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