modulator. In some embodiments, 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. In some embodiments, an HDAC inhibitor is sodium butyrate and a cell cycle modulator is a cyclin dependent kinase inhibitor. In some embodiments, a cyclin dependent kinase inhibitor is Dinaciclib.

[0027] In some embodiments, a first enhancer agent is an epigenetic modulator and a second enhancer agent is cell cycle modulator. In some embodiments, 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. In some embodiments, an epigenetic modulator is 5 '-Azacytidine and a cell cycle modulator is a cyclin dependent kinase inhibitor. In some embodiments, an epigenetic modulator is 5'-aza-2'-deoxycytidine (decitabine) and a cell cycle modulator is a cyclin dependent kinase inhibitor. In some embodiments, a cyclin dependent kinase inhibitor is Dinaciclib.

[0028] In some embodiments, a first enhancer agent is a DNA repair modulator and a second enhancer agent is an endosomal release promoting agent. In some embodiments, 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. In some embodiments, a DNA repair modulator is caffeine and an endosomal release promoting agent is pseudouridine. In some embodiments, 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. In some embodiments, 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. In some embodiments, a DNA repair modulator is caffeine and a nuclear localization promoting agent is putrescine. In some embodiments, a DNA repair modulator is theophylline and a nuclear localization promoting agent is putrescine. In some embodiments, a DNA repair modulator is theobromine and a nuclear localization promoting agent is putrescine. In some embodiments, a DNA repair modulator is hydroxychloroquine and a nuclear localization promoting agent is putrescine.

[0029] In some embodiments, a first enhancer agent is a DNA repair modulator and a second enhancer agent is an epigenetic modulator. In some embodiments, 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). In some embodiments, a DNA repair modulator is caffeine and an epigenetic modulator is 5-Azacytidine. In some embodiments, 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.

[0030] In some embodiments, a first enhancer agent is a DNA repair modulator and a second enhancer agent is a cell cycle modulator. In some embodiments, 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. In some embodiments, a DNA repair modulator is caffeine and a cell cycle modulator is a cyclin dependent kinase inhibitor. In some embodiments, 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.

[0031] In some aspects, the disclosure provides a cell culture media additive comprising sodium butyrate, and bufexamac and / or tubacin. In some aspects, the disclosure provides a cell culture media additive comprising panobinostat. In some aspects, the disclosure provides a cell culture media additive comprising vorinostat. In some aspects, the disclosure provides a cell culture media additive comprising panobinostat and mocetinostat. In some embodiments, the cell culture media additive further comprises caffeine. In some embodiments, the cell culture media additive further comprises sodium pentanoate. In some embodiments, the cell culture media additive further comprises chloroquine and / or 2’ aminopurine. In some embodiments, the cell culture media additive further comprises nicotinamide.

[0032] In some embodiments, a cell culture media additive is formulated as an aqueous solution. In some embodiments, a cell culture media additive is formulated as a solid.

[0033] In some aspects, the disclosure provides a composition comprising a population of mammalian host cells; and a composition or cell culture media additive as described by the disclosure.

[0034] In some embodiments, a population of mammalian host cells comprises HEK293 cells or derivatives thereof.

[0035] In some embodiments, host cells comprise one or more viral vector production components, for example lentivirus production vectors, AAV production vectors, adenovirus production vectors, gamma-retrovirus production vectors, etc. In some embodiments, host cells comprise one or more of the following: a lentivirus transfer vector, a lentivirus packaging vector, and / or a lentivirus envelope vector.

[0036] In some aspects, the disclosure provides a cell culture composition comprising cell culture media additive comprising between about 5 pM and about 15 pM panobinostat, and between about 5 pM and about 15 pM mocetinostat; and aqueous mammalian cell culture media.

[0037] In some embodiments, the mammalian cell culture media is complete. In some embodiments, the mammalian cell culture media is chemically defined. In some embodiments, the cell culture composition lacks serum. In some embodiments, the serum comprises human serum albumin (HSA). In some embodiments, the panobinostat and mocetinostat are present at a ratio of 1:1. In some embodiments, the cell culture composition comprises 10 pM panobinostat and 10 p M mocetinostat.

[0038] In some aspects, the disclosure provides a method of culturing mammalian cells, the method comprising contacting a population of mammalian cells with a composition or cell culture media additive as described by the disclosure.

[0039] In some embodiments, the method further comprises culturing the population of mammalian cells in the presence of the composition or cell culture media additive, under conditions under which recombinant viral particles are produced. In some embodiments, the recombinant viral particles are recombinant lentivirus particles. In some embodiments, culturing the population of mammalian cells using methods described herein results in an increased titer of recombinant viral particles (e.g., recombinant lentivirus particles) being produced by the population of mammalian cells relative to a population of mammalian cells that has not been cultured in the presence of the composition or cell culture media additive.

[0040] In some embodiments contacting the mammalian cells with the composition occurs between about 1 minute before and about 24 hours after the mammalian cells are contacted with one or more viral packaging vectors.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGs. 1A-1B are molecular structures of exemplary cell culture media additives.

[0043] FIG. 1A is the chemical structure of panobinostat. FIG. IB is the chemical structure of sodium butyrate.

[0044] FIGs. 2A-2B show that small molecules enhancers are effective in increasing lentiviral vector titer from HEK293 suspension cells. Supernatant was harvested 72h post-transfection from HEK293 suspension cells (4xl06cells / mL in LV max media) transfected with a mix of 3:1 PEI:DNA(pL:pg) and treated with no enhancing agents (“Control”), sodium butyrate (5mM or lOmM), panobinostat (lOpM), or vorinostat (lOpM) were added at Ohrs or 24 hrs relative to transduction. Virus titer (TU / mL) was calculated from GFP or CD 19 GFP expression in Jurkat cells using FACS, 5 days after transduction with filtered viral supernatant. Panobinostat transduction control is also shown. FIG. 2A (top, bottom) shows results from two representative experiments in which viral titer was calculated from CD 19 GFP expression in Jurkat cells 24 hours after transduction with filtered viral supernatant harvested from HEK293 suspension cells treated with lOpM panobinostat or lOpM vorinostat during (0 hours) or 24 hours after transfection. FIG. 2B (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 lOpM panobinostat or lOpM vorinostat 24 hours after transfection.

[0045] FIGs. 3A-3B show that small molecules enhancers are effective in increasing lentiviral vector titer from adherent 293T cells. Supernatant was harvested 72h post-transfection from adherent 293T cells (60-80% confluent in DMEM media) transfected with a lentiviral master mix of 3:1 PEEDNA and treated with no enhancing agents (“Control”), sodium butyrate (5mM or lOmM), panobinostat (lOpM), or vorinostat (lOpM) were added at Ohrs and / or 24 hrs relative to transfection. Virus titer (TU / mL) was calculated from GFP or CD 19 GFP expression in Jurkat cells using FACS, 5 days after transduction with filtered viral supernatant. FIG. 3A (top, middle, bottom) shows results from three representative experiments in which viral titer was calculated from CD19 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 lOpM vorinostat during or 24 hours after transfection. Panobinostat transduction control is also shown. FIG. 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.

