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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
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
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
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
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
T HE SHOWS AN EXAMPLE OF GENE GUN METHOD BEING APPLIED TO MOUSE
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
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.
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
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
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
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
(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.
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.
balloon catheter–based and occlusion-assisted infusion