Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors

Jaalouk, Diana E.; Crosato, Milena; Brodt, Pnina; Galipeau, Jacques

doi:10.1186/1743-422x-3-27

Cited by 16 publications

(9 citation statements)

References 49 publications

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“…For pseudoparticles used to generate cells stably expressing a protein of interest, the cells were transfected with pVSV-G encoding the G protein for the Vesicular stomatitis virus (VSV), the lentiviral packaging plasmid pCMV_ΔR8-74 and the pWPI (from Didier Trono (Addgene plasmid # 12254) encoding either the wild type or mutant (TIM-1 or Axl) and a blasticidin resistance gene. Sodium butyrate was added 24 h post transfection in order to boost plasmid transcription [ 56 ]. For cell entry experiments, the pseudoparticles were generated using a plasmid encoding for a glycoprotein of the virus of interest (CHIKV, EBOV or VSV), the lentiviral packaging plasmid pCMV_ΔR8-74, and a pWPI plasmid encoding a luciferase gene as a reporter protein.…”

Section: Methodsmentioning

confidence: 99%

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Kirui

Abidine

Lenman

et al. 2021

Cells

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Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

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Section: Methodsmentioning

confidence: 99%

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Kirui

Abidine

Lenman

et al. 2021

Cells

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“…This in turn improves RNA transcription and consequently vector production. 68 However, the effect of sodium butyrate remains controversial as the gain in vector productivity is not consistent from one author to another one. For instance, Ansorge et al 65 reported a titer increase of more than 10-fold using 5-mmol/l sodium butyrate for the production of VSV-g-pseudotyped LV.…”

Section: Lentiviral Vector Productionmentioning

confidence: 99%

Production of lentiviral vectors

Merten

Hebben

Bovolenta

2016

Molecular Therapy - Methods & Clinical Development

225

221

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Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented.

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“…We further improved the volumetric titer by adding sodium butyrate to the culture medium. Sodium butyrate is a well-known histone deacetylase inhibitor in mammalian cells; by preventing DNA compaction, it improves mRNA transcription and concomitantly, protein production 40 . Despite small increases in LV production during short production periods the presence of sodium butyrate impaired virus production in long term cultures (Fig.…”

Section: Discussionmentioning

confidence: 99%

LentiPro26: novel stable cell lines for constitutive lentiviral vector production

Tomás

Rodrigues

Carrondo

et al. 2018

Sci Rep

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Lentiviral vectors (LVs) are excellent tools to promote gene transfer and stable gene expression. Their potential has been already demonstrated in gene therapy clinical trials for the treatment of diverse disorders. For large scale LV production, a stable producer system is desirable since it allows scalable and cost-effective viral productions, with increased reproducibility and safety. However, the development of stable systems has been challenging and time-consuming, being the selection of cells presenting high expression levels of Gag-Pro-Pol polyprotein and the cytotoxicity associated with some viral components, the main limitations. Hereby is described the establishment of a new LV producer cell line using a mutated less active viral protease to overcome potential cytotoxic limitations. The stable transfection of bicistronic expression cassettes with re-initiation of the translation mechanism enabled the generation of LentiPro26 packaging populations supporting high titers. Additionally, by skipping intermediate clone screening steps and performing only one final clone screening, it was possible to save time and generate LentiPro26-A59 cell line, that constitutively produces titers above 106 TU.mL−1.day−1, in less than six months. This work constitutes a step forward towards the development of improved LV producer cell lines, aiming to efficiently supply the clinical expanding gene therapy applications.

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Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors

Cited by 16 publications

References 49 publications

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Production of lentiviral vectors

LentiPro26: novel stable cell lines for constitutive lentiviral vector production

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