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
ELECTROPORATION
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=8F3A7027421841978F18BE895F87F791&tier=4&id=C18BA4201F48462C9D124298989EF593
GENE GUN
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
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.
SONOPORATION
“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
MICROBUBBLE 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
HYDRODYNAMIC GENE DELIVERY
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.
PROBLEMS AND EFFICIENCIES
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.
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