Gene transfer by physical methods

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  • 1987 when Okino and Mohri3 showed that the use of the anticancer drug bleomycin,
  • the purpose of electroporation is to assist the uptake of useful molecules such as a DNA vaccine into a cell. The biological material is injected into or applied to the surface of the target tissue and followed by the application of brief, controlled electrical pulses directed to that tissue. As shown in the pictures below, electroporation's millisecond electrical pulses temporarily create enhanced permeability of pores in the cell membrane. After a short period of time the pores reseal, leaving the cells undamaged. During the period that these pores exist, a significant quantity of the previously injected biomolecules are taken up and then trapped in the cell, enabling them to then perform their intended function.
  • http://www.inovio.com/technology/howelectroporationworks.htm
  • gene therapy experiments. By using a high-voltage plasma discharge, DNA was efficiently delivered following very short (microsecond) pulses. Compared to electroporation, the technique resulted in greatly increased efficiency and less cellular damage.-http://en.wikipedia.org/wiki/Vectors_in_Gene_Therapy#Viruses
  • http://www.google.com.ph/imgres?q=gene+gun&hl=en&client=firefox-a&hs=zCo&sa=X&rls=org.mozilla:en-GB:official&biw=1366&bih=611&tbm=isch&prmd=imvns&tbnid=bJk8LPcFBv-gTM:&imgrefurl=http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Gene_gun.html&docid=HZHdv2KdavbJRM&imgurl=http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Helios_gun.gif&w=300&h=312&ei=_TVTT7zAIuuamQX_qJW9Cg&zoom=1DNA is deposited on the surface of gold particles, which are then accelerated by pressurized gas and expelled onto cells or a tissue. The momentum allows the gold particles to penetrate a few millimeters deep into a tissue and release DNA into cells on the path. Such a simple and effective method of gene delivery is expected to have important applications as an effective tool for DNA-based immunization. Further improvements could include chemical modification of the surface of the gold particles to allow higher capacity and better consistency for DNA coating, and fine-tuning of the expelling force for precise control of DNA deposition into cells in various tissues.27
  • http://group2fls.blogspot.com/2008/04/gene-gun-gene-gun-is-one-of-non-viral.htmlBesides that, there is research shows that gene gun bombardment with DNA-coated gold particles is a potential alternative to hydrodynamics-based transfection for delivering genes into superficial hepatocytes in hepatic gene therapy. Compared with hydrodynamic-based trasnfection, gene gun bombardment resulted in minimal scattered hepatic necrosis. On the other hand, severe hepatic infarction impedes foreign gene expression in the superficial hepatocytes after hydraodynamic-based transfection.
  • transformation of organelles as well as yeast mitochondriaDNA vaccination/genetic immunization, gene therapy, tumor biology/wound healing, plant virology and many othershttp://group2fls.blogspot.com/2008/04/gene-gun-gene-gun-is-one-of-non-viral.html
  • -enables researchers to engineer organelle-encoded herbicide or pesticide resistances in crop plants and to study photosynthetic processesHelios Gene Gunhttp://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Gene_gun.html
  • Cavitation is the formation and then immediate implosion of cavities in a liquid – i.e. small liquid-free zones ("bubbles") – that are the consequence of forces acting upon the liquid. Microbubbles are bubbles smaller than one millimetre in diameter, but larger than one micrometre.
  • http://www.aapsj.org/view.asp?art=aapsj0901009
  • http://www.intechopen.com/source/pdfs/28776/InTech-Animal_models_for_hydrodynamic_gene_delivery.pdf
  • http://www.nature.com/gt/journal/v14/n2/fig_tab/3302891f1.html

