This Slide is prepared by JYOTIRMOY BHATTACHARYYA

1. PRESENTED BY
JYOTIRMOY BHATTACHARYYA
M.PHARM 2ND SEMESTER
ROLL NO. 180520011004

2. CONTENTS
 What is gene therapy ?
 Gene Transfer
 Introduction
 Gene Transfer Techniques
 Non viral delivery systems
 Physical Methods
 Chemical Methods
 Viral delivery systems
 Conclusion
 References

3. What is gene therapy ?
 Gene therapy is an Experimental technique that uses genes
to treat or prevent disease.
 In the future, this technique may allow doctors to treat a
disorder by inserting a gene into a patient’s cells instead of
using drugs or surgery.

4. Gene Transfer
 It is defined as a technique to efficiently and stably
introduce foreign into the genome of the target cells.
 The insertion of unrelated, therapeutic genetic information
in the form of DNA into target cells.

5. Introduction
 There are different reasons to do gene transfer. Perhaps
foremost these reasons is the treatment of diseases using
gene transfer to supply patients with therapeutic genes.
 There are different ways to transfer genes. Some of
methods involve the use of a vector such as a virus so it can
take the gene along with it when it enters the cell.
 It provides a novel approach for the investigation and
potential treatment of a variety of disease.

6. Gene Transfer Techniques
Based on the vectors used the gene transfer techniques can
be divided as
• Non viral methods
• Viral methods

7. Non viral delivery systems
 Non viral systems comprise all the physical and chemical
methods .
 Generally include chemical methods either chemical
methods , such as cationic liposome and polymers, or
physical methods, such as gene gun, electroporation,
particle bombardment, ultrasound and magnetofaction.

8. Physical Methods
DNA particle bombardment
by gene gun
 Gold or tungsten spherical
particles are coated with
plasmid DNA and then
accelerated to high speed by
pressurized gas to penetrate into
target tissue cells.
 Actually it is a modification
technique called “biolistic”,
originally developed for plant
transgenesis , but now used for
in vitro and in vivo gene delivery
into mammalian cells too.

9. Ultrasound
 Ultrasound can make some nanomeric pores in membrane to facilitate
intracellular delivery of DNA particles into cells of internal organs or
tumours .
 The most important limitations of the system is low efficiency of it
especially in vivo.
Electroporation
 Electroporation is temporary destabilization of the cell membrane
targeted tissue by insertion of a pair of electrodes into it.
 DNA molecules in the surrounding media of the destabilized membrane
would able to penetrate into cytoplasm and neoplasm of the cell.
 Electroporation has been used in vivo for many types of tissues, such as
skin, muscle, lung, HPRT gene delivery and tumour treatment.
 Some problems in this method are
 The difficulty in surgical procedure in the placement of electrodes into the
internal tissues
 The high voltage applied to tissue might damage the organ and affect genomic
DNA stability.

10. Magnetofaction
 In this method the magnetic
fields are used to concentrate
particles containing nucleic
acid into target cells.
 In this way, the magnetic
force allows a very rapid
concentration of the entire
applied vector dose onto
cells.
 Magnetofaction has been
adapted to all types of nucleic
acids, non viral transfection
systems and viruses.

11. Chemical methods
Cationic liposome
 Cationic liposomes are more important current non viral polycationic
systems, which compact negatively charged nucleic acids lead to the
formation of nanomeric complexes.
 Cationic liposomes have unique characteristics –
 Capability to incorporate hydrophilic and hydrophobic drugs
 Low toxicity
 No activation of immune system.
 Targeted delivery of bioactive compounds to the site of action.
Cationic liposomes are being used in gene delivery into lung, skeletal
muscles, spleen, kidney, liver, testis, heart and skin cells.

12. Cationic Polymers
 Cationic polymers have also been used extensively for gene transfer.
 Upon mixing with DNA, these polymers form nanosized complexes,
often called polyplexes.
 Among cationic polymers, PEI is considered one of the most effective
polymer-based transfection agents.
 PEI leads to an influx of chloride counter ions within the compartment
and a build up of osmotic pressure that causes the swelling and rupture
of the endosomal membrane.
 Recently, more polymers with improved biocompatibility and
biodegradability have been reported demonstrating equal or superior
performance comparing to nondegradable PEIs.

