• 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.
Researchers are testing several approaches to gene
• Replacing a mutated gene that causes disease with a healthy
copy of the gene.
• Inactivating or “knocking out,” a mutated gene that is
• Introducing a new gene into the body to help fight a disease.
• Although gene therapy is a promising treatment option for a
number of diseases, the technique remains risky and is still
under study to make sure that it will be safe and effective.
• Gene therapy is currently only being tested for the treatment
of diseases that have no other cures.
• It is defined simply as a technique to efficiently and stably
introduce foreign genes into the genome of target cells.
• The insertion of unrelated, therapeutic genetic information in
the form of DNA into target cells.
• There are different reasons to do gene transfer. Perhaps
foremost among 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 these
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.
• During the 1970’s Rogers made it became possible to introduce
exogenous DNA constructs into higher eukaryotic cells in vitro.
• Mammalian transgenesis was first achieved in the early 1980’s. The
model used in this study was mice.
• In 1990’s, first approved gene therapy case in The United States took
place on 14th September 1990, at the national institute of health, under
the direction of professor William French Anderson.
• In 2012, Glybera became the first gene therapy treatment to be approved
for clinical use in either Europe or The United States after its
endorsement by the European commission.
• Gene transfer may help treat type 1 diabetes (which is due to
failure of the pancreas to produce enough insulin).
• Among the key factors that decide whether the gene
for insulin is turned on or off is the gene PDX-1.
• Using a vector virus the PDX-1 gene has been transferred (into
mice) where the gene is expressed by pancreatic cells which now
• Gene transfer of PDX-1 may reprogram tissues other than the
pancreas to make insulin and control the abnormally high blood
sugar levels in diabetes.
Gene Transfer Techniques
Based on the vectors used the gene transfer techniques can be
• Non-viral methods.
• Viral methods.
Non-Viral Delivery Systems
• Non-viral vectors using mechanical or chemical approaches
can efficiently transfect cells in vitro.
• Mechanical methods involve direct injection or the use of
“gene gun technology” to introduce the plasmid DNA.
Low levels of gene expression.
Inability to use for systemic administration due to the presence
of serum nucleases.
• Electroporation using electrical mediated disruption of cell
membranes to effect transfection is used mainly for in vitro
• The success of non-viral delivery will be greatly dependent
on the ability to design systems that can transfect cells with
high efficiency, increased stability in presence of serum
proteins and reduced toxicity to cells both in vitro and in
• One advantage of this system is they have no constraints on
size of the gene that can be delivered.
Generally there are two approaches for DNA transfer
1. Natural methods of DNA transfer.
2. Artificial methods of DNA transfer.
• It is an efficient process to transfer DNA into cells.
• Microscopic pores are induced in biological membrane by the
application of electric field. These pores are known as
electropores which allow the molecules, ions and water to
pass from one side of the membrane to another.
• Electroporation has been reported to enhance the level of
gene expression and significantly improve immune responses
elicited to DNA vaccines in both large and small animals.
General applications of electroporation:
• Introduction of exogeneous DNA into animal cell lines, plant
protoplast, yeast protoplast and bacterial protoplast.
• Electroporation can be used to increase efficiency of
transformation or transfection of bacterial cells.
• Wheat, rice, maize, tobacco have been stably transformed with
frequency upto 1% by this method.
• Electroporation of early embryo may result in the
production of transgenic animals.
• Hepatocytes, epidermal cells, haematopoietic stem cells,
fibroblast, mouse T and B lymphocytes can be transformed
by this technique.
• Naked DNA may be used for gene therapy by applying
electroporation device on animal cells.
Advantages of electroporation
1. Method is fast.
2. Less costly.
3. Applied for a number of cell types.
4. Simultaneously a large number of cell can be treated.
5. High percentage of stable transformants can be produced.
• Microinjection where the DNA is directly injected into plant
protoplasts or cells (specifically into the nucleus or cytoplasm)
using fine tipped (0.5 - 1.0 micrometer diameter) glass needle
• This method of gene transfer is used to introduce DNA into
large cells, normally performed under a specialized optical
microscope setup called a micromanipulator.
