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Gene transfer by physical methods

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Non- VIral Gene Trasfer through Physical Methods

Publicada em: Educação, Tecnologia
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Gene transfer by physical methods

  2. 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. 3. GENE TRANSFER Electroporation Gene gun Ultrasound Hydrodynamic delivery
  4. 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
  6. 6. The Electroporation Pulse Generator EPI 2500
  7. 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. 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. 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
  11. 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. 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. 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. 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. 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. 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. 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. 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. 19. balloon catheter–based and occlusion-assisted infusion