ASIAN J. EXP. BIOL. SCI.

VOl 1 (1) 2010:208-218

Society of Applied Sciences

REVIEW ARTICLE

Gene Transfer Technologies and their Applications: Roles in

Human Diseases
*

K.H.KHAN

Medical Biotechnology Division, School of Biosciences and Technology,

VIT University, Vellore-632014, Tamil Nadu, India.

ABSTRACT
Gene is a segment of DNA that codes RNA for polypeptide molecule. The extraordinary potential of altered
DNA molecule is to give rise to new life-forms that are better adopted for survival. Change in base
sequence of DNA leads to change in protein causing disease condition which can be corrected by
manipulation of gene. This article discuses and summarizes important work in the literature regarding the
gene transfer technologies. The main techniques focused in this article are electroporation, microinjection,
macroinjection, biolistics or microprojectiles for DNA transfer, liposome mediated gene transfer, calcium
phosphate mediated DNA transfer, DAE-Dextran for gene transfer, gene transfer by polycation-DMSO,
polyethylene glycol mediated transfection and gene transfer through peptide. Much emphasis has been
given to the gene manipulation in cardiovascular diseases, parkinsons disease, lysosomal disorders,
ocular gene therapy, gene transfer in liver and osteoarthritis. This review article will let the reader to have
a retrospective study on gene transfer technologies which manipulates the gene and also cure human
diseases.
KEY WORDS: Gene, Gene transfer technologies, Gene manipulation, Human diseases

INTRODUCTION
There is no substance so important as deoxyribonucleic acid (DNA) because it carries within its
structure the hereditary information that determines the structures of protein which is a prime
molecule of life. The most important and major contribution to the biotechnology and molecular
biology comes from the genetic engineering. Considerable efficiency and skill in introduction of
exogenous DNA into organisms and their expression have been achieved. Transgenic animals and
plants can be obtained by introduction of exogeneous DNA into targeted animals and plants
accompanied by the stable expression. The techniques for the transfer of DNA into organisms differ
from organism to organisms. Generally there are two approaches for DNA transfer. In first case the
transfer of DNA take place by natural method and in second case the transfer is by artificial method.
DNA transfer by natural method is summarized in Table 1 and artificial method in Table 2.
Table 1: Natural methods of DNA transfer.
S.No DNA transfer by natural methods
1.
Conjugation
2.
Bacterial transformation
3.
Transposition
4.
Phage transduction
5.
Retroviral transduction
6.
Agrobacterium mediated transfer

The choice of methods of DNA transfer depends upon

the target cells in which transformation will be
performed. It also depends upon the objectives of gene
manipulation. The transfection may be either stable or
transient. Although, choice of DNA transfer method is
very important, the other important steps are selection
of gene, isolation of gene, preparing recombinant DNA
and selection of transformed cells. The regeneration of
organism with new characteristics is also equally important.
This article is totally based on literature survey. The author paid his attention toward the gene transfer
technologies.
Much
focus
has
been
given
to
the
techniques
of
electroporation, microinjection, macroinjection, biolistics or microprojectiles for DNA transfer,
liposome mediated gene transfer, calcium phosphate mediated DNA transfer, use of DAE-Dextran to

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K.H Khan

transfer gene, polycation-DMSO for DNA transfer, polyethylene glycol mediated transfection and gene
transfer through peptides. Special attention is given to the diseases where gene manipulation is possible
or attempts were made to cure diseases by gene transfer. Gene manipulation in cardiovascular diseases,
parkinsons disease, lysosomal disorders, ocular gene therapy, gene transfer in liver and osteoarthritis
are focused. This article will be a boon for those readers who are going to start their research career in
molecular biology related to gene manipulation.
Table 2: Artificial methods of DNA transfer.
S.No
1.
2.
3.
4.

DNA transfer by artificial methods

Physical methods
Macroinjection
Microinjection
Protoplast fusion
Biolistics transformation

The article discusses and summarizes a number

of modern techniques of gene transfer and its
various applications. Moreover its applications
for manipulating the gene in human diseases are
also explained.