• The process is frequently used as a vector in genetic
engineering and transgenetics to insert genetic material into a
• Computerized control of holding pipette, needle, microscope
stage and video technology has improved the efficiency of
Advantages of microinjection:
• Frequency of stable integration of DNA is far better as compare
to other methods.
• Method is effective in transforming primary cells as well as
cells in established cultures.
• The DNA injected in this process is subjected to less extensive
• Mere precise integration of recombinant gene in limited copy
number can be obtained.
Limitations of microinjection:
2. Skilled personal required.
3. More useful for animal cells.
4. Embryonic cells preferred for manipulation.
5. Knowledge of mating timing, oocyte recovery is essential.
6. Method is useful for protoplasts and not for the walled
Applications of microinjection
• Process is applicable for plant cell as well as animal cell but
more common for animal cells.
• Technique is ideally useful for producing transgenic animal
• Procedure is important for gene transfer to embryonic cells.
• Applied to inject DNA into plant nuclei.
• Method has been successfully used with cells and protoplast
of tobacco, alfalfa etc.
• Microinjection is potentially a useful method for
simultaneous introduction of multiple bioactive compounds
such as antibodies, peptides, RNAs, plasmids, diffusion
markers, elicitors, Ca2+ as well as nucleus and artificial
micro or Nano particles containing those chemicals into the
same target single-cells.
• Macroinjection is the method tried for artificial DNA transfer
to cereals plants that show inability to regenerate and
develop into whole plants from cultured cells.
• Needles used for injecting DNA are with the diameter
greater than cell diameter.
• DNA injected with conventional syringe into region of plant
which will develop into floral tillers.
• Around 0.3 ml of DNA solution is injected at a point above
tiller node until several drops of solution came out from top
of young inflorescence.
• Timing of injection is important and should be fourteen
days before meiosis.
• This method was found to be successful with rye plants.
• It is also being attempted for other cereals plants.
Advantages and limitations of macroinjection
• This technique does not require protoplast.
• Instrument will be simple and cheap.
• Methods may prove useful for gene transfer into cereals which
do not regenerate from cultured cell easily.
• Technically simple.
1. Less specific.
2. Less efficient.
3. Frequency of transformation is very low.
BIOLISTICS OR MICROPROJECTILES FOR DNA TRANSFER
• Biolistics or particle bombardment is a physical method that
uses accelerated micro projectiles to deliver DNA or other
molecules into intact tissues and cells.
• Biolistics transformation is relatively new and novel method
amongst the physical methods for artificial transfer of
• This method avoids the need of protoplast and is better in
efficiency. This technique can be used for any plant cells, root
section, embryos, seeds and pollen.
• The gene gun is a device that literally fires DNA into target
• The DNA to be transformed into the cells is coated onto
microscopic beads made of either gold or tungsten. Beads are
carefully coated with DNA.
• The coated beads are then attached to the end of the plastic
bullet and loaded into the firing chamber of the gene gun.
• An explosive force fires the bullet down the barrel of the gun
towards the target cells that lie just beyond the end of the
• When the bullet reaches the end of the barrel it is caught and
stopped, but the DNA coated beads continue on toward the
• Some of the beads pass through the cell wall into the
cytoplasm of the target cells.
• Here the bead and the DNA dissociate and the cells become
• Once inside the target cells, the DNA is solubilised and may
original 22-caliber biolistic gun
• DNA is bound to the microprojectiles, which impact the
tissue or immobilized cells at high speeds
General applications of biolistics
• Biolistics technique has been used successfully to transform
soyabean, cotton, spruce, sugarcane, papaya, sunflower, rice,
maize, wheat, tobacco etc.
• Genomes of subcellular organelles have been accessible to
genetic manipulation by biolistic method.
• Method can be applied to filamentous fungi and yeast
• The particle gun has also been used with pollen, early stage
embryoids, meristems and somatic embryos.
Advantages and limitations of biolistics
1. Requirement of protoplast can be avoided.
2.Walled intact cells can be penetrated.
3. Manipulation of genome of subcellular organelles can be
1. Integration is random.
2. Requirement of equipments.
LIPOSOME MEDIATED GENE TRANSFER
• Liposomes are spheres of lipids which can be used to
transport molecules into the cells.
