This document discusses gene transfer techniques, including viral and non-viral delivery systems. It describes gene therapy as using genes to treat disease by inserting a gene into a patient's cells instead of using drugs or surgery. It outlines various non-viral physical methods like gene guns, ultrasound, electroporation, and magnetofection and chemical methods like cationic liposomes and polymers to facilitate gene transfer. Viral vectors discussed include retroviruses, adenoviruses, and adeno-associated viruses. In conclusion, while progress has been made, developing safe and effective non-viral delivery systems for in vivo gene therapy remains a challenge.

1. VIRAL AND NON VIRAL GENE TRANSFER
Presented By
Sujitha Mary
M Pharm
St Joseph College Of Pharmacy

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.

10. 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.

11. 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.

12. 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.

13. 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.

15. 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.

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

18. 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
 Treatment of T-lymphocyte deficiency(ADA),Tumour
Infiltrating Lymphocytes(TIL), Bone marrow cells(ADA
deficiency, Gauchers disease), hepatocytes(LDL receptor
deficiency) and melanoma.
 In-vivo gene transfer using retro viral vectors for suicide
genes used in brain tumour.
 Ex-vivo gene therapy.

19. 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.

20. 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,.
Applications
 Major disadvantages of these vectors are complicated
process of vector production and the limited transgene
capacity of the particles.
 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.

21. 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.

22. Reference
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.