GENE TRANSFER TECHNOLOGIES

Gene transfer technology provides the ability to
1.
genetically manipulate the cells of higher
2.
animals. During the 1970s it became possible to
3.
introduce exogenous DNA constructs into higher
4.
eukaryotic cells in vitro. Mammalian (germline)
transgenesis was first achieved in the early
Electrical methods
1980s. The model used in this study was mice.
1.
Electroporation
The delivery of genes in vitro can be done by
2.
Electrofusion
treating the cells with viruses such as retrovirus
or adenovirus, calcium phosphate, liposomes, particle bombardment, fine needle naked DNA
injection, electroporation or any combination of these methods. These are the powerful tools for
research and have possible applications in gene therapy. A number of valuable techniques used to
transfer genes in animals and plants cells and their scope and contributions are explained below.
Electroporation
Electroporation uses electrical pulse to produce transient pores in the plasma membrane thereby
allowing macromolecules into the cells. 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. The pores can be recovered only if a suitable electric pulse is applied. The
electropores reseal spontaneously and the cell can recover. The formation of electropores depends
upon the cells that are used and the amplitude and duration of the electric pulse that is applied to
them. Electric currents can lead to dramatic heating of the cells that can results in cell death. Heating
effects are minimized by using relatively high amplitude, a short duration pulse or by using two very
short duration pulses [1]. Various applications of electroporation are listed in the Table 3 and its
advantages in Table 4.
In terms of mammalian transgenesis, electroporation is an effective method of introducing exogenous
DNA into embryonic stem (ES) cells [2]. This technique has recently, been used to transfer genes into
cultured mammalian embryos at defined stages of development [3]-[4]. It was reported [5] that there
is an increase from 12 to 19% of transgenic bovine blastocysts when electroporation was included in
an otherwise passive sperm-DNA uptake protocol. Similar findings were reported [6], again with
transgenic bovine blastocysts. Fish species were also reported to be genetically manipulated in this
way [7]-[8]. Electroporation has been reported to enhance the level of gene expression and
significantly improve immune responses elicited to DNA vaccines in both large [9] and small [10[[11] animals.
Chemical methods
DNA transfer by calcium phosphate method
Transfer of DNA by use of polyethene glycol
Use of DEAE-Dextran for DNA transfer
Liposome mediated transfer

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Table 3: General applications of electroporation.

S.No
1.
2.
3.
4.
5.
6.
7.
8.

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.
Genes encoding selectable marker may be used to introduce genes
using electroporation.
To study the transient expression of molecular constructs.
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.
Table 4: Advantages of electroporation.
S.No Advantages
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
In microinjection DNA can be introduced into cells or protoplast with the help of very fine needles or
glass micropipettes having the diameter of 0.5 to 10 m. Some of the DNA injected may be taken up
by the nucleus. Computerized control of holding pipette, needle, microscope stage and video
technology has improved the efficiency of this technique. The advantages, limitations and
applications are listed in the Table 5, 6 and 7 respectively.
Table 5: Advantages of microinjection.
S.No
1.
2.
3.
4.

S.No
1.
2.
3.
4.
5.
6.
7.
8.

Advantages
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
modifications.
Mere precise integration of recombinant gene in limited copy
number can be obtained.
Table 6: Limitations of microinjection.
Limitations
Costly.
Skilled personal required.
More useful for animal cells.
Embryonic cells preferred for manipulation.
Knowledge of mating timing, oocyte recovery is essential.
Method is useful for protoplasts and not for the walled cells.
Process causes random integration.
Rearrangement or deletion of host. DNA adjacent to site of
integration are common.

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S.No
1.
2.
3.
4.
5.

K.H Khan

Table 7: Applications of microinjection.

Applications
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 quickly.
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 [12]-[13], peptides [14], RNAs [1]-16], plasmids [17]-[18], diffusion
markers [19]-[20], elicitors [21], Ca2+ [22] as well as nucleus [23] and artificial micro or
nanoparticles containing those chemicals [24-25] into the same target single-cells.
Macroinjection
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 [41].
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 limitation of macroinjections are listed in the Table
8.
Table 8: Advantages and limitations of macroinjection.
S.No
1.
2.
3.
4.
1.
2.
3.

