RESUMO
To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.
Assuntos
Adenoviridae , Vetores Genéticos , Linhagem Celular , Adenoviridae/genética , Peptídeo Hidrolases/genéticaRESUMO
Lentiviral vectors (LVs) are important for cell therapy because of their capacity to stably modify the genome after integration. This study describes a novel and relatively simple approach to generate packaging cells and producer clones for self-inactivating (SIN) LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). A novel gene regulation system, based on the combination of the cumate and coumermycin induction systems, was developed to ensure tight control for the expression of cytotoxic packaging elements. To accelerate clone isolation and ensure monoclonality, the packaging genes were transfected simultaneously into human embryonic kidney cells (293SF-3F6) previously engineered with the induction system, and clones were isolated after limiting dilution into nanowell arrays using a robotic cell picking instrument with scanning capability. The method's effectiveness to isolate colonies derived from single cells was demonstrated using mixed populations of cells labeled with two different fluorescent markers. Because the recipient cell line grew in suspension culture, and all the procedures were performed without serum, the resulting clones were readily adaptable to serum-free suspension culture. The best producer clone produced LVs expressing GFP at a titer of 2.3 × 108 transduction units (TU)/mL in the culture medium under batch mode without concentration.
RESUMO
The homeodomain transcription factor insulin promoter factor (IPF)-1/pancreatic duodenal homeobox (PDX)-1 plays a crucial role in both pancreas development and maintenance of beta-cell function. Targeted disruption of the Ipf1/Pdx1 gene in beta-cells of mice leads to overt diabetes and reduced Ipf1/Pdx1 gene expression results in decreased insulin expression and secretion. In humans, mutations in the IPF1 gene have been linked to diabetes. Hence, the identification of molecular mechanisms regulating the transcriptional activity of this key transcription factor is of great interest. Herein we analyzed homeodomain-interacting protein kinase (Hipk) 2 expression in the embryonic and adult pancreas by in situ hybridization and RT-PCR. Moreover, we functionally characterized the role of HIPK2 in regulating IPF1/PDX1 transcriptional activity by performing transient transfection experiments and RNA interference. We show that Hipk2 is expressed in the developing pancreatic epithelium from embryonic d 12-15 but that the expression becomes preferentially confined to pancreatic endocrine cells at later developmental stages. Moreover, we show that HIPK2 positively influences IPF1/PDX1 transcriptional activity and that the kinase activity of HIPK2 is required for this effect. We also demonstrate that HIPK2 directly phosphorylates the C-terminal portion of IPF1/PDX1. Taken together, our data provide evidence for a new mechanism by which IPF1/PDX1 transcriptional activity, and thus possibly pancreas development and/or beta-cell function, is regulated.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Homeodomínio/genética , Pâncreas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/genética , Envelhecimento , Animais , Primers do DNA , Regulação da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Glutationa Transferase/metabolismo , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Ilhotas Pancreáticas/fisiologia , Camundongos , Proteínas Nucleares/metabolismo , Pâncreas/crescimento & desenvolvimento , Biossíntese de Proteínas , RNA/genética , RNA/isolamento & purificação , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismo , Transcrição GênicaRESUMO
Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14×106 cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54×1010 Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100-150nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing.
Assuntos
Células HEK293 , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vacinas contra Influenza/isolamento & purificação , Tecnologia Farmacêutica/métodos , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/ultraestrutura , Vacinas contra Influenza/imunologia , Neuraminidase/genética , Plasmídeos , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura , Proteínas Virais/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150-200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Feminino , Células HEK293 , Testes de Hemaglutinação , Humanos , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Transfecção , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
ß-catenin plays a dual role both as a key effector in the regulation of adherens junctions and as a transcriptional coactivator. Tyrosine phosphorylation of ß-catenin is implicated as a means for its release from E-cadherin complexes and correlates with enhanced transcriptional activity. However, it remains unclear whether or not tyrosine phosphorylated ß-catenin degrades slower or faster than its unphosphorylated form or transactivates the downstream target genes differently. We have recently demonstrated that tyrosine phosphatase SHP-1 negatively regulates the nuclear transcriptional function of ß-catenin. The mechanism by which SHP-1 specifically inhibits ß-catenin/TCF transcriptional activity remains, however, to be elucidated. Herein, we demonstrate that inhibition of tyrosine phosphatases with pervanadate induced both c-src-dependent tyrosine phosphorylation and nuclear translocation of ß-catenin. Moreover, ectopic expression of SHP-1 but not the inactive form of SHP-1 (C453S) inhibited src-induced tyrosine phosphorylation of ß-catenin on tyrosines 86 and 654. SHP-1 expression and mutations of tyrosine-86 and tyrosine-654 to phenylalanine significantly and similarly decreased the transactivation potential of ß-catenin on the TOPFLASH reporter. SHP-1 expression as well as mutations of tyrosine-86 and tyrosine-654 to phenylalanine also significantly interfered with the association of ß-catenin with TBP. Mutations of tyrosine-86 and/or tyrosine-654 did not markedly alter ß-catenin stability whereas SHP-1 expression promoted proteasomal ß-catenin degradation through a GSK3ß-dependent mechanism. In conclusion, SHP-1 negatively regulates ß-catenin transcriptional activity i) by dephosphorylating ß-catenin on tyrosines 86 and 654, ii) by impairing its capacity to interact with the basal transcriptional factor TBP and iii) by promoting ß-catenin degradation in a GSK3ß-dependent manner.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , beta Catenina/metabolismo , Animais , Proteína Tirosina Quinase CSK , Núcleo Celular/metabolismo , Células Cultivadas , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , Ubiquitinação , Vanadatos/farmacologia , beta Catenina/genética , Quinases da Família srcRESUMO
SHP-1 is expressed in the nuclei of intestinal epithelial cells (IECs). Increased SHP-1 expression and phosphatase activity coincide with cell cycle arrest and differentiation in these cells. Suspecting the tumor-suppressive properties of SHP-1, a yeast two-hybrid screen of an IEC cDNA library was conducted using the full-length SHP-1 as bait. Characterization of many positive clones revealed sequences identical to a segment of the Cdk2 cDNA sequence. Interaction between SHP-1 and Cdk2 was confirmed by co-immunoprecipitations whereby co-precipitated Cdk2 phosphorylated SHP-1 protein. Inhibition of Cdk2 (roscovitine) or proteasome (MG132) was associated with an enhanced nuclear punctuate distribution of SHP-1. Double labeling localization studies with signature proteins of subnuclear domains revealed a co-localization between the splicing factor SC35 and SHP-1 in bright nucleoplasmic foci. Using Western blot analyses with the anti-SHP-1 antibody recognizing the C terminus, a lower molecular mass species of 45 kDa was observed in addition to the full-length 64-65-kDa SHP-1 protein. Treatment with MG132 led to an increase in expression of the full-length SHP-1 protein while concomitantly leading to a decrease in the levels of the lower mass 45-kDa molecular species. Further Western blots revealed that the 45-kDa protein corresponds to the C-terminal portion of SHP-1 generated from proteasome activity. Mutational analysis of Tyr(208) and Ser(591) (a Cdk2 phosphorylation site) residues on SHP-1 abolished the expression of the amino-truncated 45-kDa SHP-1 protein. In conclusion, our results indicate that Cdk2-associated complexes, by targeting SHP-1 for proteolysis, counteract the ability of SHP-1 to block cell cycle progression of IECs.