[0046] 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 (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 or lOpM panobinostat 24 hrs after transfection. FIG. 4B (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 panobinostat, or 40pM panobinostat 24 hrs after transfection. FIG. 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.

[0047] 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.

[0048] DETAILED DESCRIPTION OF INVENTION

[0049] Despite the promise of lentiviral vectors for gene therapies, standard methods for viral vector (e.g., lentiviral, AAV, etc.) productions have often had limited success in producing titers high enough for practical use outside of small research settings, especially for vectors comprising large genes. Current methods typically include optimizing packaging plasmid transduction procedures and / or harvesting techniques (e.g., ion exchange and tangential flow filtration). Described herein are 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. The disclosure is 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.

[0050] Typically, viral replication and packaging in a mammalian host cell comprise several stages. Retroviruses (e.g., lentiviruses, y-retroviruses), for example, convert their viral genome into a double-stranded DNA (dsDNA) molecule with reverse transcriptase; the resulting dsDNA is then transported to the nucleus, where it is integrated into the host cell genome by an integrase. Integrated retroviral genes are transcribed by host cell machinery into retroviral RNA, which is exported from the nucleus. Once in the cytoplasm, some retroviral RNA is translated into retroviral peptides; the resulting RNA and peptides are then packaged into retroviral particles (e.g., capsids) and, optionally enveloped. 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.

[0051] Disclosed herein are viral 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.

[0052] Enhancer Agents

[0053] Aspects of the disclosure relate to titer enhancing 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). As used herein, 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.

[0054] In some aspects, the disclosure provides compositions, for example cell culture media and / or cell culture media additives, comprising two or more enhancer agents. In some embodiments, a composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more enhancer agents. In some embodiments, 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. In some embodiments, a composition comprises 2 or more (e.g., 2, 3, 4, 5, or more) endosomal release promoting agents. In some embodiments, a composition comprises 2 or more (e.g., 2, 3, 4, 5, or more) nuclear localization promoting agents. In some embodiments, 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.

[0055] In some embodiments, an enhancer agent is an endosomal release promoting agent. As used herein, 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. In some embodiments, an endosomal release promoting agent promotes endosomal membrane fusion. In some embodiments, an endosomal release promoting agent promotes destabilization of an endosomal membrane. In some embodiments, an endosomal release promoting agent promotes the formation of endosomal pores. In some embodiments, 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.

[0056] In some embodiments, 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. In some embodiments, a nuclear localization promoting agent stabilizes cargo for transport into the nucleus. In some embodiments, a nuclear localization promoting agent promotes the formation of nuclear pore complexes. In some embodiments, 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. In some embodiments, 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. In some embodiments, a nuclear localization promoting agent increases the availability of guanine triphosophates (GTP) in a host cell (e.g., a vector producing cell). In some embodiments, a nuclear localization promoting agent increases the availability of GTPase enzymes in a host cell (e.g., vector producing cell) or a target cell. In some embodiments, a nuclear localization promoting agent increases the availability of Ran in a host cell. In some embodiments, 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. Typically, polyamines are polycations. A polyamine may be naturally occurring or synthetic. In some embodiments, a polyamine is a small molecule. In some embodiments, a polyamine interacts with negatively charged molecules, for example DNA, RNA, and / or negatively charged peptides. Without wishing to be bound by any particular theory, 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). In some embodiments, a nuclear localization promoting agent is a polyamine. In some embodiments, an enhancer agent is a polyamine selected from putrescine, spermine, and spermidine.

[0057] In some embodiments, 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. In some embodiments, a chromatin modifier enables the incorporation of histone variants into the organization of chromatin. In some embodiments, a chromatin modifier remodels chromatin. In some embodiments, a chromatin modifier regulates post translational modifications to histones (e.g., phosphorylation, acetylation, methylation, ubiquity lation). In some embodiments, a chromatin modifier is a histone deacetylase (HD AC) inhibitor. Human histone deacetylases are generally grouped into several classes. Class I HDACs include HDAC1, HDAC2, HDAC3, and HDAC8. Class IIA HDACs include HDAC4, HDAC5, HDAC7, and HDAC9. Class IIB HDACs include HDAC6 and HDAC10. Class III HDACs are referred to as sirtuins and include SIRT1-SIRT7. Class IV HD AC includes only HDAC11. HD AC inhibitors are a class of small molecules that inhibit histone deacetylases, which remove the acetyl groups from the lysine residues of core histones and mediate formation of a condensed and transcriptionally silenced chromatin. A HD AC inhibitor may be a direct inhibitor (e.g., an inhibitor that binds directly to an HD AC protein or nucleic acid encoding an HD AC protein) or an indirect inhibitor (e.g., an inhibitor that binds to a modulator of an HD AC protein). In some embodiments, an HDAC inhibitor is a selective HDAC inhibitor. A “selective HD AC inhibitor” binds to and inhibits a single HDAC protein (e.g., HDAC6). In some embodiments, an HDAC inhibitor is a non-selective HDAC inhibitor. A “non-selective HDAC inhibitor” binds to and inhibits two or more of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11. Examples of chromatin modifiers include sodium butyrate, sodium propionate, vorinostat (also known as suberoylanilide hydroxamic acid (SAHA)), romidepsin (FK228), belinostat (PXD101), givinostat (ITF2357), panobinostat (EBH589), mocetinostat (MGCD0103), valproic acid, trichostatin A (TSA), bufexamac, and / or tubacin. In some embodiments, 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. In some embodiments, 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. In some embodiments, a composition comprises panobinostat and another chromatin modifier. In some embodiments, a composition comprises panobinostat and vorinostat. In some embodiments, a compositions comprises panobinostat and mocetinostat.

[0058] In some embodiments, HDAC6 and HDAC10 have been observed to have anti-HIV activity. Without wishing to be bound by any particular theory, inhibition of certain HDACs (e.g., HDAC6 and / or HD AC 10) may result in increased viral particle production. Thus, in some embodiments, a composition comprises an enhancer agent (e.g., HD AC inhibitor) that inhibits HDAC6 and / or HD AC 10, for example bufexamac or panobinostat.

[0059] In some embodiments, 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.