Advantages
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.
Limitations
Less specific.
Less efficient.
Frequency of transformation is very low.

Biolistics or microprojectiles for DNA transfer

Biolistics or particle bombardment is a physical method that uses accelerated microprojectiles to
deliver DNA or other molecules into intact tissues and cells [26]. The method was developed initially
to transfer genes into plants by Sanford [27]. Biolistics transformation is relatively new and novel
method amongst the physical methods for artificial transfer of exogenous DNA. 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 cells [28]. 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 barrel. When the bullet reaches the end of
the barrel it is caught and stopped, but the DNA coated beads continue on toward the target cells.
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 transformed. Once inside the target cells, the DNA is
solubilised and may be expressed [29].

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This approach, sometimes known as biolistics, was originally developed for plant transgenesis but
has been shown to be effective for transferring transgenes into mammalian cells in vivo [30]. The gene
gun is particularly useful for transforming cells that are difficult to transform by other methods, e.g.
plant cells. It is also gaining in use as a method for transferring DNA construct into whole animals.
The general applications of biolistics are listed in the Table 9. Advantages and limitations are listed in
Table 10.
Table 9: General applications of biolistics.
S.No
1.
2.
3.
4.
5.

Applications
Biolistics technique has been used successfully to transform soyabean,
cotton, spruce, sugarcane, papaya, corn, sunflower, rice, maize, wheat,
tobacco etc.
Genomes of subcellular organelles have been accessible to genetic
manipulation by biolistic method.
Mitochondria of plants and chloroplast of chlamydomonas have been
transformed.
Method can be applied to filamentous fungi and yeast (mitochondria).
The particle gun has also been used with pollen, early stage
embryoids, meristems and somatic embryos.
Table 10: Advantages and limitations of biolistics.

S.No
1.
2.
3.
1.
2.

Advantages
Requirement of protoplast can be avoided.
Walled intact cells can be penetrated.
Manipulation of genome of subcellular organelles can be achieved.
Limitations
Integration is random.
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 [31][33]. 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 (N-1((2,3-dioleoyloxy) propyl)-N,N,N-trimethylammoniumethyl sulphate) and DOTMA (N-(1-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride) [34]. These are commercially available
lipids that are sold as an in vitro-transfecting agent, as lipofectin. Advantages are listed in the Table
11.
Table 11: Advantages of liposome mediated DNA transfer.
S.No
1.
2.
3.
4.

Advantages
Simplicity.
Long term stability.
Low toxicity.
Protection of nucleic acid from degradation.

Liposomes for use as gene transfer vehicles are prepared by adding an appropriate mix of bilayer
constituents to an aqueous solution of DNA molecules. In this aqueous environment, phospholipid
hydrophilic heads associate with water while hydrophobic tails self-associate to exclude water from
within the lipid bilayer. This self-organizing process creates discrete spheres of continuous lipid

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bilayer membrane enveloping a small quantity of DNA solution. The liposomes are then ready to be
added to target cells [35]-[36]. Germline transgenesis is possible with liposome mediated gene
transfer and ES cells have been successfully transfected by liposomes also [37].
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 % [38]. It can
be increased if very high purity DNA is used and the precipitate allowed to form slowly [39].
Procedures have been developed where cell taking up exogenous DNA could be upto 20%. This
technique is used for introducing DNA into mammalian cells. The limitation of this method is
explained in Table 12. ES cells can be transfected by co-precipitation [40] also.
Table 12: Limitations of calcium phosphate mediated DNA transfer.
S.No
1.
2.
3.
4.
5,

