Presentation
The VVTG platform is a University Paris Cité platform, located in the Faculty of Health on the Necker site.
Historically, we have provided custom viral production services for researchers in French academic research laboratories.
The platform was awarded the "IBISA" label on 8 December 2022, thanks to the quality of its services and the many developments it has implemented since it was set up in 2009.
The recombinant gene transfer virus offering covers the production of research-grade Lentiviruses, Retroviruses and Adenoviruses that are defective for replication.
Viral vectors have the ability to transfer genetic material into infected target cells. These powerful and specific tools can be used to modulate gene expression using genetic sequences for gene overexpression or repression (shRNA, miRNA), or by introducing modifications into the genome of infected cells using molecular scissors (CRISPR-CAS-9 system). These viral gene transfer vectors provide a better understanding of cellular molecular mechanisms. They are an invaluable aid in the search for treatments for diseases, particularly through gene therapy.
The four employees of the VVTG platform are experts in all aspects of L2 class 2 virus production and titration, offering :
1) multiple viral types, covering a wide range of in vitro and in vivo gene transfer applications,
2) production packaging adapted to research-grade gene transfer applications,
3) quality control, through viral test productions and cellular controls,
5) standard viral tools (GFP, CRE, ß-gal, GFP-CRE) for different viruses.
Since 2018 and 2021, the VVTG platform has also offered an immortalisation service for human B lymphocytes (LBs) and human T lymphocytes (LTs) using EBV and Saïmiri viruses.
LB and LT immortalisation can be carried out using the same human or murine blood sample. This service is suitable for research into rare diseases affecting the immune system.
IMPORTANT: contact us in advance of your projects so that we can assess your requirements and send you the information, documents and methodologies you need to set up your services.
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
General contact for the platform and gene transfer virus services : sylvie.fabrega@inserm.fr
Contact for B and T lymphocyte immortalisation services:
Staff
Laure Nay
AI-Université Paris Cité
Alicia Fernandes
AI-Inserm
Redouane Si-bouazza
AI-Université Paris Cité
Equipement
The VVTG platform has confined rooms L1, L2 and L3 dedicated to its activities.
An L2 room is reserved for the production of group 2 class 2 viral GMOs with type A inserts, and for the culture of primary human cells and cell lines by platform staff. This L2 is fully equipped for the activities carried out there.
L2 of VVTG plateform
The "L3 SFR Necker" is a confined BCL3 room that complies with standards, set up and managed by the VVTG platform within the SFR Necker.
It represents a new technical platform open to the work of academic research teams involving class 3 microorganisms and their GMOs.
This 83 m2 L3, including 52 m2 of work rooms, will open at the end of 2023.
L3 manager: sylvie.fabrega@inserm.fr
IMPORTANT: contact the L3 manager as soon as possible to anticipate any planned activity involving class 3 microorganisms, as well as any production of class 3 viral gene transfer GMOs by platform staff.
NB: for recombinant viruses produced in L3, downgrading of use to L2 is authorised.
Any activity will require users to have an up-to-date class 3 GMO authorisation for their project in L3
Services
EXPERTISE AND SERVICES FOR THE PRODUCTION OF GENE TRANSFER VIRUSES
The platform provides advice on:
- Viral vectors and expression vectors,
- Classification of DNA inserts as category A or B, depending on their dangerousness,
- Production packaging,
- Virus production methods,
- Infection methods,
- GMO regulations, installation approval and GMO authorisation procedures,
- Procedures for handling gene transfer viruses in L2 and L3,
- Regulated transport of GMOs,
- Immortalisations of B and T lymphoid cells.
IMORTANT: COMPLIANCE WITH CLASS 1, 2 AND 3 GMO REGULATIONS
The VVTG platform has its own group II, class 2 GMO approval to produce L2 viruses for its own vectors.
Any user-customer of the VVTG platform must have an up-to-date GMO authorisation for their project in order to be able to use the class 2 and 3 viral GMOs produced by the platform.
The same applies to their declaration of class 1 GMOs.
To ensure compliance with the regulations on viral GMOs, the VVTG platform records :
- The characteristics of the viral vectors to be produced (information from the association table),
- The GMO project authorisation number (class 2 and 3) of the user's laboratory
- The date of authorisation.
For all GMO informations: sylvie.fabrega@inserm.fr
Production and amplification of class 2 GMO viruses in L2:
I - LENTIVIRUS AND RETROVIRUS
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Third generation ΔU3 INSS lentivirus, VSV‐G envelope
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MLV retrovirus, VSV‐G or Ecotrope envelopes
The viral particles are obtained in HEK-293T production cells by co-transfection of a viral expression vector (lentiviral or retroviral) containing the sequences to be expressed (inserts) in the presence of trans-complementation vectors supplying the complementary viral material required to obtain viral particles. Viruses are pseudotyped with the VSV-G pantropic envelope.
The viruses collected in the culture medium were filtered, concentrated by ultracentrifugation and stored in aliquots at -80°C.
Infection of human HCT116 or murine NIH3T3 cells (retroviruses only) with different dilutions of the viruses produced enables the viruses to be assayed (titration). Their presence in the cell genome is detected either by the expression of fluorophores (FACS analysis), by the selection of a resistance gene using drugs (puromycin, blasticidin, hygromycin) or by the detection of pro-viral sequences (Q-PCR analysis).
The production-titration process for Lentivirus and Retrovirus takes around 15 days.
Production packaging, volume and viral titels delivered - contact us for further details.
The quality of the production cells is regularly analysed.
The quality of the viruses produced is checked by the viral title of lentiviral and retroviral test vectors.