[0060] In some embodiments, an enhancer agent is an epigenetic modulator. An “epigenetic modulator” is a small molecule which alters the activity of methyltransferases. In some embodiments, an epigenetic modulator inhibits the activity of methyltransferases, thereby inhibiting DNA methylation. In some embodiments, an “epigenetic modulator” is not a “chromatin modifier.” In some embodiments, an enhancer agent is an epigenetic modulator selected from 5 '-azacytidine, 5 '-aza-2'-deoxy cytidine (decitabine), and dimethyl sulfoxide (DMSO). In some embodiments, a composition comprises 2, 3, 4, 5, or more epigenetic modulators, for example 5 '-azacytidine and 5'-aza-2'-deoxycytidine (decitabine), etc. In some embodiments, an enhancer agent is a DNA repair modulator. As used herein, a “DNA repair modulator” is a small molecule which modulates the activity of DNA repair mechanisms and / or frequency in a host cell. In some embodiments, DNA repair modulators alter the activity or availability of enzymes and proteins which mediate base excision repair mechanisms. In some embodiments, DNA repair modulators alter the activity or availability of enzymes and proteins which mediate nucleotide excision repair mechanisms. In some embodiments, 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. In some embodiments, an enhancer agent is 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. In some embodiments, a composition comprises 2, 3, 4, 5, or more DNA repair modulators, for example caffeine and chloroquine, caffeine and a PARP inhibitor, etc.

[0061] In some embodiments, an enhancer agent is a cell cycle modulator. A “cell cycle modulator” is a small molecule which alters the normal (e.g., non-cancerous) transition of a host cell from one stage of the cell cycle to another. In some embodiments, a cell cycle modulator arrests a host cell in one stage of the cell cycle (e.g., S-phase). In some embodiments, a cell cycle modulator arrests a host cell in the transition from one stage of the cell cycle to another (e.g., G2-M transition). In some embodiments, cell cycle modulators inhibit cellular proliferation. In some embodiments, cell cycle modulators inhibit apoptotic factors. Examples of cell cycle modulators include but are not limited to hydroxyurea, nocodazole, Akti-1, Akti-2, and cyclin dependent kinase inhibitors. As used herein “cyclin dependent kinase inhibitor” or “CDK inhibitor” refers to a small molecule that inhibits cyclin dependent kinases (CDKs), which are serine threonine kinase proteins that require a cyclin domain to function. Typically, CDKs play an important role in cell cycle progression. Without being bound by any particular theory, inhibition of CDKs promotes viral replication and packaging by slowing cell growth and maintaining host cells in a phase amenable for viral reproduction. 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). In some embodiments, a CDK inhibitor is a selective CDK inhibitor. A “selective CDK inhibitor” binds to and inhibits a single CDK protein (e.g., CDK2). In some embodiments, 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. In some embodiments, the cyclin dependent kinase inhibitor is selected from dinaciclib, NU 6140, NU 2058, and Ro 3306. In some embodiments, cell cycle modulator is selected from hydroxyurea, nocodazole, Akti-1, and Akti-2.

[0062] In some embodiments, 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.

[0063] 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.

[0064] In some embodiments, a composition comprises a total of IpM to lOOpM 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 IpM to 5pM, IpM to lOpM, 5pM to lOpM, IpM to 15pM, 5pM to 15pM, lOpM to 15pM, IpM to 20pM, 5pM to 20pM, lOpM to 20pM, 15pM to 20pM, IpM to 25pM, 5pM to 25pM, lOpM to 25pM, 15pM to 25pM, 20pM to 25pM,lpM to 30pM, 5pM to 30pM, lOpM to 30pM, 15pM to 30pM, 20pM to 30pM, 25pM to 30pM. In some embodiments, a composition comprises 30pM to 50pM, 40pM to 60pM, 50pM to 70pM, 60pM to 80pM, or 80pM to lOOpM 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0065] In some embodiments, 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). In some embodiments, a composition comprises a total of 5pM of panobinostat. In some embodiments, a composition comprises a total of lOpM of panobinostat. In some embodiments, a composition comprises a total of 15pM of panobinostat. In some embodiments, a composition comprises a total of 20pM of panobinostat. In some embodiments, 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.

[0066] In some embodiments, 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). In some embodiments, a composition comprises a total of 5pM of vorinostat. In some embodiments, a composition comprises a total of lOpM of vorinostat. In some embodiments, a composition comprises a total of 15pM of vorinostat. In some embodiments, a composition comprises a total of 20pM of vorinostat. In some embodiments, 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.

[0067] In some embodiments, 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). In some embodiments, a composition comprises a total of 5pM of mocetinostat. In some embodiments, a composition comprises a total of lOpM of mocetinostat. In some embodiments, a composition comprises a total of 15pM of mocetinostat. In some embodiments, a composition comprises a total of 20pM of mocetinostat. In some embodiments, 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.

[0068] In some embodiments, 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). In some embodiments, a composition comprises a total of 5pM of panobinostat and mocetinostat. In some embodiments, a composition comprises a total of lOpM of panobinostat and mocetinostat. In some embodiments, a composition comprises a total of 15pM of panobinostat and mocetinostat. In some embodiments, 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.

[0069] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 more. In some embodiments, a titer enhancing composition further comprises four, five, six, seven, eight, nine, ten, or more enhancer agents.

[0070] In some embodiments, 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. In some embodiments, a second enhancer agent is an endosomal release promoting agent, a nuclear localization promoting agent, an epigenetic modulatory, or a cell cycle modulator. In some embodiments, 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.

[0071] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, a composition comprises a ratio (e.g., a molar ratio) of panobinostat:mocetinostat:a third enhancer ratio at a ratio of 1 : 1 : 1.

[0072] The presence of 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,

[0073] 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7- fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold,

[0074] 3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold,

[0075] 4.8-fold, 4.9-fold, 5-fold, 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8- fold, 5.9-fold, 6-fold, 6.1-fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold, 6.6-fold, 6.7-fold, 6.8-fold,

[0076] 6.9-fold, 7-fold, 7.1-fold, 7.2-fold, 7.3-fold, 7.4-fold, 7.5-fold, 7.6-fold, 7.7-fold, 7.8-fold, 7.9- fold, 8-fold, 8.1-fold, 8.2-fold, 8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold, 8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10- fold, 10.5-fold, 11-fold, 11.5-fold, 12-fold, 12.5-fold, 13-fold, 13.5-fold, 14-fold, 14.5-fold, 15- fold, 15.5-fold, 16-fold, 16.5-fold, 17-fold, 17.5-fold, 18-fold, 18.5-fold, 19-fold, 19.5-fold, 20- fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100- fold, 200-fold, 500-fold, or more relative to the same number of packaging cells in their absence. In some embodiments, combinations of enhancer agents act synergistically (e.g., result in a combined effect greater than the sum effect of each enhancer agent alone) or in an additive or sub-additive manner, but higher than can be achieved by each factor individually.

[0077] Titer-enhancing Compositions

[0078] In some embodiments, a titer enhancing composition comprises an endosomal release promoting agent selected from pseudouridine, N1 -methylpseudouridine (mlT), and 5-methyl- cytidine. In some embodiments, a titer enhancing composition comprises pseudouridine. In some embodiments, a titer enhancing composition comprises N1 -methylpseudouridine (m I T). 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.

[0079] In some embodiments, a titer enhancing composition comprises two or more endosomal release promoting agents. In some embodiments, a titer enhancing composition comprises pseudouridine and N1 -methylpseudouridine (mlT). In some embodiments, a titer enhancing composition comprises pseudouridine and 5-methyl-cytidine. In some embodiments, a titer enhancing composition comprises N1 -methylpseudouridine (m I T) and 5-methyl-cytidine.