Limitations
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.
Integration with host cell chromosome is random.
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 present. 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 transfectants. If DEAE-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO)
shock, then upto 80% transformed cell can express the transferred gene. It is known that serum
inhibits this transfection so cells are washed nicely to make it serum free [41]. Stable expression is
very difficult to obtain by this method. Treatment with chloroquinine increases transient expression of
DNA. 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.
Transfer of DNA by polycation-DMSO
As calcium phosphate method of DNA transfer is reproducible and efficient but there is narrow range
of optimum conditions. DNA transfer by polycation, polybrene is used to increase the adsorption of
DNA to the cell surface followed by a brief treatment with 25-30% DMSO to increase membrane
permeability and enhance uptake of DNA. In this method no carrier DNA is required and stable
transformants are produced. This method works with mouse fibroblast and chick embryo [41].
Polyethylene glycol mediated transfection
This method is utilized for protoplast only. Polyethylene glycol stimulates endocytosis and therefore
DNA uptake occurs. Protoplasts are kept in the solution containing polyethylene glycol (PEG). The
molecular weight of PEG used is 8000 dalton having the final concentration of 15%. Calcium chloride
is added and sucrose and glucose acts as osmotic buffering agent. To reduce the effects of nuclease
present, the carrier DNA from salmon or herring sperm may also be added. 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 [41]. PEGbased vehicles were less toxic and more resistant to nonspecific protein adsorption making them an
attractive
alternative
for
nonviral
gene
delivery
[42].
PEG PBLG nanoparticle mediated HSV TK/GCV gene therapy for oral squamous cell carcinoma was
also reported [43].
Gene transfer through peptide

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A variety of peptide sequences are there which are able to bind to, and condense, DNA to make it
more amenable for entry into cells; e.g. the tetrapeptide Serine - proline-lysine-lysine (present on
the C- terminus of the histone H1 protein) helps in DNA transfer [44]. Lysine is a positively charged
amino acid. The positively charged lysine amino acid side chains help to counteract the negatively
charged phosphate DNA backbone and allow the DNA molecules to pack closely to each other.
Rational design of peptide sequences has also been used to develop synthetic DNA binding peptide.
Tyrosine-lysine-alanine-(Lysine)s-tryptophan-lysine is a peptide which is very effective to form
complexes with DNA [45]. DNA binding peptides that can be coupled to cell specific ligands can also
be synthesized. It allows receptor mediated targeting of the peptide/DNA complexes to specific cell
types.
Gene transfer by retroviruses
The relatively low efficiency of foreign DNA integration into animal cells, combined with the lack of
naturally occurring plasmids, led to the manipulation of viruses as potential vectors for gene transfer.
Retroviruses are found in many species including most mammals [46]. The genome of retroviruses
can be manipulated to carry exogenous DNA. The advantages of using retrovirus based vectors arise
from the stability of the integration of the viral genome into the host. The integration of a single copy
of the viral DNA at a random location within the hosts genome allows for the long term expression
of the integrated foreign gene. Additionally, retroviruses represent a highly efficient mechanism for
the transfer of DNA into cells. Virus uptake is effective for many somatic cell lines, however
germline cells are infected at low frequency, due to a high level of mosaicism [47]-[48]. Virus can be
used for highly developed tissues, such as those of foetuses, juveniles or adults [49]. This holds great
promise in the context of somatic gene therapy. Retroviral vectors have also been used to introduce
transgenes into the ES cell genome [50]. However, retroviral vectors are limited or problematic in a
number of respects. They exhibit random nature of integration process, which may have deleterious
effects on the host cell, and the general requirement that retrovirus have to infect only dividing cells.
APPLICATIONS TO HUMAN DISEASES
Gene therapy is a potential therapeutic modality that needs effective gene delivery into live cells.
Gene transfer methods have been used in gene therapy attempts on humans since 1990. Gene therapy
is particularly attractive for diseases that currently do not have satisfactory treatment options, and is
probably easier for monogenic disorders than for complex diseases. Gene transfer has its application
in wide range of diseases like cardiovascular disease, parkinsons disease, lysosomal disorders, ocular
gene therapy, liver disorder, osteoarthritis and a number of other diseases. A number of diseases
where gene transfer proved to be beneficial are discussed below.
Cardiovascular disease
Cardiovascular disease (CVD) is still the leading cause of death in industrialized countries, despite the
substantial advances made during the last few years in the prevention and treatment of cardiovascular
events. CVD are the greatest health threat to the United States today. Application of gene therapy in
the field of cardiovascular disorders has been the subject of intensive work over the recent period.
Recent human clinical trials have shown that injection of naked DNA encoding vascular endothelial
growth factor promotes collateral vessel development in patients with critical limb ischemia or
chronic myocardial ischemia. Promising studies in animals have also fueled enthusiasm for treatment
of human restenosis by gene therapy, but clinical applications are warranted. Application of gene
transfer to other cardiovascular diseases will require the coordinated development of a variety of new
technologies, as well as a better definition of cellular and gene targets [51].
Parkinsons disease
After nearly 20 years of preclinical experimentation with various gene delivery approaches in animal
models of Parkinsons disease (PD), clinical trials are finally underway. The risk/benefit ratio for
these procedures is now generally considered acceptable under approved protocols. The current
vehicle for gene delivery to the human brain is recombinant adeno-associated viral vector, which is
nonpathogenic and nonself-amplifying. Candidate genes tested in PD patients encode glutamic acid
decarboxylase, which is injected into the subthalamic nucleus to catalyze biosynthesis of the
inhibitory neurotransmitter -aminobutyric acid and so essentially mimic deep brain stimulation of
this nucleus. It also encodes aromatic L-amino acid decarboxylase, which converts L-dopa to
dopamine and also neurturin, a member of the glial cell line-derived neurotrophic factor family [52].
Lysosomal disorders