• These are artificial vesicles that can act as delivery agents for
exogenous materials including transgenes.
• They are considered as sphere of lipid bilayers surrounding
the molecule to be transported and promote transport after
fusing with the cell membrane.
• Cationic lipids are those having a positive charge are used
for the transfer of nucleic acid.
• These liposomes are able to interact with the negatively
charged cell membrane more readily than uncharged
liposomes, with the fusion between cationic liposome and
the cell surface resulting in the delivery of the DNA directly
across the plasma membrane.
• Cationic liposomes can be produced from a number of
cationic lipids, e.g. DOTAP and DOTMA.
• These are commercially available lipids that are sold as an in
vitro-transfecting agent, as lipofectin.
• Liposomes for use as gene transfer vehicles are prepared by
adding an appropriate mix of bilayer constituents to an
aqueous solution of DNA molecules.
• The liposomes are then ready to be added to target cells.
• Germline transgenesis is possible with liposome mediated
gene transfer and ES cells have been successfully transfected
by liposomes also.
Advantages of liposome mediated DNA transfer
2. Long term stability.
3. Low toxicity.
4. Protection of nucleic acid from degradation.
CALCIUM PHOSPHATE MEDIATED DNA TRANSFER
• The process of transfection involves the admixture of isolated DNA
(10-100ug) with solution of calcium chloride and potassium
phosphate under condition which allow the precipitate of calcium
phosphate to be formed.
• Cells are then incubated with precipitated DNA either in solution or
in tissue culture dish. A fraction of cells will take up the calcium
phosphate DNA precipitate by endocytosis.
• Transfection efficiencies using calcium phosphate can be quite low,
in the range of 1-2 %. It can be increased if very high purity DNA is
used and the precipitate allowed to form slowly.
Limitations of calcium phosphate mediated DNA transfer
• Frequency is very low.
• Integrated genes undergo substantial modification.
• Many cells do not like having the solid precipitate adhering
to them and the surface of their culture vessel.
• Due to above limitations transfection applied to somatic gene
therapy is limited.
DNA TRANSFER BY DAE-DEXTRAN METHOD
• DNA can be transferred with the help of DAE Dextran also. DAE-Dextran
may be used in the transfection medium in which DNA is
• This is polycationic, high molecular weight substance and
convenient for transient assays in cos cells.
• It does not appear to be efficient for the production of stable
• If DEAE-Dextran treatment is coupled with Dimethyl Sulphoxide
(DMSO) shock, then upto 80% transformed cell can express the
• It is known that serum inhibits this transfection so cells are
washed nicely to make it serum free.
• Stable expression is very difficult to obtain by this method.
• Treatment with chloroquinine increases transient expression
• The advantage of this method is that, it is cheap, simple and
can be used for transient cells which cannot survive even
short exposure of calcium phosphate.
POLYETHYLENE GLYCOL MEDIATED TRANSFECTION
• This method is utilized for protoplast only. Polyethylene
glycol stimulates endocytosis and therefore DNA uptake
• Protoplasts are kept in the solution containing PEG.
• Calcium chloride is added and sucrose and glucose acts as
osmotic buffering agent.
• After exposure of the protoplast to exogenous DNA in
presence of PEG and other chemicals, PEG is allowed to get
removed. Intact surviving protoplasts are then cultured to
form cells with walls and colonies in turn.
• After several passages in selectable medium frequency of
transformation is calculated. PEG based vehicles were less
toxic and more resistant to nonspecific protein adsorption
making them an attractive alternative for non-viral gene
Viral Delivery Systems
• Viruses are naturally evolved vehicles that efficiently
transfer their genes into host cells.
• This ability has made them attractive as tools for gene
• Viral vectors that have been extensively studied and
genetically manipulated for safety concerns in laboratory
research and for in vivo gene transfer protocols include
retroviruses, adenoviruses, herpes simplex viruses,
lentiviruses, adeno associated viruses and Sindbis viruses.
• Each of the viral vectors has their own individual
advantages, problems, and specific applications.