13.  Some Polymers Commonly Used for Gene Transfer
Polymer Abbreviation Unique feature
Poly(ethylene)glyc
ol
PEG Inert
Polyethylenimine PEI cationic
Poly(L-lysine) PLL Cationic
Poly(N-
vinylpyrrolidone)
PVP Neutral
Poly(propylenimin
e)
PPI Dendromer
Poly(amidoamine) PAMAM Dendromer
Triethylenetetrami
ne
TETA Cationic
Poly(allylamine) Cationic
Poly(phosphazene
)s
PPZ Biodegradable

14. Viral delivery systems
 Viruses are naturally evolved vehicles that efficiently
transfer their genes into host cells.
 Choice of viral vector is dependent on gene transfer
efficiency, capacity to carry foreign genes, toxicity, stability,
immune responses towards viral antigens and potential
viral recombination.
 There are a wide variety of vectors used to deliver DNA or
oligo nucleotides into mammalian cells, either in vitro or in
vivo.
 The most common vector system based on retroviruses,
adenoviruses, herpes simplex viruses, adeno associated
viruses.

16. The three commonly used viral gene transfer systems are-
• Retro virus(RV)
• Adeno virus(AV)
• Adeno Associated Virus(AAV)

17. Retro Virus Vector
 Commonly employed vectors , derived from Murine Leukemia
Virus(MuLV).
 Virus genome has two single copy RNA molecules, complexed with viral
core proteins, surrounded by lipid envelope.
Applications
 Ex-vivo gene therapy.
 In-vivo gene transfer using retro viral vectors for suicide genes used in
brain tumour.
 Treatment of T-lymphocyte deficiency(ADA),Tumour Infiltrating
Lymphocytes(TIL), Bone marrow cells(ADA deficiency, Gauchers
disease), hepatocytes(LDL receptor deficiency) and melanoma.

18. Adeno Virus Vectors
 These are non enveloped DNA viruses, linear genome and double
stranded DNA molecule of about 36kb.
 Adeno viral vectors have been isolated from a large number of different
species and more than 100 different serotypes have been reported.
 Adeno viruses type2 and type 5 can be utilized for transferring both
dividing and non dividing cells and have low host specificity.
Applications
 In vivo gene therapy – transduce non dividing and terminally
differentiated cells.
 Transfect cells in vivo in the intact organ.
 Gene therapy for cystic therapy.
 Gene therapy of muscle in liver and therapy of disease of CNS.

19. Adeno Associated Virus Vector
 Members of Parvo virus family.
 Heat stable and resistant to various chemicals.
 Depend on virus – cannot replicate its own, another virus is necessary
for replicate,.
 Major disadvantages of these vectors are complicated process of vector
production and the limited transgene capacity of the particles.
Applications
 Used in haematopoietic stem cells for treatment of ß- thalassemia and
sickle cell anaemia.
 ß-thalassemia erythrocyte contains insufficient ß- globin chain
whereas, mutant ß- globin chains are produced in sickle cell.

20. Conclusion
Although numerous viral and non viral gene delivery systems have been
developed in the last 3 decades, all of them have some disadvantages
that have made some limitations in their clinical application and yet no
delivery system has been designed that can be applied in gene therapy
of all kinds of cell types in vitro and in vivo with no limitation and side
effects. So it seems that the process of developing successful delivery
systems, especially non viral systems, for use in in vivo is still in its
adolescence and more efforts are needed. Totally, key steps effective in
improving the currently available systems include the following: (1)
improving extracellular targeting and delivery, (2) enhancing
intracellular delivery and long-time expression, and (3) reducing
toxicity and side effects on human body.

21. References
1. Nayerossadat N et.al, “Viral and nonviral delivery systems for gene
delivery”, Advanced Biomedical Research. 2012 ;1(2):1-12.
2. Stone D. Novel viral vector systems for gene therapy. Viruses
2010;2:1002-7.
3. Katare DP, Aeri V. Progress in gene therapy: A review. I.J.T.P.R
2010;1:33.
4. ASIAN J.EXP.BIOL.SCI.Vol1(1)2010:208-218.
5. Smith KR. Gene Therapy: The Potential Applicability of Gene Transfer
Technology to the Human Germline. Int J Med Sci 2004;1:76-91.

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