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
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
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
18.
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.
22.
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.
1987 when Okino and Mohri3 showed that the use of the anticancer drug bleomycin,
the purpose of electroporation is to assist the uptake of useful molecules such as a DNA vaccine into a cell. The biological material is injected into or applied to the surface of the target tissue and followed by the application of brief, controlled electrical pulses directed to that tissue. As shown in the pictures below, electroporation's millisecond electrical pulses temporarily create enhanced permeability of pores in the cell membrane. After a short period of time the pores reseal, leaving the cells undamaged. During the period that these pores exist, a significant quantity of the previously injected biomolecules are taken up and then trapped in the cell, enabling them to then perform their intended function.
gene therapy experiments. By using a high-voltage plasma discharge, DNA was efficiently delivered following very short (microsecond) pulses. Compared to electroporation, the technique resulted in greatly increased efficiency and less cellular damage.-http://en.wikipedia.org/wiki/Vectors_in_Gene_Therapy#Viruses
http://www.google.com.ph/imgres?q=gene+gun&hl=en&client=firefox-a&hs=zCo&sa=X&rls=org.mozilla:en-GB:official&biw=1366&bih=611&tbm=isch&prmd=imvns&tbnid=bJk8LPcFBv-gTM:&imgrefurl=http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Gene_gun.html&docid=HZHdv2KdavbJRM&imgurl=http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Helios_gun.gif&w=300&h=312&ei=_TVTT7zAIuuamQX_qJW9Cg&zoom=1DNA is deposited on the surface of gold particles, which are then accelerated by pressurized gas and expelled onto cells or a tissue. The momentum allows the gold particles to penetrate a few millimeters deep into a tissue and release DNA into cells on the path. Such a simple and effective method of gene delivery is expected to have important applications as an effective tool for DNA-based immunization. Further improvements could include chemical modification of the surface of the gold particles to allow higher capacity and better consistency for DNA coating, and fine-tuning of the expelling force for precise control of DNA deposition into cells in various tissues.27
http://group2fls.blogspot.com/2008/04/gene-gun-gene-gun-is-one-of-non-viral.htmlBesides that, there is research shows that gene gun bombardment with DNA-coated gold particles is a potential alternative to hydrodynamics-based transfection for delivering genes into superficial hepatocytes in hepatic gene therapy. Compared with hydrodynamic-based trasnfection, gene gun bombardment resulted in minimal scattered hepatic necrosis. On the other hand, severe hepatic infarction impedes foreign gene expression in the superficial hepatocytes after hydraodynamic-based transfection.
transformation of organelles as well as yeast mitochondriaDNA vaccination/genetic immunization, gene therapy, tumor biology/wound healing, plant virology and many othershttp://group2fls.blogspot.com/2008/04/gene-gun-gene-gun-is-one-of-non-viral.html
-enables researchers to engineer organelle-encoded herbicide or pesticide resistances in crop plants and to study photosynthetic processesHelios Gene Gunhttp://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Gene_gun.html
Cavitation is the formation and then immediate implosion of cavities in a liquid – i.e. small liquid-free zones ("bubbles") – that are the consequence of forces acting upon the liquid. Microbubbles are bubbles smaller than one millimetre in diameter, but larger than one micrometre.