• Choice of viral vectors is dependent on gene transfer
efficiency, capacity to carry foreign genes, toxicity, stability,
immune responses towards viral antigens and potential viral
• There is a wide variety of vectors used to deliver DNA or
oligo nucleotides into mammalian cells, either in vitro or in
• The most common vector systems are based on retroviruses,
adeno -associated virus (AAV), adenovirus, herpes simplex
virus (HSV), cationic liposomes, and receptor-mediated
• Other viral vectors that are currently under development are
based on lenti viruses, human cytomegalovirus (CMV),
Epstein-Barr virus (EBV), poxviruses, negative-strand RNA
viruses (influenza virus), alpha viruses etc.
The three commonly used viral gene transfer systems are
1. Retrovirus (RV).
2. Adenovirus (AV).
3. Adeno Associated Virus (AAV).
Retro Virus Vectors
• Commonly employed vectors.
• Derived from Murine Leukemia Virus (MuLV).
• Not associated with pathology in humans.
• Virus genome has two single copy RNA molecules,
complexed with viral core proteins, surrounded by lipid
• Infect wide variety of cells.
• Proviral copy – stable integration into the host cell –
• Viral replication sequences – cis and Trans acting
elements – generation of replication defective
Contains two building blocks
• Retroviral Vector (Transfer Gene).
• Retrovirus Packing Cell (Replication Defective Virus
• The packing signal and other cis acting sequences are
• Transfected into the packing cells.
• Grown in culture medium, released into the medium.
• Harvested and added to the cells to be genetically corrected.
• Ex-vivo gene therapy.
• Treatment of T-lymphocyte deficiency (ADA), Tumor
Infiltrating Lymphocytes (TIL), Bone marrow cells (ADA
deficiency, Gauchers disease), hepatocytes (LDL receptor
deficiency), and melanoma.
• In-vivo gene transfer using retroviral vectors for suicide
genes - used in Brain Tumor.
Adeno Virus Vectors
• These are non-enveloped DNA viruses, linear genome and
double stranded DNA molecule of about 36kb.
• 49 distinct subtypes (serotypes).
• Genome Regions: Distinguished into,
• Early (E), and Late (L) – transcription of regions – prior to
or after onset of DNA replication.
• The extremity – consists of a short sequence, The Inverted
Terminal Repeat (ITR) - viral replication and for
• The Early (E1) genes – Trans activation of other E genes (E2 &
E4) – shut down host cell protein synthesis – starts replication
of adenovirus DNA.
• Late genes L2 – L5 – activated – code for structural proteins of
• New virion particles are released by host cell lysis.
• These virus enters the Nucleus and remains extra
chromosomally and will not integrate into the host cell
• Biology of Adenovirus – characterized.
• Not associated with severe human pathology.
• Efficient in introducing its DNA into host cells.
• Can infect a variety of cells & have a broad host range.
• Replication of defective recombinant Adenoviruses, lacking
E1 region can be propagated in-vitro in human cells
harboring E1 sequence in the genome.
• In-vivo gene therapy – transduce non-dividing and
terminally differentiated cells.
• Transfect cells in-vivo in the intact organ.
• Gene therapy of Cystic Fibrosis.
• Gene therapy of muscle in liver (Blood clotting disorders)
and therapy of diseases of CNS.
Adeno Associated Virus Vectors (AAV)
• Members of Parvo virus family.
• Lack envelope.
• Heat stable and resistant to various chemicals.
• Single stranded DNA molecule.
• Depend on virus – cannot replicate on its own – another virus
is necessary for replicate, uses Adeno/Herpes virus for
• Establishes latent infection – integrates its genome into the
host cell DNA.
• Used in hematopoietic stem cells for treatment of -
Thalassemia and sickel cell anaemia.
• - Thalassemic erythrocyte contains insufficient - globin
chain whereas, mutant - globin chains are produced in
• Daan J. A. Crommelin and Robert D. Sindelar, “
Pharmaceutical Biotechnology”, 1st Edition, 1997, Harwood
Academic Publishers, page no-167 – 181.
• James D. Watson, Michael Gilman, Jan Witkowski, Mark
Zoller, “Recombinant DNA”, 2nd Edition, Scientific American
books, 1998, Page no-567 –579.
• ASIAN J. EXP. BIOL. SCI. VOl 1 (1) 2010:208-218.