Presentation

The VVTG platform, part of platform of University Paris Cité, is located on the site of the Faculty of Necker. The PF proposes viral productions for researchers of the Campus, and it is also open to national academic teams as part of the GIS-IBISA.

Our production service can provide viral vectors derived from Lentiviruses, Retroviruses and Adenoviruses, research grade and defectives for replication.

Viral vectors have the ability to transfer genetic material into infected target cells. These powerful and specific tools make it possible to modulate the expression of genes using genetic sequences for the overexpression or repression (shRNA, miRNA) of genes, but also by introducing modifications into the genome of infected cells using molecular scissors (CRISPR-CAS-9 system). 

These viral vectors of gene transfer provide a better understanding of cellular molecular mechanisms. They are a valuable aid in the search for treatments for diseases, particularly through gene therapy.

To implement these activities, the platform relies on specific know-how :

  • developing tools and processes for the production, purification and characterization of research grade viral vectors

  • produce pre-clinical grade viral vectors (research grade)

  • advising on the choice of vectors (viral vector design) and their use

The VVTG platform also offers a service for immortalizing human B lymphocytes (LB) and human T lymphocytes (LT) using EBV and Saïmiri viruses. The immortalization of LB and LT can be carried out from the same patient blood sample. This service is essential for research on immune cells and in particular on rare diseases affecting the immune system.

Localisation 

Plateforme Vecteurs Viraux et Transfert de gènes (VVTG)

Faculté de Santé Paris Centre, Université Paris Cité

156-160 rue de Vaugirard

75015 Paris

 

Tél : 01 40 61 54 54 ou 40 61 54 57

Contact : sylvie.fabrega@inserm.fr

 

Contact immortalisation de lymphocytes B et T :

alicia.fernandes@inserm.fr

ebv-vvtg.sfrnecker@inserm.fr

 

The platform VVTG is located at the first floor of the faculty building Necker.

Staff

 
Sylvie Fabrega v2_edited.jpg
Fabrega Sylvie, PhD

Head- IR INSERM

sylvie.fabrega@inserm.fr

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Franck Oury, PhD

Scientific referent

franck.oury@inserm.fr

Laure Nay

AI-Université Paris Cité

Laure.nay@inserm.fr

Alicia Fernandes
Redouane Si-bouazza

AI-Université Paris Cité

redouane.si-bouazza@inserm.fr

Equipement

 

The VVTG platform includes L1, L2 and L3 dedicated to its activities. A room L2 is reserved for the production of class 2 viral GMOs with type A inserts, the culture of primary human cells and cell lines. This L2 is fully equipped for these activities.

The VVTG platform is managing to open, during summer 2022, its L3 technical laboratory, which will be proposed to any user of the University Paris Cité, who has experiments to carry on class 3 GMOs, and also on group 3 pathogenic microorganisms, such as the HIV-1 virus and Sars-Cov-2. The platform will carry out its own productions of viral GMOs with type B inserts, as well as cellular immortalizations.

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L2 of VVTG  plateform

Services

EXPERTISE AND SERVICES FOR THE PRODUCTION OF GENE TRANSFER VIRUSES

The platform provides advice on:

  • Viral vectors, expression vectors,

  • The classification of DNA inserts in category A or B, according to their dangerousness,

  • Production packaging,

  • The methods of production of viruses,

  • The modalities of infections,

  • GMO regulations,

  • The methods of handling viruses in L2,

  • Transport of GMOs

  • The immortalization of B and T cells.

 

Production and amplification of class 2 GMO viruses in L2:

I - LENTIVIRUS AND RETROVIRUS

  • Third generation ΔU3 INSS lentivirus, VSV‐G envelope 

  • MLV retrovirus, VSV‐G or Ecotrope envelopes

 

The viral particles are obtained in HEK-293T production cells, by the co-transfection of a viral expression vector (lentiviral or retroviral) containing the sequences to be expressed (inserts) with trans-complementation vectors providing the complementary viral material necessary to obtain the viral particles. Pseudotyping of viruses is performed using the VSV-G pantropic envelope.

 

The viruses collected in the culture medium are filtered, concentrated by ultracentrifugation, stored in aliquots at - 80 ° C.

The infection of human cells HCT116 or murine NIH3T3 by different viral dilutions allows the determination of viruses whose presence in the cellular genome is detected either by the expression of fluorophores (FACS analysis), or by the selection of a resistance gene using drugs (puromycin, blasticidine, hygromycin) or by the detection of pro-viral sequences (Q-PCR analysis).

The production-titration process is about 15 days.

 
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The quality of the production cells is regularly tested.

The quality of the viruses produced is verified by the viral titer of lentiviral vectors and retroviral tests.