[0080] In some embodiments, a titer enhancing composition comprises an endosomal release promoting agent and another (e.g., one or more) enhancer agent. In some embodiments, a titer enhancing composition comprises pseudouridine and another (e.g., one or more) enhancer agent. In some embodiments, a titer enhancing composition comprises N1 -methylpseudouridine (mlT) and another (e.g., one or more) enhancer agent. In some embodiments, a titer enhancing composition comprises 5-methyl-cytidine and another (e.g., one or more) enhancer agent.

[0081] In some embodiments, 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.

[0082] In some embodiments, 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.

[0083] In some embodiments, an enhancer agent is a poly amine selected from putrescine, spermine, and spermidine. In some embodiments, a titer enhancing composition comprises putrescine. In some embodiments, a titer enhancing composition comprises spermine. In some embodiments, a titer enhancing composition comprises spermidine. In some embodiments, a titer enhancing composition comprises putrescine, spermine, spermidine, or a combination thereof.

[0084] In some embodiments, 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.

[0085] In some embodiments, 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.

[0086] In some embodiments, 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. In some embodiments, a titer-enhancing composition comprises sodium butyrate. In some embodiments, a titer-enhancing composition comprises sodium propionate. In some embodiments, a titer-enhancing composition comprises vorinostat. In some embodiments, 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.

[0087] In some embodiments, a titer-enhancing composition comprises two or more chromatin modifiers. In some embodiments, a titer-enhancing composition comprises sodium butyrate and another (e.g., one or more) chromatin modifier. In some embodiments, a titer-enhancing composition comprises panobinostat and another (e.g., one or more) chromatin modifier. In some embodiments, a titer-enhancing composition comprises vorinostat and another (e.g., one or more) chromatin modifier. In some embodiments, a titer-enhancing composition comprises mocetinostat and another (e.g., one or more) chromatin modifier. In some embodiments, a titerenhancing composition comprises panobinostat and mocetinostat.

[0088] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0089] In some embodiments, 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.

[0090] In some embodiments, a titer enhancing composition comprises an epigenetic modulator selected from 5-azacytidine, 5'-aza-2'-deoxycytidine (decitabine), and dimethyl sulfoxide (DMSO). In some embodiments, a titer-enhancing composition comprises 5-azacytidine. In some embodiments, a titer-enhancing composition comprises 5'-aza-2'-deoxycytidine (decitabine). In some embodiments, a titer-enhancing composition comprises DMSO. In some embodiments, a titer-enhancing composition comprises 5-azacytidine, 5'-aza-2'-deoxycytidine (decitabine), dimethyl sulfoxide (DMSO), or a combination thereof.

[0091] In some embodiments, a titer enhancing composition comprises two or more epigenetic modulators. In some embodiments, a titer-enhancing composition comprises 5-azacytidine and 5'-aza-2'-deoxycytidine (decitabine). In some embodiments, a titer-enhancing composition comprises DMSO and 5-azacytidine. In some embodiments, a titer-enhancing composition comprises 5'-aza-2'-deoxycytidine (decitabine) and DMSO.

[0092] In some embodiments, a titer enhancing composition comprises an epigenetic modulator and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises 5-azacytidine and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises 5'-aza-2'-deoxycytidine (decitabine) and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises DMSO and another (e.g., one or more) enhancer agent.

[0093] In some embodiments, 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. In some embodiments, a titer-enhancing composition comprises caffeine. In some embodiments, a titerenhancing composition comprises nicotinamide. In some embodiments, a titer-enhancing composition comprises chloroquine diphosphate. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0094] In some embodiments, a titer enhancing composition comprises two or more DNA repair modulators. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, a titer-enhancing composition comprises hydroxychloroquine and one or more of NU7026, JQ1, theophylline, theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU-60019, KU- 559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, a titer-enhancing composition comprises NU7026 and one or more of JQ1, theophylline, theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, a titer-enhancing composition comprises JQ1 and one or more of theophylline, theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, a titer-enhancing composition comprises theophylline and one or more of theobromine, adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU- 60019, KU-559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, a titerenhancing composition comprises theobromine and one or more of an adenosine receptor agonist, wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, a titer-enhancing composition comprises an adenosine receptor agonist and one or more of wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, a titer-enhancing composition comprises wortmannin and one or more of CP-466722, KU-55933, KU-60019, KU-559403, PARP inhibitors, olaparib, and etoposide. In some embodiments, 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. In some embodiments, a titer-enhancing composition comprises KU-559403 and one or more of a PARP inhibitor, olaparib, and etoposide. In some embodiments, a titer-enhancing composition comprises a PARP inhibitor and one or more of olaparib, and etoposide. In some embodiments, a titerenhancing composition comprises olaparib and etoposide.

[0095] In some embodiments, a titer enhancing composition comprises a DNA repair modulator and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises caffeine and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises nicotinamide and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises chloroquine diphosphate and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises hydroxychloroquine and another (e.g., one or more) enhancer agent. In some embodiments, 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. In some embodiments, a titer-enhancing composition comprises wortmannin and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises CP- 466722 and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises KU-55933 and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises KU-60019 and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises KU- 559403 and another (e.g., one or more) enhancer agent. In some embodiments, 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.

[0096] In some embodiments, a titer enhancing composition comprises a cell cycle modulator selected from a cyclin dependent kinase modulator, hydroxyurea, nocodazole, Akti-1 and Akti- 2. In some embodiments, a titer-enhancing composition comprises a cyclin dependent kinase modulator. In some embodiments, a titer-enhancing composition comprises hydroxyurea. In some embodiments, a titer-enhancing composition comprises nocodazole. In some embodiments, a titer-enhancing composition comprises Akti-1. In some embodiments, a titerenhancing composition comprises Akti-2. In some embodiments, a titer-enhancing composition comprises cyclin dependent kinase modulator, hydroxyurea, nocodazole, Akti-1, Akti-2, or a combination thereof.

[0097] In some embodiments, 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. In some embodiments, 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.

[0098] In some embodiments, 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. In some embodiments, a titer-enhancing composition comprises a cyclin dependent kinase modulator and another (e.g., one or more) enhancer agent. In some embodiments, a titerenhancing composition comprises hydroxyurea and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises nocodazole and another (e.g., one or more) enhancer agent. In some embodiments, 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.

[0099] In some embodiments, 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.

[0100] In some embodiments, 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.

[0101] In some embodiments, a titer enhancing composition comprises a cell cycle modulator and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises dinaciclib and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises NU 6140 and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises NU 2058 and another (e.g., one or more) enhancer agent. In some embodiments, a titer-enhancing composition comprises Ro 3306 and another (e.g., one or more) enhancer agent.

