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Electropermeabilization
Jean-Michel Escoffre
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
∆Ψi = |f r E cos(θ) |
| ∆Ψ0+ ∆Ψi | = 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-α � Arthritis: Inoue et al., 2005– X11-α and X11-β � Alzheimer disease: Xie et al., 2005
� shRNA:
– PnNOS � Erectile dysfunctionnement: Magee et al., 2007
– β-catenin/HIF-1α� 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
� hTNFαR � 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-γ):– 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