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Lysosomal storage disorders comprise a group of more than 50 different diseases (Table 13).
Table 13: Lysosomal diseases.
S.No
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.

Diseases
Krabbes disease
Fabrys disease
Schindlers and Kanzakis disease
Gauchers disease
Niemann Pick A or B disease
Farbers disease
Wolmans disease
Austins disease
Aspartylglucosaminuria
Fucosidosis
-mannosidosis
-mannosidosis
GM1 gangliosidosis: Landings disease
variant B or B1: Tay-Sachs disease

Enzyme deficiency
Galactosylceramidase
galactosidase A
acetylgalactosaminidase or galactosidase B
- glucosidase
Sphingomyelinase
Ceramidase
Acid lipase
Multiple sulfatases deficiency
N acetyl glucosaminidase
fucosidase
mannosidase
-mannosidase
galactosidase
Hexosaminidase A

They are due to the deficiency of specific lysosomal enzymes, hydrolases involved in the catabolism
of macromolecules (glycolipids, glycoproteins, glycosaminoglycans, etc.), resulting in the
intracellular accumulation of storage material. They are transmitted in an autosomal recessive manner,
except for Hunters and Fabrys diseases, which are X-linked. Lysosomal storage diseases are
monogenic metabolic disorders resulting from a deficiency in intra lysosomal enzymes involved in
macromolecule catabolism. Various groups have been delineated according to the affected pathway
and the accumulated substrate: mucopolysaccharidoses, lipidoses, glycoproteinoses and glycogenosis
type II. Their clinical severity and the absence of efficient therapy for the majority of these disorders
justify the development of gene transfer methods. Most of the genes encoding the normal lysosomal
enzymes have been cloned and recently numerous animal models have been obtained for nearly all
these diseases. Due to the clinical heterogeneity of lysosomal diseases, showing multivisceral
involvement or affecting predominantly the reticuloendothelial system, muscle or central nervous
system, various gene therapy strategies have to be developed [53].
Recent developments in ocular gene therapy
The eye exhibits unique features for the development of successful gene therapy. It is easily
accessible and is an immune-privileged site. An extra benefit of using the eye for gene therapy is the
possibility of assessing the success of the treatment in a noninvasive manner by directly measuring
visual function. Delivery of exogenous genes to the eye in vivo has the potential of treating ocular
diseases using genes as drugs. Genes have many additional advantages over conventional drugs. Once
the genes get inside the cells, are capable to express their products for longer periods of time that
greatly exceed the duration of action of currently available drugs. Because of the possibility of
utilizing specific promoters, the expression of a desired transgene can also be targeted. Restricted
expression of a gene to a specific cell type would preclude affecting neighboring tissues and diminish
secondary effects. Further, the expression of a gene can be regulated. Considering that many eye
diseases are chronic and progressive, like macular degeneration and glaucoma, or that some are
caused by an elevated number of mutations, like retinal degeneration, this facet of gene therapy holds
an important potential. Gene transfer of plasmid DNA to the rat corneal stroma was achieved by
a combination of corneal injection and application of electric pulses [54]. The expression of the
reporter LacZ gene peaked at 6 days and no inflammation developed. In a different study, naked
plasmid DNA was injected into the cornea of mice and persisted for 10 days [55].
Photoreceptors are the retinal neurons which are specialized in converting the light entering the eye
into a neural signal. Retinitis pigmentosa (RP) is a common form of retinal degeneration causing loss
of vision. It refers to a group of inherited diseases where deaths of retinal photoreceptors take place.
One out of three thousand people are suffering from this disease. There is no cure or successful
treatment. Attempts to slow the death of photoreceptors by gene-mediated therapy have been