II - ADENOVIRUS serotype 5, ΔE1 / ΔE3
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- Successive amplifications of existing adenoviral particles,
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- Obtaining new viral particles by transfecting the linearised adenoviral expression vector (40 kb) into production cells, followed by successive amplifications of the virus. AdEasy kit technique.
The amplified viral particles are purified by double ultracentrifugation on a caesium chloride gradient, desalted on a column and then stored in aliquots at -80°C.
Infectious viral particles are assayed by infection of HEK 293A cells using the lysis plate technique.
The production and titration process for existing adenoviral particles takes approximately 45 days.
III- B AND T LYMPHOCYTE IMMORTALISATION EXPERTISE AND SERVICES
From blood samples :
- Isolation of lymphocytes on a Ficoll gradient,
- Selection of B or T lymphocyte proliferation,
- Immortalisation using EBV (LB) or HVS (LT) viruses,
- Amplification of cell lines, freezing of ampoules of established cell lines,
- Delivery of immortalised lymphocyte cultures.
Products delivered :
- immortalised B-EBV or T-HVS lines in culture (T25 flask)
- ampoules of B-EBV or T-HVS lines (3 ampoules)
To ensure compliance with regulations, the VVTG platform records :
- The characteristics of the samples to be processed,
- A copy of the patient's consent,
- The investigator's authorisation to work on human biological samples.
IMPORTANT: blood and cell samples received by the platform must be anonymised beforehand.
Publications
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Co-Transplantation of Barcoded Lymphoid-Primed Multipotent (LMPP) and Common Lymphocyte (CLP) Progenitors Reveals a Major Contribution of LMPP to the Lymphoid Lineage. Michaels V, Chalabi S, Legrand A, Renard J, Tejerina E, Daouya M, Fabrega S, Megret J, Olaso R, Boland A, Deleuze JF, Battail C, Tronik-Le Roux D, Ezine S. Int J Mol Sci. 2023 Feb 22;24(5):4368. doi: 10.3390/ijms24054368. PMID: 36901798
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Glioblastoma cell motility depends on enhanced oxidative stress coupled with mobilization of a sulfurtransferase. Saurty-Seerunghen MS, Daubon T, Bellenger L, Delaunay V, Castro G, Guyon J, Rezk A, Fabrega S, Idbaih A, Almairac F, Burel-Vandenbos F, Turchi L, Duplus E, Virolle T, Peyrin JM, Antoniewski C, Chneiweiss H, El-Habr EA, Junier MP. Cell Death Dis. 2022 Oct 30;13(10):913. doi: 10.1038/s41419-022-05358-8. PMID: 36310164
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MiR-324-5p and miR-30c-2-3p Alter Renal Mineralocorticoid Receptor Signaling under Hypertonicity Vu TA, Ingrid Lema, Imene Hani, Lydie Cheval, Laura Atger-Lallier, Vilayvane Souvannarath, Julie Perrot, Mélanie Souvanheuane, Yannick Marie, Sylvie Fabrega, Anne Blanchard, Jérôme Bouligand, Peter Kamenickỷ, Gilles Crambert, Laetitia Martinerie, Marc Lombès and Say Viengchareun.. Cells 2022, 11, x. doi: 10.3390/cells11091377.
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Machine learning-driven identification of drugs inhibiting cytochrome P450 2C9.Elodie Goldwaser, Catherine Laurent, Nathalie Lagarde, Sylvie Fabrega, Laure Nay , Bruno O Villoutreix , Christian Jelsch , Arnaud B Nicot , Marie-Anne Loriot , Maria A Miteva. PLoS Comput Biol. 2022 Jan 26;18(1):e1009820., doi: 10.1371/journal.pcbi.1009820.
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Combination of lentiviral and genome editing technologies for the treatment of sickle cell disease. Ramadier S, Chalumeau A, Felix T, Othman N, Aknoun S, Casini A, Maule G, Masson C, De Cian A, Frati G, Brusson M, Concordet JP, Cavazzana M, Cereseto A, El Nemer W, Amendola M, Wattellier B, Meneghini V, Miccio A. Mol Ther. 2022 Jan 5;30(1):145-163. doi: 10.1016/j.ymthe.2021.08.019. Epub 2021 Aug 19.PMID: 34418541
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Biallelic mutations in the SARS2 gene presenting as congenital sideroblastic anemia Elia Colin, Geneviève Courtois, Chantal Brouzes, Juliette Pulman, Marion Rabant, Agnès Rötig, Hélène Taffin, Mathilde Lion-Lambert, Sylvie Fabrega, Lydie Da Costa, Mariane De Montalembert, Rémi Salomon, Olivier Hermine and Lucile Couronné. Haematologica 2021, 106
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Pharmacological Premature Termination Codon Readthrough of ABCB11 in Bile Salt Export Pump Deficiency: An In Vitro Study. Amzal R, Thébaut A, Lapalus M, Almes M, Grosse B, Mareux E, Collado-Hilly M, Davit-Spraul A, Bidou L, Namy O, Jacquemin E, Gonzales E. Hepatology. 2021 Apr;73(4):1449-1463. doi: 10.1002/hep.31476. PMID: 32702170
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Functional rescue of an ABCB11 mutant by ivacaftor: A new targeted pharmacotherapy approach in bile salt export pump deficiency Mareux E, Lapalus M, Amzal R, Almes M, Aït-Slimane T, Delaunay JL, Adnot P, Collado-Hilly M, Davit-Spraul A, Falguières T, Callebaut I, Gonzales E, Jacquemin E. Liver Int. 2020 Aug;40(8):1917-1925. doi: 10.1111/liv.14518. Epub 2020 Jun 8. PMID: 32433800.
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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
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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
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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
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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.
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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
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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