INTERNATIONAL JOURNAL OF

Int J Med Rev 2018 Sep;5(3):106-117

MEDICAL
doi 10.29252/IJMR-050304
REVIEWS
Narrative Review

Gene Therapy: A New Approach in Modern Medicine

Azam Yazdani1, Zahra Alirezaie1, Mohammad Javad Motamedi2, Jafar Amani3*
1
Department of Biology, Faculty of Basic Science, Shahed University, Tehran, Iran
2
Green Gene Company, Tehran, Iran
3
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences,
Tehran, Iran

Corresponding Author: Jafar Amani, Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah
University of Medical Sciences, Vanak Sq., Molasadra St., Tehran, Iran. Tel: +98-21-82482568, Fax: +98-21-88068924, Email: jafar.
amani@gmail.com

Received June 10, 2018; Accepted August 15, 2018; Online Published September 30, 2018

Abstract
In general, gene therapy is the transfer of a genetic material to treat a disease, or at least to improve the clinical status of a patient. One way
gene therapy works is to turn viruses into genetic vectors that carry the gene of interest to the target cells. Based on the genome’s nature,
these vectors are divided into RNA-based or DNA-based viral vectors. Most RNA-based vectors are derived from simple retroviruses, such
as the murine leukemia virus. One major drawback of these viruses is that they are not transferred to non-dividing cells (post-mitotic cells).
This problem can be solved by using new retroviral vectors derived from lentiviruses, such as the human immunodeficiency virus (HIV).
DNA-based vectors originate from adeno-viruses and adeno-associated viruses (AAVs). An example of gene deletion due to gene therapy
is the deletion of the human CCR5 gene in T cells (which control HIV infection). Although available vector systems have the ability to
transfer genes to living cells (in the human body), an ideal vector for gene delivery has not yet been found. Therefore, the current viral
vectors should be used with great caution in human cases. Moreover, the development of new vectors is necessary.
Keywords: Gene Therapy, Lentivirus Vectors, Viral Vectors, Cancer Therapy
Citation: Yazdani A, Alirezaie Z, Motamedi MJ, Amani J. Gene therapy: a new approach in modern medicine. Int J Med Rev. 2018;5(3):106-117.
doi:10.29252/IJMR-050304.

Introduction in gene therapy for human diseases include the development

Gene therapy has provided treatments for incurable
diseases that previously had only temporary remedies. Gene
therapy has not been successful for a long time; however,
in recent years, effective and long-term cured cases have
been reported. Promising results have been achieved for a
wide range of genetic diseases including blood disorders,
immune deficiency, eye problems, regeneration of nerve
cells, metabolic disorders, and various types of cancer.1 To
date, about 2000 clinical trials have been carried out or are
in progress on different patients, and many others are in the
process of preparation. On the other hand, the design of gene
therapy vectors and their clinical development are progressing
rapidly.2 In this review, after introducing the gene therapy
process, some of the considerable recent achievements of
clinical gene therapies are examined by presenting different
approaches and introducing general tools, including vectors
as the most important gene therapy tool.
Gene therapy has the potential to treat diseases that cannot
be treated with conventional medicine. It is applied by
transferring one or more nucleic acids into a patient’s cells or
by modifying a defective gene. The main factors of investment
of gene therapy vectors, optimization of gene delivery under in
vivo and in vitro conditions, and enhancement of the clinical
experience. Gene therapy, as an advanced technology, goes
beyond the modification of genetic disorders and has spread to
a wide range of applications. In fact, promising progress made
in the treatment of leukemia using modified chimeric antigen
receptors (CAR) of T-cells encouraged Science magazine to
select cancer immunotherapy as the most important scientific
achievement of 2013.3 Effective approaches to clinical
gene therapy include gene delivery to non-dividing cells
and tissues (post-mitotic cells) in vivo, or gene delivery to
autologous cells out of the body (ex vivo) in which the gene
is transferred to the patient through adoptive transfer (Figure
1). Among viral vectors, the adeno-associated viruses (AAVs)
have shown the highest clinical success in in vivo gene
transfer (Figure 2). Owing to the wide range of serotypes and
capsids, they can target a variety of cells and tissues. Clinical
gene therapy in vitro focuses on gene transfer to autologous
hematopoietic stem cells (HSCs) for the treatment of various
diseases, especially hematologic ones, and to other blood cells
such as different types of T lymphocytes for immunotherapy.

