Physical methods can be used to transfer genes into cells by transiently permeabilizing cell membranes. This allows naked DNA to enter cells. Key physical methods include electroporation, gene guns, ultrasound, and hydrodynamic delivery. Electroporation uses short electrical pulses to create temporary pores in the cell membrane through which DNA can enter. Gene guns use compressed gas to accelerate DNA-coated gold or tungsten particles into cells. Ultrasound combines microbubbles and acoustic cavitation to permeabilize membranes for DNA uptake. Hydrodynamic delivery involves rapid injection of a large DNA solution volume to generate pressure forcing DNA into organ cells like hepatocytes. These methods show promise but also have limitations like tissue damage, shallow

1. G ENE T RANSFER BY P HYSICAL
M 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

6. H OW DOES THE
ELECTROPORATION PROCESS
WORK ?

9. The Electroporation Pulse Generator EPI 2500

10. 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

11. 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

12.  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

13. T HE SHOWS AN EXAMPLE OF GENE GUN METHOD
BEING APPLIED TO MOUSE

14. 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

15. 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.

16. 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

17. 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

19. 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

20. 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

21.  (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.

23. 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.

24. balloon catheter–based and occlusion-assisted infusion