 

The VVTG platform has its own group II GMO approval, class 2 to carry out viral production in L2. For compliance with the regulations on viral GMOs, the VVTG platform records the characteristics of the vectors to be produced. All users will have their GMO approval up to date to use the GMOs produced.

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II - ADENOVIRUS serotype 5, ΔE1 / ΔE3

  • Successive amplifications of existing adenoviral particles, 

  • Obtaining new viral particles by transfection of the linearized adenoviral expression vector (40 kb) in production cells, then successive amplifications of the virus.

 

The amplified viral particles are purified by a double ultracentrifugation on cesium chloride gradient, desalinated on a column and then stored in aliquots at - 80 ° C. 

Infectious viral particles are assayed by infection of HEK 293A cells according to the technique of lysis ranges.

 

The production-titration process is about 45 days.

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EXPERTISE AND SERVICES FOR IMMORTALIZATION OF B AND T LYMPHOCYTES

From blood: isolation of ficoll gradient lymphocytes, selection of B or T lymphocytes, immortalization using EBV (LB) or HVS (LT) viruses, amplification of lines, freezing of ampoules of established lines and delivery of immortalized lymphocyte cultures.

Products delivered:

• B-EBV or T-HVS lines immortalized in culture (T25 bottle)

• ampoules of lymphocytes

• bulbs of line B-EBV or T-HVS (3 ampoules)

 

For compliance with the regulations, the VVTG platform records the characteristics of the samples to be treated, requests a copy of the patient's consent and that the investigator has his authorization to operate on human biological samples.

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Publications

 
  1. The role of MHC class I recycling and Arf6 in cross-presentation by murine dendritic cells. Montealegre S, Abramova A, Manceau V, de Kanter AF, van Endert P. Life Sci Alliance. 2019 Nov 18;2(6):e201900464. DOI: 10.26508/lsa.201900464

  2. High-throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts. Emma Cretin, Priscilla Lopes, Elodie Vimont, Takashi Tatsuta, Thomas Langer, Anastasia Gazi, Martin Sachse, Patrick Yu-Wai-Man, Pascal Reynier, Timothy Wai. EMBO Mol Med (2021) e13579  https://doi.org/10.15252/emmm.202013579

  3. The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression. Hélène Lazareth, Carole Henique, Olivia Lenoir, Victor G. Puelles, Martin Flamant, Guillaume Bollée, Cécile Fligny, Marine Camus, Lea Guyonnet, Corinne Millien, François Gaillard, Anna Chipont, Blaise Robin, Sylvie Fabrega, Neeraj Dhaun, Eric Camerer, Oliver Kretz, Florian Grahammer, Fabian Braun, Tobias B. Huber, Dominique Nochy, Chantal Mandet, Patrick Bruneval, Laurent Mesnard, Eric Thervet, Alexandre Karras, François Le Naou, Eric Rubinstein, Claude Boucheix, Antigoni Alexandrou, Marcus J. Moeller, Cédric Bouzigues & Pierre-Louis Tharaux. Nature  Communications (2019) 10:3303 https://doi.org/10.1038/s41467-019-11013-2

  4. Persistence of Integrase-Deficient Lentiviral Vectors Correlates with the Induction of STING-Independent CD8+ T Cell Responses. Celine Cousin, Marine Oberkampf, Tristan Felix, Pierre Rosenbaum, Robert Weil, Sylvie Fabrega, Valeria Morante, Donatella Negri, Andrea Cara, Gilles Dadaglio and Claude Leclerc Cell Reports 2019 : 26, 1242–1257. https://doi.org/10.1016/j.celrep.2019.01.025.

  5. Changes in chromatin state reveal ARNT2 at a node of a tumorigenic transcription factor signature driving glioblastoma cell aggressiveness. Alexandra Bogeas · Ghislaine Morvan‑Dubois · Elias A. El‑Habr1· François‑Xavier Lejeune · Matthieu Defrance ·Ashwin Narayanan · Klaudia Kuranda · Fanny Burel‑Vandenbos· Salwa Sayd · Virgile Delaunay·Luiz G. Dubois · Hugues Parrinell7· Stéphanie Rialle · Sylvie Fabrega · Ahmed Idbaih · Jacques Haiech ·Ivan Bièche · Thierry Virolle · Michele Goodhardt · Hervé Chneiweiss · Marie‑Pierre Junier. Acta Neuropathologica February 2018, Volume 135, Issue 2, pp 267–283   https://doi.org/10.1007/s00401-017-1783-x

  6. ZRF1 is a novel S6 kinase substrate that drives the senescence programme. Manuela Barilari, Gregory Bonfils, Caroline Treins, Vonda Koka, Delphine De Villeneuve, Sylvie Fabrega & Mario Pende. The EMBO Journal Mar 15;36(6):736-750. 2017. DOI: 10.15252/embj.201694