[0102] Media Additives and Cell Culture Media

[0103] Aspects of the disclosure relate to compositions comprising an aqueous mammalian cell culture media. In some embodiments, the aqueous mammalian cell culture media is a complete media. As used herein, “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. In some embodiments, the aqueous mammalian cell culture media is a serum-free media. Examples of mammalian cell culture media include but are not limited to Minimum Essential Media (MEM), Dulbecco’s MEM (DMEM), RPMI 1640, F12, etc. In some embodiments, a mammalian cell culture media is chemically defined (e.g., does not contain any animal, plant, or yeast-derived products).

[0104] In some aspects, the 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. In some embodiments, a cell culture media additive is formulated as a solid. For example, a cell culture media additive may be a composition comprising two or more enhancer agents in a powdered (e.g., lyophilized) form. In some embodiments, media additives are formulated in an aqueous solution. For example, two or more enhancer agents may be dissolved in a solvent, such as water, ethanol, DMSO, etc.

[0105] In some embodiments, 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. In some embodiments, 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. In some embodiments, a cell culture media additive comprises sodium butyrate, bufexamac, tubacin, and nicotinamide. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, and sodium pentanoate. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, and chloroquine. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, and 2’ aminopurine. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, and nicotinamide. In some embodiments, 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. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, sodium pentanoate, chloroquine, and nicotinamide. In some embodiments, a cell culture media additive sodium butyrate, bufexamac, tubacin, caffeine, sodium pentanoate, chloroquine, 2’ aminopurine, and nicotinamide.

[0106] In some embodiments, media additives described herein are added to a basal medium

[0107] (e.g., MEM, DMEM, GMEM) as a supplement. In some embodiments the compositions described herein are formulated as complete cell culture media. In some embodiments, a complete cell culture medium comprises amino acids, carbon sources, vitamins, minerals, growth factors, hormones, inorganic salts, and / or trace metals. In some embodiments, 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. In some embodiments, a complete cell culture medium comprises one or more carbon source selected from sugars or sugar alcohols, including glucose, galactose, maltose, fructose, and inositol. In some embodiments, 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. In some embodiments, a complete cell culture medium comprises one or more minerals selected from iron, zinc, manganese, copper, aluminum, molybdenum, vanadium, nickel, and / or tin. In some embodiments, a complete cell culture comprises growth factors, including serums (e.g., fetal bovine serum, bovine calf serum, equine serum, and / or porcine serum). In some embodiments, a complete cell culture medium comprises one or more minerals selected from calcium, zinc, iron, folate, and / or magnesium. In some embodiments, a complete cell culture medium comprises inorganic salts, including, but not limited to sodium chloride, lithium chloride, sodium selenite, and ammonium meta-vanadate. In some embodiments, a complete cell culture medium comprises one or more trace metals selected from iron, zinc, manganese, copper, aluminum, molybdenum, vanadium, nickel, and / or tin. In some embodiments, a complete cell culture comprises growth factors, including serums (e.g., fetal bovine serum, bovine calf serum, equine serum, and / or porcine serum). In some embodiments, the cell culture media formulations described herein are suitable for use with antibiotics.

[0108] In some embodiments, 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. In some embodiments, 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. In some embodiments, the complete cell culture medium formulations described herein are acceptable for pharmaceutical use.

[0109] The 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. Non-limiting examples of 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).

[0110] Viral Packaging Cells

[0111] The 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. As used herein, a “host cell” is a cell capable of receiving or being transformed by a heterologous nucleic acid molecule (e.g., packaging or helper plasmids). In some embodiments, a host cell receives (e.g., is transfected, transduced, or otherwise contacted by) a heterologous nucleic acid. In some embodiments, a heterologous nucleic acid is a vector (e.g., a helper vector or helper plasmid).

[0112] A “vector” is a vehicle for the transfer of genetic material (e.g., nucleic acids) from a first environment into a second environment. In some embodiments, 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. Non-limiting examples of suitable transfection methods include electroporation, heat shock, liposomes, nanoparticles, microinjection, sonoporation, photoporation, magnetofection, hydroporation, biolistics, continuous infusion, and impalefection.

[0113] In some embodiments, a vector is a virion or viral particle comprising packageable nucleic acids and structural and / or functional genetic elements derived primarily from a virus. In some embodiments, 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). In some embodiments, 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). In some embodiments a viral vector is a gammaretroviral vector (GW), comprising structural and / or functional genetic elements derived primarily from gamma-retroviruses. 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). Notably, the terms “retrovirus” and “retroviral vector / RVV” include lentiviruses and gamma-retroviruses, and their corresponding vectors, respectively. In some embodiments, 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. In some embodiments, a viral vector comprises some or all of a viral genome. In some embodiments, a viral vector comprises a plasmid. In some embodiments, a viral vector comprises an envelope. In some embodiments, a viral vector comprises an envelope comprising viral surface proteins. In some embodiments, a viral vector comprises and envelope comprising viral surface proteins of a different virus.

[0114] A vector may be a packaging vector. As used herein, a “packaging” or “helper” vector is a vector (e.g., plasmid) comprising some or all of the components necessary to produce and package viral vectors. Packaging vectors may comprise one, two, three, four, or more genes of viral genome. In some embodiments, packaging or helper vectors comprise viral transcription, encapsulating, and / or enveloping sequences. In some embodiments, packaging vectors are retroviral packaging vectors, and comprise some or all of a retroviral genome (e.g., the genes gag, pro, pol, env). In some embodiments, retroviral packaging or helper vectors further comprise regulatory genes (i.e., the genes tat and rev). In some embodiments, retroviral packaging or helper vectors comprise accessory genes (e.g., vif, vpr, vpu, vpx, nef, and / or other retroviral genes). In some embodiments, retroviral packaging or helper vectors comprise env genes encoding envelope proteins of a different virus (e.g., vesicular stomatitis virus). In some embodiments, packaging vectors are adeno-associated viral packaging or helper vectors and comprise some or all of an adeno-associated viral genome (e.g., the genes rep and cap). In some embodiments, adeno-associated viral packaging vectors comprising adenovirus helper function genes (e.g., E4, E2a, VA). In some embodiments, packaging vectors comprise other viral components (e.g., proteins) and / or host cell components. In some embodiments, a packaging vector is a plasmid. In some embodiments, a packaging vector is a viral particle. In some embodiments, packaging vectors enable host cells to produce replication defective viruses.

[0115] In some embodiments, a host cell is transformed into a viral packaging cell after being contacted by (e.g., transfected, transduced) one or more packaging vectors, using methods well known by those of skill in the art. In some embodiments, a host cell is sequentially contacted by a plurality of packaging vectors and is transformed into a viral packaging cell. In some embodiments, a host cell is simultaneously contacted by a plurality of packaging vectors and is transformed into viral packaging cell. In some embodiments, a host cell is co-transfected with packaging vectors and vectors comprising transgenes. In some embodiments, nucleic acids delivered by packaging vectors are not integrated into a host cell genome. In some embodiments, a viral packaging cell generates RVVs. In some embodiments, 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.

