-Jacqueline De Vera

GENE TRANSFER BY PHYSICAL METHODS

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

GENE TRANSFER

Electroporation

Gene gun

Ultrasound

Hydrodynamic delivery

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

HOW DOES THE ELECTROPORATION PROCESS

WORK?

The Electroporation Pulse Generator EPI 2500

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

THE 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.

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.

balloon catheter–based and occlusion-assisted infusion