Lentiviral vectors. RNA delivery for Cell and Tissue Engineering. Pascale Bouillé, PhD, CEO
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1 Lentiviral vectors RNA delivery for Cell and Tissue Engineering Pascale Bouillé, PhD, CEO
2 Companyprofile Privately owned company founded in 2005, Vectalys is specialized in the production of lentiviral tools dedicated to cell and tissue engineering: Plasmids Vectors Cells Animals Construction Production QC Production Concentration Purification Titration Immortalized Primary cells Stem cells Injection Cell engrafment Transgenesis A multidisciplinary team of 14 scientists expert in: Molecular biology and bioinformatics Production processes and vector testing Models engineering 2
3 Companyprofile Strong expertise in the field carried out by the Founder, Pascale Bouillé, PhD» more than 15 years of research experience in virology (HIV) at Institut Gustave Roussy(Pr. Claude Paoletti), Institut Pasteur(Pr. Luc Montagnier, 2008 Nobel Price) and in vectorology (rlv, raav, radv) at Genethon(Pr. Olivier Danos) The comprehensive vectorology platform is the core of: Customlentiviral vectors Premade lentiviral vectors Cell & Animal services New vector design for cell engineering 3
4 Agenda What sa good lentiviral tool?» Highly concentrated» Highly pure» Functional units A non integrative tool dedicated to» T cell engineering» genome editing(cripr, CRE) 4
5 What s a good lentiviral tool? A toolthatleads to 100% of modifiedcells» Stable expression without antibiotic selection» Efficiency on immortalized, primary and stem cells»one toolfor in vitro and in vivo experiments A toolthatprotectscellsfromdamages» No interference with the cell phenotype» No effect on proliferation or metabolism» No replicative system A tool that provides reproducible results» Titration of functional particles
6 Why using a concentrated product? The transduction efficiencyand the expression levelare higherwithvectalys lentiviral vectors than with competitor vectors Vectalys Competitor Cells are seeded in 24-wells multiplate at cells/well, incubated 24h at 37 C / 5% CO 2 Cells are transduced at MOI 100 (allowing to reach the best transduction efficiency) according to the titer given by each lentiviral vector producer. Cells are incubated for 4 days at 37 C/5%CO 2.
7 Whyusinga purifiedproduct? «Vectalys vectorsare of high quality& suitablefor transduction of hes» «We can recover clonally transduced hes cell colonies following single cell dissociation» Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors. Yang G, Si-Tayeb K, CorbineauS, Vernet R, Gayon R, DianatN, Martinet C, Clay D, Goulinet-MainotS, Tachdjian G, Tachdjian G, Burks D, Vallier L, Bouillé P, Dubart-Kupperschmitt A, Weber A. BMC Biol Jul 19;11:86. doi: /
8 A tool dedicated to primary cells PRODUCTION CLARIFICATION CONCENTRATION - PURIFICATION Transient tri-transfection Of 293T cells Production in serum-free medium Centrifugation Filtration 0,45µm Tangential ultrafiltration Diafiltration Chromatography >>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>> VectaCell Tangential Ultrafiltration Titer: 10 9 TU/ml Specificactivity> 10 8 TU/ml PP/TU<400 Hard to transfect cells Delicate cells i.v. and local injection
9 T cellsengineering Naive and activated human total T lymphocytes Naive phenotype is maintained Naive and activated murine CD4+ T lymphocytes Murine CD4+ regulatory T lymphocytes 28.3 tdtomato Foxp3
10 Agenda What s a good lentiviral tool?» Highly concentrated» Highly pure» Functional particles A non integrative tool dedicated to» T cell engineering» genome editing(cripr, CRE) 10
11 Lentiviral delivery tools An integrative lentiviral vector ILV as: An analytic tool Uses: cell labeling tissue/cell type stage differentiation phenotype identification A non integrative lentiviral particle NILP as: A preparative tool Uses: cell expression Cellreprogramming, Celldifferentiation, Genome engineering CRISPR/CRE An efficient and stableexpression: Viral particle+ viral genome+ «gene» of interest Genomic signature An efficient and transient expression: Viral particle+ non viral genome+ «gene» of interest No genomic signature
12 New non integrative engineered particle: NILP A new particle providing strong and transient expression of heterologous RNAs Viral protein Bacteriophage protein Protein synthesis or Hairpin repeats non viral RNA Genome editing (ie: CRISPRs, etc...) non viral RNA or RNA silencing encapsidationof heterologous non-viral RNA no viral sequence(ltr, Ψ, integraseor RT...) no integration event
13 A strong & transient reporter expression NILP expression lasts about 24 hours and reaches its maximum at 8 hours post-transduction
14 NILP use for in vivo experiments NILP is fully functional for in vivo experimentations, and provide a tool for strong and transient expression Mice were vein tail-injected with NILP-Luc (n=5) or Integrative lentiviral vector-luc (n=3) and luciferase was tested 5, 8, 24, 48 and 72 hours post-injection.
15 A strong & transient CRE expression NILP-Cre is an efficient tool to use the Cre-Lox system Flow cytometry analysisof HCT116-Lox-dsRed-Lox cells 6 days posttransduction/transfection with RLP-MS2-Cre, ILV-Cre, or pcre % dsred+ cells NT +ILV-Cre +plv-cre +RLP-MS2-Cre dsred intensity (RFU) HCT116 HCT116-Lox-dsRed-Lox T2 cl14 % dsred+ cells dsred intensity
16 GFP silencing with RLP-CRISPR HCT116-GFP HCT116-GFP + MS2RLP-Cas9-WPRE HCT116-GFP + ILV-Cas9-WPRE
17 Human T Cells engineering % of ZsGreen+ T cells human T Cells Transduction by ZsGreen expressing particles NT MS2 RLP 10pg P24 ILV MOI 25 ILV MOI 50 Naives Activated Relative Fluorescence Intensity NT MS2RLP 20pg P24 ILV MOI 25 ILV MOI 50 Naives Activated
18 Thank you for your attention Pascale Bouillé, PhD»Founder& CEO of Vectalys» 18
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