[0116] In some embodiments, a viral packaging cell is a transient packaging cell. A “transient” packaging cell is a host cell which has been contacted by packaging vectors and which transiently generates viral particles in the absence of replication competent viruses. In some embodiments, a transient producer cell generates RVVs. In some embodiments, a transient producer cell generates LVVs. In some embodiments, a transient producer cell generates GRVs. In some embodiments, a transient producer cell generates AAVs. In some embodiments, a transient packaging cell generates viral particles (e.g., LVVs) comprising one or more transgenes.

[0117] In some embodiments, a packaging cell line is a stable packaging cell. A “stable” packaging cell is a host cell which has been engineered, using methods known to those of ordinary skill in the art, to stably express one or more of the components (e.g., viral genes) required to package viral particles. In stable cell lines, genes required to package a viral particle (e.g., LVVs) are integrated into the host cell genome, such that progeny can also produce viral particles. Genes required to package viral particles may be under the control of an inducible promoter, constitutive promoter, or tissue- specific promoter. In some embodiments, a viral packaging cell is the progeny of a stable packaging cell which received (e.g., was transfected, transduced, or otherwise contacted by) one or more packaging vectors. In some embodiments, a viral packaging cell is part of a cell line derived from an ancestral or progenitor cell which has received (e.g., was transfected, transduced, or otherwise contacted by) one or more packaging or helper vectors. In some embodiments, a stable packaging cell generates RVVs. In some embodiments, a stable packaging cell generates LVVs. In some embodiments, a transient producer cell generates GRVs. In some embodiments, a transient producer cell generates AAVs.

[0118] Any host cell capable of generating viral particles or being transformed into a viral packaging cell may be suitable for practicing the compositions and methods described herein . In some embodiments, a host cell is a mammalian cell. In some embodiments, a mammalian cell is a human cell. In some embodiments, a host cell is a cell from an immortalized cell line. In some embodiments, a host cell is a HEK 293 cell or any derivative thereof. A “HEK293 cell” or “293 cell” refers to an immortalized human embryonic kidney cell that comprises a human adenovirus 5 nucleotide sequences 1-4344 inserted in human chromosome 19. Examples of 293 cells include but not limited to 293 cells, HEK 293T cells, and HEK293F cells. In some embodiments, a host cell is a Chinese hamster ovary (CHO) cell, or any derivative thereof. In some embodiments, a host cell is a Jurkat cell, or any derivative thereof.

[0119] In some embodiments, a host cell has been received (e.g., been transfected, transduced, or otherwise contacted by) one or more heterologous nucleic acids. In some embodiments, a host cell is the progeny of an ancestral host cell which received (e.g., was transfected, transduced, or otherwise contacted by) one or more heterologous nucleic acids. In some embodiments, a host cell is part of a cell line derived from an ancestral or progenitor cell which received (e.g., was transfected, transduced, or otherwise contacted by) one or more packaging vectors. As used herein, a “cell line” is a population of cells capable of continuous growth and division in vitro. It is understood in the art that the progeny of a single parental cell may not be completely identical in morphology or in genomic content as the original parent due to natural, accidental, or deliberate mutation. It is also known in the art that spontaneous changes to cell morphology or genomic content may occur during storage or transfer of clonal populations of a cell line. Therefore, cells in a cell line include variants which are not precisely identical to an ancestral or progenitor cell.

[0120] Methods of Producing Vectors

[0121] Aspects of the disclosure relate to methods for producing viral particles (e.g., lentiviral particles). The disclosure is based, in part, on compositions comprising two or more enhancer agents that, when added to a host cell culture during a particular period, result in increased production (e.g., titer) of viral particles relative to host cells that have not been treated with the compositions.

[0122] The time at which the host cell culture is contacted with the composition (e.g., cell culture media or cell culture media additive) may vary. In some embodiments, host cells are contacted with the media additives and / or cell culture compositions described herein during a transformative process (e.g., transfection or transduction with viral packaging vectors). In some embodiments, host cells are first contacted with a composition described herein, then contacted with a mixture of polyethylenimine (PEI) and viral packaging vectors. In some embodiments, host cells are first contacted (e.g., transduced or transfected) with a mixture of PEI and viral packaging or helper vectors, then contacted with a composition described herein, for example, in a cell culture medium.

[0123] In some embodiments, 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.

[0124] In some embodiments, 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 of the contacting with the PEI and viral packaging or helper vectors. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0125] In some embodiments, 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. In some embodiments, 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.

[0126] In some embodiments, 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. In some embodiments, 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.

[0127] In some embodiments, transduced and / or transfected host cells (i.e., packaging 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. In some embodiments, 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. In some embodiments, packaging cells are contacted with a composition 1, 2, 3, 4, 5, or more times. In some embodiments, a cell is contacted with a second, third, fourth, etc. composition by changing the culture media. In some embodiments, a cell is contacted with a second, third, fourth, etc. composition without changing the culture medium.

[0128] Collection of Viral Vectors

[0129] As described herein, titer enhancing compositions may comprise packaging cells. In some embodiments, following culturing of packaging cells with the titer enhancing compositions described herein, the packaging cells and used compositions (e.g., supernatant) may comprise viruses, viral particles, viral vectors, and / or components thereof (e.g., nucleic acids, proteins). Such 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. In some embodiments, viruses, viral particles, viral vectors, and / or components thereof are collected from the cells and / or supernatant in a single harvest. In some embodiments, 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.

[0130] 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. 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 102TU / mL, 103TU / mL , 104TU / mL, 105 TU / mL, 106TU / mL, 107TU / mL IO8TU / mL, IO9TU / mL, IO10TU / mL, IO11TU / mL, 1012TU / mL, IO13TU / mL, 1014TU / mL, 1015TU / mL, 1016TU / mL, 1017TU / mL, IO18TU / mL, IO19TU / mL, or IO20TU / 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.

[0131] EXAMPLES

[0132] Example 1: Treatment with sodium butyrate, panobinostat, or vorinostat improves viral titer during transfection of suspension cells

[0133] Sodium butyrate, an HD AC inhibitor, is widely used as a single agent for improving lentiviral particle titer; however, sodium butyrate has only been shown to be effective for increasing viral titer when added at specific concentrations and during specific time points. Though several alternative HD AC inhibitors exist, their contribution to lentiviral particle titer in vector production had not previously been shown, and their chemical structure differs dramatically, which may alter their inhibitory potency and / or specificity towards HD AC isoforms. For example, though considered a potent HD AC inhibitor generally, 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). Its effects on viral titer were previously unknown. In this Example, the effects of panobinostat, and vorinostat (i.e., SAHA) on lentiviral titer produced by lentiviral producer suspension cells were compared to sodium butyrate, the industry standard.

[0134] On Day 1, 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 4xl06cells / 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. 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.

[0135] On Day 1, Jurkat cells (2.75xl05cells) 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. On Day 5, 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.