Transcript

  • 1. G ENE T RANSFER BY P HYSICALM ETHODS
  • 2. PHYSICAL METHOD Naked DNA  Minimal immune response than DNA encapsulated in lipids transient injuries or defects on cell membranes, so that DNA can enter the cells by diffusion. In vitro in vivo
  • 3. GENE TRANSFER Electroporation Gene gun Ultrasound Hydrodynamic delivery
  • 4. E LECTROPORATION 1970s, 1990 versatile method – in vivo (skin and muscles) short pulses of high voltage to carry DNA across the cell membrane to assist the uptake of useful molecules such as a DNA vaccine into a cell Parameters  electrical field strength [V/cm]  pulse length http://www.inovio.com/technology/howelectroporationworks.htm
  • 5. H OW DOES THEELECTROPORATION PROCESS WORK ?
  • 6. The Electroporation Pulse Generator EPI 2500
  • 7. DRAWBACKS Limited effective range of ~1 cm between the electrodes Surgical procedure is required to place the electrodes deep into the internal organs High voltage applied to tissues can result in irreversible tissue damage as a result of thermal heating electron-avalanche transfection http://www.drugdeliverytech.com/ME2/dirmod.asp?nm=Back+Issues&type=Publishing&mod=Publications%3A%3AArticle&mid=8F3A7027421841978F18B E895F87F791&tier=4&id=C18BA4201F48462C9D124298989EF593
  • 8. G ENE G UN simplest method of direct introduction of therapeutic DNA into target cells looks like a pistol but works more like a shotgun “Golden pellets” first described as a method of gene transfer into plants John Sanford at Cornell University in 1987 Particle bombardment -physical method of cell transformation in which high density and sub-cellular sized particles are accelerated to high velocity in order to carry DNA or RNA into living cells
  • 9.  DNA (or RNA) become “sticky,” adhers to biologically inert particles such as metal atoms (usually tungsten or gold) accelerating this DNA-particle complex in a partial vacuum and placing the target tissue within the acceleration path gathers the DNA cells that take up the desired DNA, identified through the use of a marker gene are then cultured to replicate the gene and possibly cloned most useful for inserting genes into plant cells such as pesticide or herbicide resistance
  • 10. T HE SHOWS AN EXAMPLE OF GENE GUN METHOD BEING APPLIED TO MOUSE
  • 11. OVERALL EFFICIENCY Temperature, amount of cells, and their ability to regenerate adjust the length of the flight path of the particles type of gun used:  helium powered vs. gun-powder, hand-held vs. stand-alone
  • 12. MAJOR LIMITATIONS shallow penetration of particles associated cell damage the inability to deliver the DNA systemically the tissue to incorporate the DNA must be able to regenerate and the equipment itself is very expensive.
  • 13. S ONOPORATION “ultrasonic frequencies” known as cellular sonication modifying the permeability of the cell plasma membrane employs the acoustic cavitation of microbubbles to enhance delivery of these large molecules Similar to electroporation low-frequency (<MHz) ultrasound has been demonstrated to result in complete cellular death (rupturing) sonoporator
  • 14. M ICROBUBBLE AGENT Optison (Perflutren Protein-Type A Microspheres Injectable Suspension, USP) is a sterile non-pyrogenic suspension of microspheres of human serum albumin with perflutren for contrast enhancement during the indicated ultrasound imaging procedures (GE) transfection efficiency- the frequency, the output strength of the ultrasound applied, the duration of ultrasound treatment, and the amount of plasmid DNA used become an ideal method for noninvasive gene transfer into cells of the internal organs major problem for ultrasound-facilitated gene delivery is low gene delivery efficiency
  • 15. H YDRODYNAMIC G ENE D ELIVERY naked plasmid DNA into cells in highly perfused internal organs with an impressive efficiency  anatomic structure of the organ  injection volume  speed of injection used to express proteins of therapeutic value such as hemophilia factors( blood) generates high hydrodynamic pressure in the circulation refluxing to the target organ defects (pores) arebeen created on the cell defects are restoring, trapping inside the cytoplasm the infused molecules
  • 16. VEHICLE FOR THE MOLECULES  Normal Saline,  Ringer’s Solution  Phosphate Buffered  Saline and the dosage range from 0.1 to 10 mg/kg, depending on the application The main application of the hydrodynamic delivery is the therapy studies, especially genes encoding secretory proteins which can be even isolated and purified
  • 17.  (a) For simplicity, fenestrated endothelium in the center. (b) injected solution (bright green) is forced out of the endothelium and into impacted hepatocytes. (c) Physical expansion of the liver showing stretched endothelium and swollen hepatocytes due to entry of DNA solution into cell interior. (d) Architecture of the liver showing recovered endothelium and transfected hepatocytes.
  • 18. P ROBLEMS AND E FFICIENCIES how to translate this simple and effective procedure to one that is applicable to humans?  Rat liver can be transfected similarly through tail vein injection using an injection volume equivalent to 8% to 9% of body weight  7.5 L of saline at a high rate- humans  However, successful liver transfection has been achieved using balloon catheter–based and occlusion-assisted infusion to specific lobes in rabbit and swine models, indicating that with modification, hydrodynamic gene delivery can become a clinically relevant procedure.
  • 19. balloon catheter–based and occlusion-assisted infusion