Physical methods for gene delivery - M2P VTV - Univ. P. Sabatier

Physical methods for gene delivery

Jean-Michel Escoffre [email protected]

Outline   Plasmid DNA

  Physical methods:   Direct injection   Micro-injection   Hydrodynamic injection   Jet-injection   Sonoporation   Gene Gun   Laser-beam gene transduction   Magnetofection

  Combination of physical methods


  Physical methods:   Direct injection   Micro-injection   Hydrodynamique injection   Jet-injection   Sonoporation   Gene Gun   Laser-beam gene transduction   Magnetofection


Plasmid DNA   Nucleic acid

  Size   Conventional molecule: 3-15 kb   Mini-circle: 2-3 kb

  Closed circular molecule   Non associated to proteins

  Prokaryotes sequences   Replication origin   Resistance gene (Kanamycin, Ampicillin…)

  Eukaryotic sequences   Enhancer   Promoter

  Ubiquitous: Viral (CMV, SV40…), non-viral (EF1α, CAG…)   Tissue-specific: Muscle (Desmin), skin (Kératine 14)

  Intron   polyA sequence   Transgene   DNA Targeting Sequence (DTS): Increase the nuclear uptake   S/MAR: long-lasting transgene expression

Gill et al., Gene Therapy, 2009

Plasmid DNA   Advantages

  Easier to mass-produce   Low immunogenicity   Cost

  Limitations   Level of gene expression   Low kinetics of gene expression   Safe and efficient delivery methods

  Applications   Gene therapy   Genetic vaccination   Anti-cancer therapy

Gill et al., Gene Therapy, 2009




Direct injection   Principle: Direct and localized injection of pDNA

  Mechanism: pDNA/receptors interactions

Wolff et al., Science, 1990 ; Budker et al., J. Gene Med., 2000

Injection intra-musculaire Injection intra-dermique

Direct injection   Advantages

  Simple   Rapid   Low cost

  Limitations   Low level of gene expression   Low kinetics of gene expression   Low biodistribution   Inter-individual variability   Access to deeper tissues

  Applications   Genetic vaccinations

Kawase et al., J. Pharm Sci., 2003 ; André et al., Gene Ther., 2003

Intramuscular injection 50 µg p85A-EGFP (BCG)

50 µg p85A (BCG) 50µL PBS

Th1 Response




Micro-injection   Principle: Direct injection of pDNA in embryo or cell

  Mechanism: Membrane ruptures

Zhang et al., Curr. Opin. Biotechnol., 2008

Micro-injection   Advantages

  100% efficiency   Safe

  Limitations   Clever   Cost   In-vitro gene delivery

  Applications   Transgenesis (KI, KO…)

  Study the gene function   Create in-vivo model of human diseases

Zhang et al., Curr. Opin. Biotechnol., 2008

Beta-galactosidase expression under the control of neurogenin-1 promoter

Micro-injection 0.5 mg/mL pDNA

Injection: 145 hPa for 0.3s




Hydrodynamic injection   Principle : Rapid injection of large volume of pDNA

  Mechanism:   Mechanical destabilization of plasma membrane   Pore formation: Hydroporation

Zhang et al., Gene Therapy, 2004

Hydrodynamic injection   Advantages

  Simple   Rapid   Low cost

  Limitations   Tissues: Liver, Muscle   Perturbation of cardiac function   Translation to clinical applications

  Injection of 8 L (10% bw) saline buffer at high speed: Tolerance problem   Solution: Perfusion with catheter or with occlusion

  Applications   Genetic vaccination   Liver pathologies

  Cancer   Hepatitis

  Muscular pathologies   Muscular dystrophies Herweijer and Wolff, Gene Therapy, 2007

Hydrodynamic injection Injection site: i.v. tail vein

Volume of injection: 10% bw of souris Duration: 5s

pDNA: β-glucuronidase

Liver = Secretory organ

Biodistribution of β-glucuronidase Liver, spleen, brain, muscle, kidney, heart, lungs

 Production of lysosomales enzymes β-hexoaminidase A & α-galactosidase

  Accumulation of GAGs

Correcting the pathology Vacuolated cells (liver, spleen, bone marrow): (-)

Joint damage (+/-) Neuropathologies (+)




Jet-injection   Principle: Injection of small volume of pDNA (3-10 µL)

at high speed (>300 m/s)

  Mechanism:   Transient destabilization of plasma membrane

Walther et al., Gene Therapy, 2001 ; Walther et al., Clinical Cancer Research, 2008

Jet-injection   Advantages

  Simple   Rapid   Safe   Gene transfer:

  Targeted   Depth: modulation of pressure   Comparable efficacy to electroporation, gene gun

  Limitations   Unknown mechanism   Deeper organs

  Applications   Anticancer gene therapy   Genetic vaccination

Cartier et al., Anal. Biochem., 2000 ; Stein et al., Molecular Therapy, 2008 ; Sawamura et al., Gene Therapy, 1999