[0136] As an additional control, supernatant was produced from suspension cells only treated with Panobinostat, to isolate medium containing Panobinostat without virus and elucidate the impact Panobinostat, if any, may have upon transduction.

[0137] On day 1, suspension HEK293 suspension cells were expanded in a 10 cm culture dish in lOmL LV Max (10% FBS, 1% PS) culture media. On Day 2 (24 hours post-expansion), cells were dispensed into T125 flasks at 20ml volume with a cell density of 4xl06cells / ml. On Day 3 (48 hours post-expansion), cells were treated with lOpM Panobinostat (in DMSO). On Day 4 (24 hours post-treatment), 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.

[0138] On Day 1 of transduction, Jurkat cells (2.75xl05cells) were plated in a flat-bottom 96 well plate (200pl / well), transduced with O.lpL-lpL of IRC GFP lentivirus vectors and 4-5ul of the filtered cell suspension, and incubated. On Day 5, expression of GFP was analyzed using fluorescent activated cell sorting (FACS) method. Titer was calculated using standard formulas in the art. Seven repetitions of the above-described experiments were performed; two representative results of the above experiments are shown in FIG. 2A (for CD 19 GFP expression) and FIG. 2B (for GFP expression). Overall, addition of sodium butyrate, panobinostat, and vorinostat were found to improve viral titer relative to Control in lentiviral producer suspension cells. Notably, 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.

[0139] No significant change was found in transduction or GFP expression for panobinostat transfection control, indicating that residual panobinostat at the transduction step does not impact transduction in suspension cells. Accordingly, panobinostat and vorinostat may be useful for increasing viral titer in lentiviral producer suspension cells.

[0140] Example 2: Treatment with sodium butyrate, panobinostat, or vorinostat improves viral titer during transfection of adherent cells

[0141] In this Example, the effects of panobinostat, and vorinostat on lentiviral titer lentiviral producer adherent cells were compared to sodium butyrate, the industry standard.

[0142] On Day 1, adherent HEK 293T cells (seeded at 80,000 cells / cm2) 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 / cm2of 3:1 (pL:pg) ratio of PEEDNA. 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.

[0143] On day 2 (with transfection) and / or day 3 (24 hours post-transfection) cells were treated with: 5mM sodium butyrate (in water), 10pm Panobinostat (in DMSO), or 10pm vorinostat (in DMSO). Viral supernatant was collected from the cells on Day 5 (72hrs post-transfection) and filtered through a 0.45 -micron filter. Filtered viral supernatant was frozen as aliquots of 1 ml or greater. Panobinostat transduction controls were prepared and viral titer was assessed as described in Example 1.

[0144] Seven repetitions of the above-described experiments were performed; two representative results of the above experiments are shown in in FIG. 3A (for GFP expression) and FIG. 3B (for GFP and CD 19 GFP expression). Overall, as shown for suspension cells, addition of sodium butyrate, panobinostat, and vorinostat were found to improve viral titer relative to Control in lentiviral producer adherent cells. 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.

[0145] Again, no significant change was found in transduction or GFP expression for panobinostat transfection control, indicating that Panobinostat itself does not impact transduction in adherent cells. Accordingly, panobinostat and vorinostat may be useful for increasing viral titer in lentiviral producer suspension cells.

[0146] Example 3: Higher doses of panobinostat, or vorinostat improves viral titer during transfection of suspension cells and adherent cells

[0147] 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.

[0148] Suspension cells (HEK 293 suspension cells) and adherent cells (HEK 293T cells) were prepared as described in Examples 1 and Examples 2, respectively, differing only by concentrations of panobinostat (lOpM, 20pM, or 40pM); however, viral supernatant volumes used for transduction in 20pM and 40pM panobinostat conditions were reduced (0.05pL-0.5pL) due to potential toxicity of panobinostat. Generally, 1 to O.lpl virus volume was used for transfection after producer cells were treated with 20 to 100 pM panobinostat.

[0149] Panobinostat transduction controls were prepared and viral titer was assessed as described in Example 1.

[0150] FIG. 4A (TOP, BOTTOM) 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

[0151] 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.

[0152] Table 2. Panobinostat-induced viral titer changes in lentiviral producer HEK 293T adherent cells

[0153] Together, these findings suggest that unlike sodium butyrate, broader ranges of panobinostat are useful for increasing viral titer in lentiviral producer suspension cells.

[0154] Example 4: Suspension Cell Transfection

[0155] In this Example, the effects of higher doses of panobinostat and mocetinostat, alone or in combination, on viral titer during transfection were assessed.

[0156] 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.

[0157] As shown in FIG. 5 and in Table 3 below, combinations of 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. Surprisingly, the viral titer increases induced by both panobinostat and mocetinostat is greater than additive, suggesting a synergistic effect of the combination.

[0158] Table 3. Panobinostat- and / or mocetinostat- induced viral titer changes in lentiviral producer HEK293 suspension cells

[0159] Overall, these findings indicate panobinostat and mocetinostat in combination significantly improve lentiviral titer over the leading enhancer agent, sodium butyrate.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising:(i) 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;(ii) 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(iii) an aqueous mammalian cell culture media.

2. The composition of claim 1, wherein the mammalian cell culture media is complete.

3. The composition of claim 1 or 2, wherein the mammalian cell culture media is chemically defined.

4. The composition of any one of claims 1 to 3, wherein the composition lacks serum, optionally wherein the serum comprises human serum albumin (HSA).

5. The composition of any one of claims 1 to 4, wherein the endosomal release promoting agent comprises pseudouridine, N1 -methylpseudouridine (m I T) or 5-methyl-cytidine.

6. The composition of any one of claims 1 to 5, wherein the nuclear localization promoting agent comprises a polyamine, optionally wherein the polyamine comprises putrescine, spermine, and / or spermidine.

7. The composition of any one of claims 1 to 6, wherein the chromatin modifier comprises a histone deacetylase (HD AC) inhibitor.

8. The composition of claim 7, wherein the HDAC 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).

9. The composition of any one of claims 1 to 8, wherein the epigenetic modulator comprises 5 '-Azacytidine or 5'-aza-2'-deoxycytidine (decitabine).

10. The composition of any one of claims 1 to 9, wherein the DNA repair modulator comprises caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist.

11. The composition of claim 10, wherein the adenosine receptor agonist comprises wortmannin, CP-466722, KU-55933, KU-60019, KU-559403, or a PARP inhibitor (e.g., Olaparib).

12. The composition of any one of claims 1 to 11, wherein the cell cycle modulator comprises a cyclin dependent kinase inhibitor, hydroxyurea, or nocodazole.

13. The composition of any one of claims 1 to 12, further comprising 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.

14. The composition of claim 1, wherein each of the first enhancer agent and second enhancer agent is an endosomal release promoting agent, optionally wherein the first enhancer agent is pseudouridine, or N1 -methylpseudouridine (m IT), and the second enhancer agent is 5- methyl-cytidine.