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increasingly successful [56]. Various repots of gene transfer associated with glaucoma, cornea, retinal
degeneration were also made [56].
Gene transfer in liver
Hepatic gene therapy is a good approach for the treatment of metabolic defects or serum protein
deficiencies. However, treating genetic diseases by therapeutic gene delivery poses a number of
problems, including the need for a stable, therapeutic expression of the transferred genes. Gene
delivery using retroviruses results in long term expression because they integrate into cell
chromosomes. However, the use of retroviruses for in vivo gene transfer has been hampered by the
need for cell proliferation since retroviruses only integrate into dividing cells [57]. Follistatin
administered intraportally to 50% hepatectomized rats accelerated liver regeneration, increased the
number of hepatocytes cycling, and was quite effective in promoting gene delivery with retroviral
vectors [58].
Osteoarthritis
Osteoarthritis is a bone disease that affects over 43 million Americans, a number that is predicted to
rise to 60 million by the year 2020 [59]-[60]. This disease is not only incurable and its treatment
inadequate, but it is also very debilitating, leading to physical impairment, reduction in quality of life
and lost working days. Gene transfer to the synovial linings of affected joints is a promising strategy
for achieving sustained, therapeutic, intraarticular concentrations of antiarthritic gene
products. Reports were made regarding the progress in achieving direct, in vivo intraarticular gene
delivery and expression. Numerous non-viral vectors have been evaluated for their ability to transfect
the synovia of experimental animals following intraarticular injection. None have given more than
low levels of temporary transgene expression and many are inflammatory. Several viral vectors,
however, are very effective in this regard and successfully treat experimental models of osteoarthritis.
Adeno-associated virus has been used in a phase I study for the gene therapy of rheumatoid arthritis.
Its use in a clinical trial for treating osteoarthritis is pending [61].
CONCLUSION
Thus there are a number of ways by which the gene can be introduced into the cells. With the advent
of molecular tools and technologies it is now comparatively easy to introduce gene into cells without
loosing its integrity and biological activity. Moreover the recent development in molecular biology
has made the transfer of gene with great accuracy to the target cell. The transfer of gene through
different gene transfer technologies has cured a number of diseases. Research is on progress to cure
those diseases which cannot be cured by using drugs. Moreover the treatment of diseases by gene
transfer provides better result for a prolong period of time. It is the need of hour to discover new and
cheap method of gene transfer technologies so to make the treatment of the diseases a little easier and
affordable. Improvements in gene transfer are required in terms of transgene design to permit gene
targeting, and in terms of transfection approaches to allow improved transgene uptake efficiencies.
ACKNOWLEDGEMENTS
The first and corresponding author Dr. Kishwar Hayat Khan presently working as an Assistant
Professor in Medical Biotechnology Division, School of Biosciences and Technology, VIT
University, Vellore, Tamil Nadu, India wishes to thank Dr. G. Vishwanathan (Founder & Chancellor),
S. Vishwanathan, (Pro-Chancellor), GV. Selvam (Pro-Chancellor) of the University for their help and
support. The name of Prof. S.K.Jain is highly acknowledged.
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CORRESPONDING AUTHOR: Dr. Kishwar Hayat Khan, Assistant Professor, Medical

Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore-632014,
Tamil Nadu, India. Mob: +91-9944327624, Phone: +91-416-2202536 (O), Fax: +91-4162243092/2240411 Email: hamkishwar191@yahoo.co.in, kishwar@vit.ac.in

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