2. LEARNING OBJECTIVES
• Introduction to gene therapy.
• Gene Transfer Techniques.
• Application and features perspectives of
gene therapy.
• Conclusion.
3. INTRODUCTION
As per WHO, Gene therapy is defined as the treatment
of disorder or disease through transfer of engineered
genetic material into human cells.
On January 19, 1989 Dr. James A. Wyngarden,
approved the first clinical protocol to insert a foreign
gene into the immune cells of persons with cancer.
4. GOALS OF GENE THERAPY
Gene therapy involves
1. Suppression of Gene Expression.
2. Overexpression of Gene.
3. Modification of Gene.
5. Ideal characteristics of a successful
Gene delivery comprises of :-
1. It should shield the transferred gene from
intracellular nucleases .
2. It should be able to carry the transferred gene
from cell membrane to the target nucleus .
3. It should be capable of overcoming the barriers
involved at each phase of gene delivery process.
4. It should not cause any toxic effect and should
have minimal inflammatory response.
6. Ideal Technique for Gene Transfer
Techniques.
• For the success of gene therapy, it becomes important to
select safe and efficient method to transfer the desired gene
in targeted cell.
7. Methods of Gene Transfer
Physical Methods Chemical Methods Biological Methods
1-Electroporation
2-Thermal assisted gene
transfer
3- Biolistic
4- Microinjection
5- Laser-assisted gene
transfer
6-Ultrasound-assisted gene
transfer
7-Hydrodynamic gene
transfer
1-Calcium Phosphate-
mediated transinfection
2- DEAE- Dextran
mediated transinfection
3- Cationic lipid -
mediated transinfection
4- Polymers
Cationic Dendritic
Transduction using
viral vectors
8. Physical Methods
1-Electroporation
• Electroporation is a process in which brief electrical pulses
create transient pores in the plasma membrane that allow
exogenous nucleic acids to enter the cellular cytoplasm.
• It is cost effective and highly reliable.
Disadvantage of Electroporation
• Limited effective range of working electrodes is 1 cm thus
a surgical procedure is needed to place these electrodes deep
inside organs ,where genes have to be delivered.
• Death rate of Cells subjected to this method is high.
• As high voltage is applied on target cell ,it causes thermal
heating resulting in irreversible damage to the tissues.
9. Thermal Assisted Gene Transfer
• It is hypothetical method .
• Change in the surrounding temperature of cell causes
increases in membrane permeability under external force,
• The desired gene with suitable vector gets inside the host cell
and produced desired effect.
Disadvantage of thermal assisted gene transfer
High voltage applied during elctrotransfer of gene may cause
tissue damage.
To overcome this disadvantage heat can be supplied
exogenously ,so that same effect is obtained in lower voltage
thus targeted application of heat could also help in plasmid
localization.
10. Biolistic
• It involves transfer of gene by using gene gun .
• It involves bombardment of DNA-coated particles.
• It comprises of a DNA cassette that carries all necessary
information for proper expression within target cell.
Mechanism of transfer of gene
Placing DNA-Particle on surface of target tissue
Powerfully accelerated shot into the tissue is given
Thus introducing the DNA effectively into target cell
11. The Advantage of gene gun over in
vivo delivery
• Toxic chemicals and biological systems are not used.
• Delivery of gene is not governed by receptor interaction.
• DNA fragments irrespective of size are transported
within the cell with high reproducibility.
• The only disadvantage of gene gun over in vivo delivery
is :-
* Short term expression.
** Low amount expression.
12. Microinjection
• It is direct physical method of gene delivery.
• Micro needling is needed for gene delivery.
• Done only into single cell.
• Microneedles are made from silicone and biodegradable
polymer.
• Microinjection can be used both in vitro and in vivo gene
delivery.
• Microinjection is important technique for delivering the gene
to embryonic cells.
13. Laser assisted transfection
• LASER : Light Amplification by stimulated emission of
radiation.
• The underlying principal of this method is the transient
modulation of the cell membrane permeability is possible
through a laser pulse .
• The subsequent pore formation leads to difference in the
osmotic pressure between medium and cytosol .
• Facilitating the entry of genetic materials and
macromolecules into the desired cells.
14. Ultrasound-assisted gene transfer
• Aka Sonoporation .
• Ultrasound wave forms cavitation of bubbles in cell
membrane.
these microbubbles expands and contract under
Ultrasound influence.
Collapsing of the bubble and rupturing cell
membrane.
Formation of pore & facilitating entry of gene into
cell.
• It is potentially a suitable method of gene delivery ,as it
supports real time tracking of irradiated fields .
15. Disadvantages of Ultrasound-assisted gene
transfer
• Causes cell Injury.
• This procedure has low –transfection efficacy.
• It is based on hydrodynamic principal of blood flow within
capillaries.
• Injecting large volume of genetic material solution creates
hydrodynamic pressure causing Inc in permeability forming
pore in membrane .
• Membrane pores are closed and molecules are retained
within the cell.
This method of gene delivery is superior due to its simplicity
versatility and efficiency.
Hydrodynamic gene transfer
16. Chemical Methods
• Chemical methods include use of chemical agents as carriers
for intracellular gene delivery.
• 1-Calcium Phosphate-mediated transfection
• 2- DEAE- Dextran mediated transfection
• 3- Cationic lipid - mediated transfection
• 4- Polymers
a) Cationic Polymers
b) Dendritic Polymers
17. Calcium Phosphate-mediated trans
infection
• This method involves mixing the DNA solution with CaCl2,
and a phosphate buffer to form a fine coprecipitate of
calcium phosphate and DNA.
• These complexes bind to the cell membrane and are
phagocytosed within the cell. This method can be used for
both transient and stable transfection.
• This method has a low-transfection efficiency, but the
advantage is that this method is inexpensive.
Cationic lipid - mediated transfection
These are easy to synthesize ,overall positive in charge
These also have low immunogenicity
18. DEAE-dextran-mediated transfection
• It is a polycationic derivative of the carbohydrate polymer
dextran with high molecular weight.
• this enters the cell through endocytosis .
• This method is simple , cheap, and can be employed in
transient cells that cannot survive even a short exposure to
calcium phosphate.
• Disadvantages of DEAE-dextran-mediated transfection
this polymer is it is very toxic, cannot be used to transfect
cells that are sensitive to serum-free media, and is
inefficient for the production of stable transfectants.
19. • Polymers :
Cationic polymers are long chained
DNA material have higher transfection efficiency.
Higher Molecular weight makes it non biodegradable and
toxic
ADVANTAGES OF POLYMERS
Have increased transfection efficacy due to its higher
polymer to nucleic acid change ratio.
It is economically cheap .
But low trans infection efficacy ,cytotoxicity limits its use in
gene therapy
20. Dendritic Polymers
• Possess large molecules.
• Possess large no terminal function groups on surface.
• Highly efficient in target delivery of the genetic material
• Dendritic are repeating branched and large molecules .
22. Application and future
perspective of Gene Therapy
• Gene therapy can be a permanent alternative for a
patient born with genetic diseases to live a healthy
life.
For eg-
Hemophilia A an AAV mediated gene therapy called
valrox has been approved for treatment in patients
with Hemophilia A in 2020
23. Conclusion
• Proves to be a promising therapeutic strategy.
• It circumvents the limitations of pharmacological
treatments .
• However the still the challenge lies in the delivery
of plasmid gene to the cell of interest .