15. The composition of claim 1, wherein the first enhancer agent is an endosomal release promoting agent and the second enhancer agent is a nuclear localization promoting agent,optionally wherein the endosomal release promoting agent is pseudouridine, mlT, or 5-methyl- cytidine and the nuclear localization promoting agent is putrescine, spermine, or spermidine.

16. The composition of claim 1, wherein the first enhancer agent is an endosomal release promoting agent and the second enhancer agent is an epigenetic modulator, optionally wherein the endosomal release promoting agent is pseudouridine, m I , or 5-methyl-cytidine and the epigenetic modulator is 5 '-Azacytidine or 5'-aza-2'-deoxycytidine (decitabine).

17. The composition of claim 1, wherein the first enhancer agent is an endosomal release promoting agent and the second enhancer agent is a cell cycle modulator, optionally wherein the endosomal release promoting agent is pseudouridine, m I , or 5-methyl-cytidine and the cell cycle modulator is a cyclin dependent kinase inhibitor, hydroxyurea, or nocodazole.

18. The composition of claim 1, wherein the first enhancer agent is a nuclear localization promoting agent and the second enhancer agent is an epigenetic modifier, optionally wherein the nuclear localization promoting agent is putrescine, spermine, or spermidine and the epigenetic modifier is 5 '-Azacytidine or 5'-aza-2'-deoxycytidine (decitabine).

19. The composition of claim 1, wherein the first enhancer agent is a nuclear localization promoting agent and the second enhancer agent is a cell cycle modulator, optionally wherein the nuclear localization promoting agent is putrescine, spermine, or spermidine and the cell cycle modulator is a cyclin dependent kinase inhibitor, hydroxyurea, or nocodazole.

20. The composition of claim 1, wherein the first enhancer agent is a chromatin modifier and the second enhancer agent is an endosomal release promoting agent, optionally wherein the 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 the endosomal release promoting agent is pseudouridine, N1 -methylpseudouridine (m I T) or 5-methyl-cytidine.

21. The composition of claim 1, wherein the first enhancer agent is a chromatin modifier and the second enhancer agent is a nuclear localization promoting agent, optionally wherein the 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 the nuclear localization promoting agent is putrescine, spermine, or spermidine.

22. The composition of claim 1, wherein the first enhancer agent is a chromatin modifier and the second enhancer agent is an epigenetic modulator, optionally wherein the 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 the epigenetic modulator is 5 '-Azacytidine or 5 '-aza-2 '-deoxycy tidine (decitabine) .

23. The composition of claim 1, wherein the first enhancer agent is a chromatin modifier and the second enhancer agent is cell cycle modulator, optionally wherein the 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 the cell cycle modulator is a cyclin dependent kinase inhibitor, hydroxyurea, or nocodazole.

24. The composition of claim 1, wherein the first enhancer agent is an epigenetic modulator and the second enhancer agent is cell cycle modulator, optionally wherein the epigenetic modulator is 5 '-Azacytidine or 5'-aza-2'-deoxycytidine (decitabine) and the cell cycle modulator is a cyclin dependent kinase inhibitor, hydroxyurea, or nocodazole.

25. The composition of claim 1, wherein the first enhancer agent is a DNA repair modulator and the second enhancer agent is an endosomal release promoting agent, optionally wherein the DNA repair modulator is caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist and theendosomal release promoting agent is pseudouridine, N1 -methylpseudouridine (mlT) or 5- methyl-cytidine.

26. The composition of claim 1, wherein the first enhancer agent is a DNA repair modulator and the second enhancer agent is a nuclear localization promoting agent, optionally wherein the DNA repair modulator is caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist and the nuclear localization promoting agent is putrescine, spermine, and / or spermidine.

27. The composition of claim 1, wherein the first enhancer agent is a DNA repair modulator and the second enhancer agent is an epigenetic modulator, optionally wherein the DNA repair modulator is caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist and the epigenetic modulator is 5 '-Azacytidine or 5'-aza-2'-deoxycytidine (decitabine).

28. The composition of claim 1, wherein the first enhancer agent is a DNA repair modulator and the second enhancer agent is a cell cycle modulator, optionally wherein the DNA repair modulator is caffeine, nicotinamide, chloroquine diphosphate, hydroxychloroquine, NU7026, JQ1, caffeine, theophylline, theobromine, or an adenosine receptor agonist and the cell cycle modulator is a cyclin dependent kinase inhibitor, hydroxyurea, or nocodazole.

29. A cell culture media additive comprising sodium butyrate, and bufexamac and / or tubacin.

30. A cell culture media additive comprising panobinostat.

31. A cell culture media additive comprising vorinostat.

32. A cell culture media additive comprising panobinostat and mocetinostat.

33. The cell culture media additive of any one of claims 29 to 32, further comprising caffeine.

34. The cell culture media additive of any one of claims 29 to 33, further comprising sodium pentanoate.

35. The cell culture additive of any one of claims 29 to 34, further comprising chloroquine and / or 2’ aminopurine.

36. The cell culture additive of any one of claims 29 to 35, further comprising nicotinamide.

37. The cell culture additive of any one of claims 29 to 36, wherein the additive is formulated as an aqueous solution.

38. The cell culture additive of any one of claims 29 to 37, wherein the additive is formulated as a solid.

39. A composition comprising:(i) a population of mammalian host cells; and(ii) the composition of any one of claims 1 to 38.

40. A composition comprising:(i) a population of mammalian host cells; and(ii) the cell culture media additive of any one of claims 29 to 38.

41. The composition of claim 39 or 40, wherein the population of mammalian host cells comprises HEK293 cells or derivatives thereof.

42. The composition of any one of claims 39 to 41, wherein the host cells comprise one or more of the following: a lentivirus transfer vector, a lentivirus packaging vector, and / or a lentivirus envelope vector.

43. A cell culture composition comprising:(i) cell culture media additive comprising between about 5 pM and about 15 p M panobinostat, and between about 5 pM and about 15 pM mocetinostat; and(ii) aqueous mammalian cell culture media.

44. The cell culture composition of claim 43, wherein the mammalian cell culture media is complete.

45. The cell culture composition of claim 43 or 44, wherein the mammalian cell culture media is chemically defined.

46. The cell culture composition of any one of claims 43 to 45, wherein the composition lacks serum, optionally wherein the serum comprises human serum albumin (HSA).

47. The cell culture composition of any one of claims 43 to 46, wherein the panobinostat and mocetinostat are present at a ratio of 1:1.

48. The cell culture composition of any one of claims 43 to 47, comprising 10 pM panobinostat and 10 pM mocetinostat.

49. A method of culturing mammalian cells, the method comprising contacting a population of mammalian cells with the composition of any one of claims 1 to 28, the cell culture additive of any one of claims 29 to 38, or the cell culture composition of any one of claims 43 to 48.

50. The method of claim 49, wherein the contacting occurs between about 1 minute before and about 24 hours after the mammalian cells are contacted with one or more viral packaging vectors.

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