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Electropermeabilization - M2P VTV - Univ. P. Sabatier

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Text of Electropermeabilization - M2P VTV - Univ. P. Sabatier

  • Electropermeabilization

    Jean-Michel Escoffre

    escoffre@ipbs.fr

    Professionnal MasterVectorology, Gene Therapy, Vaccinology

    2006-2007

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • Plasma membrane:a too selective barrier !

    Membrane destabilisation is required Electropermeabilization

    Therapeutic molecules(DNA, siRNA, anti-tumoral molecules)

    Targets(Nucleus)

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • Electropermeabilization

    i = |f r E cos() |

    | 0+ i | = 200-300 mV

    Transient Permeant StructuresEscoffre et al., Mol. Biotechnol., 2008

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • Electrogenetransfer

    Escoffre et al., Mol. Biotechnol., 2008

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • Electrochemotherapy (I)Introduction

    Sersa et al., EJSO, 2008

  • Electrochemotherapy (II)Applications

    Before

    After

    IT: Cisplatin + IL-12

    Before

    After

    IV: BleomycinRols et al. Melanoma Res., 2001 Rols et al. Bioelectrochem. (2002)

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • RNA interference (I)Introduction

    Advantages: Specificity Efficiency Stable inhibition

    Limits Resistance Transfection:

    Physical methods Chemical methods Viral methods

    Agami et al, Curr.Opin. Chem. Biol., 2002

  • RNA interference (II)Applications

    siRNA:

    Mitf Melanoma: Nakai et al., 2007

    TNF- Arthritis: Inoue et al., 2005 X11- and X11- Alzheimer disease: Xie et al., 2005

    shRNA:

    PnNOS Erectile dysfunctionnement: Magee et al., 2007

    -catenin/HIF-1 Melanoma: Takahashi et al., 2006

    Myostatin Myopathies: Magee et al., 2006

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • Skeletal muscles

    Easy access

    High vascularization Secretion of therapeutic proteins

    Quiescent fibers with long life Long lasting gene expression

    Polynucleated structures High level of gene expression

    Aihara et al., Nature Biotechnol., 1998 ; Mir et al., PNAS, 1999

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • Therapeutic proteins (I)Introduction

  • Therapeutic proteins (II)Applications

    BMP4 Demineralization of bone matrix : Kotajima et al.,

    2006

    hTNFR Uveitis : Bloquel et al., 2006

    FVIII Hemophilia A : Long et al., 2005

    Pro-opiomelanocortin Chronic Constriction Injury : Wu et

    al., 2004

    Plasminogen K5 Corneal neovascularization induced by

    alkalin burns : Yu et al., 2003

  • Summary

    Plasma membrane

    Electropermeabilization

    Electrogenetransfer

    Applications:

    Electrochemotherapy

    RNA interference

    Skeletal muscles

    Secretion of therapeutic proteins

    Genetic vaccine

  • Genetic vaccines (I)Introduction

    Principle: Injection of plasmid encoding vaccinal protein under the control of eukaryotic promoter

    Comparison with gene therapy:

    Low gene expression in few cells

    Transient gene expression

  • Genetic vaccine (II)Th-1 and Th-2 responses

  • Genetic vaccine (III)Plasmid vector

    Composition:

    Double stand DNA

    supercoiled structure

    Sequences:

    Bacterial replication origin

    Resistance gene

    Antigen gene

    Regulation sequences of gene expression (Promoter, enhancer)

    Immunostimulations sequences (CpG, cytokine gene, costimulation

    molecules gene, T-helpers epitopes)

    Administration ways:

    Intraveinous, intramuscular

    Cutaneaous, mucosal, oral

  • Genetic vaccine (IV)Stimulation and orientation of IR

    ISS sequences (such as CpG):

    Adjuvant role: Maturation and activation of DC

    Th-1 response

    Stimulation of innate immunity

    Production of IL-6, IL-12 and IFN

    Cytokines and costimulation molecules (IL-2, IFN-): Intensity of immune response

    Th-1 and/or Th-2 responses

    Intensity of immune response:

    Efficiency of antigen presentation

    Long lasting antigen expression

    Adjuvant effects of CpG

  • Genetic vaccine (V)Applications

    Intramuscular delivery:

    HA and NA of H9N2 virus: Qiu et al., 2006

    Ag85A and ESAT-6 of Mycobacterium Tuberculosis: Li et al.,

    2006

    L-HDAg and S-HDAg of HDV virus: Shiau et al., 2006

    PcrV andPilA of Pseudomonas Aeruginosa: Saha et al., 2006

    Intradermal delivery:

    HBsAg of HBV virus: Medi et al., 2005

    VEGF-165: Pedron-Mazoyer et al., 2007

    PSA of prostate cancer: Roos et al., 2006

  • Bibliographic work

    Read 5 scientific publications

    Oral presentation of publications

    2 or 3 students per publication

    10 min per publication

    The presentation is evaluated on 20 points

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