Jet-Injection Injection site: intra-tumoral Injection volume: 10 µL PB

pDNA: 40 µg pCMV-Cytosine Déaminase Procedure: 4 i.t. à 3.0 bars

Drug: 500 mg/kg 5-FluoroCytosine




Sonoporation   Principle: Co-injection of contrast agents with pDNA

followed by the controlled application of US

  Mechanism: Pores??   Microbubble oscillations   Microbubble destruction   Intact implosion: Micro-jets

Lentacker et al., Soft Matter, 2009

Sonoporation   Advantages

  Simple   Safe   Gene transfer:

  Efficacy (comparable to electroporation)   Targeted

  Limitations   Mechanism

  Applications   Chemotherapy   Anticancer gene therapy   Genetic vaccine

Newman and Bettinger, Gene Therapy, 2007

Sonoporation Injection site: intravenous pDNA: 500 µg VEGF-165

Procedure: 1.3 MHz, 0.9 W, cationic MBs

  Vessel density   Blood flow of microvessels

 Arteriogenesis

Correction of lower limb ischemia




Gene gun   Principle: Bombardment of gold particles coated with

pDNA

  Mechanism: Rupture of plasma membrane

Cheng et al., PNAS, 1993 ; Dileo et al., Hum. Gene Ther., 2003

Gene gun   Advantages

  Simple   Rapid   Targeted

  Limitations   Low power of penetration (few mm)   Access to deeper organs   Fate of gold particles   Cost

  Applications   Genetic vaccinations   Anti-viral and anti-cancer immunotherapies

Ghochikyan et al., Eur. J. Immunol., 2003 ; Dietrich et al., Cancer Biother. Radiopharm.,2006

Gene Gun Injection site: intradermal (Abdomen) pDNA: 0.5µg vaccinia virus L1 protein

Procedure: pDNA+spermidin+CaCl2+2 µm gold particles




Laser-beam gene transduction   Principle: pDNA injection followed by the application of

femtosecond IR laser-beam (5s, 30mW)

  Mechanism: Pore formation

Kurata et al., Exp. Cell Res., 1986 Zeira et al., FASEB J., 2007

Laser-beam gene transduction   Advantages ???

  Limitations   Power of laser penetration laser (2 mm for skin)   Limited applications to the skin and muscle   Access to the deeper organs

  Applications   Genetic vaccination

Zeira et al., Mol. Ther., 2003 ; Zeira et al., FASEB J., 2007

LBGT Injection site: intradermal

pDNA: 10µg HBsAg Procedure: titanium-spahire laser, 200 fs

 Local production of proteins   Production of secreted proteins

Genetic vaccine = Engerix®

Th1 response




Magnetofection   Principle: Delivery of pDNA-coated paramagnetic nanoparticles

under magnetic field

  Mechanism: depend on nanoparticle size   > 375 nm: Endocytosis   185 nm – 240 nm: Pore formation

Scherer et al., Gene Therapy, 2002 ; Chorny et al., FASEB Journal, 2007

Magnetofection   Avantages

  Targeted transfer

  Limitations   Low gene expression   Fate of nanoparticles   Cost   Access to the deeper organs

  Applications   Cystic fibrosis   Vet clinic: felin fibrosarcoma

Xenariou et al., Gene Therapy, 2006 ; Kauma et al., Nucleic Acids Research, 2007 ; Huttinger et al., Journal of Gene Medecine, 2008

feGM-CSF

Fibrosarcome félin

 Nb infiltrating macrophages

Mageétofection Injection site: intratumoral

Volume: 500 µL pDnA: 10µg fGM-CSF+transMAGPEI (1:1 w/w)

Procedure: Neodynium-iron-boron magnet (1h)

Anticancer immune response




Electrosonoporation Injection site: intramuscular

pDNA: 100µg pCAGGS-mIL-12 Procedure (SN): 5min, 1 MHz, 50%DC, 2.0 W/cm2

Procedure (EP): 6x100 ms, 1Hz, 25V

 Serum production of mIL-12

 Serum production of IFN-γ

  Survival rate

IH-SN Injection site IP

pDNA: 0,5 mL of 100µg/mL pCAGGS-mIL-12 Procedure (IH): clamping renal vein , 5s

Procedure (SN): pDNA/Optison microbubbles (3:1)

 Local production of luciferase

 Local production of EPO

Traitment of anemia related to renal deficiencies

Take-home messages   Combination of physical force with injection of pDNA :

  Increase:   Transfection level (nb transfected cells)   Transfection efficacy (Level of gene expression)   pDNA biodistribution   Targeting of gene transfer   Targeting of gene expression

  Decrease of inter-individual variability

  Therapeutic applications:   Gene therapy (Cancer, infectious diseases…)   Genetic vaccination (Therapeutic and prophylactic vaccines)   Chemotherapy (Delivery of anticancer drugs)

Workshop   one publication per student

  Oral presentation:   10 minutes   Emphasize on physical method and on transgene

  Questions:   10 min   Score /20

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Physical methods for gene delivery - M2P VTV - Univ. P. Sabatier