Copyright © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
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Yazdani et al
which guide the insertion of the gene into certain positions of
the chromosome.1
Adenoviruses
This class of viruses has a double-stranded DNA genome
that causes respiratory, intestinal, and ocular infections in
humans. When these viruses infect a cell, they insert their
DNA molecules into the host’s; however, the genetic material
of the adenovirus does not integrate into the genetic material
of the host cell. Instead, the DNA molecule remains inside
the host cell’s nucleus, and this foreign DNA is transcribed
like any other gene in the cell. The only difference regarding
adenoviruses is that when the host cell is divided, the foreign
genes are not replicated; as a result, cells generated by cell
division will not have additional genes. Therefore, the
treatment of a growing cell population with adenoviruses
requires the re-injection of them.1
Adeno-Associated Viruses
AAVs comprise a small class of viruses with single-stranded
and non-coated DNA. They have the ability to infect both
dividing and non-dividing cells with constitutive expression.1
The ability of these viruses to be present in cells, in both
lysogenic and lytic forms, has made them a good candidate
for gene therapy. Another prominent feature of these viruses
is their non-pathogenicity for humans in the absence of
adenoviruses and herpes viruses as auxiliary viruses. In the
absence of auxiliary viruses, these viruses can insert their
genetic material into a specific location on chromosome 19.
Herpes Simplex Viruses
Viruses in this class have double-stranded DNA that infects
a specific type of neural cells. Type 1 herpesvirus infection is
a common human pathogen that causes cold sores and fever
blisters.10 Herpes Simplex Virus is a human neurotropic virus
which is used for gene transfer mainly in the nervous system.
The wild HSV-1 virus can infect neurons and escape the host’s
immune response; however, this virus may be inactivated and
produce a lytic cycle of viral replication. Therefore, a mutant
HSV-1 strain that is incapable of replication is typically used.11
Non-viral Methods
Today, non-viral methods are more beneficial than viral ones.
Ease of production in a high scale and lower immune responses
by the host (host immune system responses) are only 2 of
their advantages. In the past, the level of transfection and low
expression of the gene were considered as disadvantages of
this method. However, recent progress in vector technology
has led to the production of molecules and techniques with
the same efficiency as the viral techniques.1
Ormasil
The use of Ormasil (silica or modified organic silicate) is
another non-viral method. The relative ease of working with
silica has made it a good option for gene delivery. The most
common method of using silica in gene therapy (due to its
low toxicity) is the use of a combination of nanoparticles with
amino silicones. However, delivery in the presence of serum,
108 International Journal of Medical Reviews. 2018;5(3):106–117
due to the reaction between serum proteins as a limiting
factor, reduces the efficiency of this method.12
Injection of Naked DNA
Injection of naked DNA is the simplest non-viral transfer
method. Although clinical trials of this method have been
successful, gene expression is much lower with it than
with other methods. In addition to tests performed with
plasmids, experiments have also been performed with
naked PCR products. The cellular uptake of naked DNA is
generally inefficient. Researchers have, therefore, focused on
improving the efficiency of DNA uptake, which has led to
the development of new methods, including electroporation,
sonoporation, and the use of the “gene gun” where DNA
coated with gold particles is introduced into a cell with helium
gas at high pressure (Figure 3).
Physical Methods for Improving DNA Transfer
Electroporation
Electroporation is a method that uses high-voltage short
pulses to transfer DNA from cell membranes. Small pores
caused by electrical shock are formed temporarily on the
surface of the membrane, which makes it permeable to
nucleic acid. Electroporation can be applied to a variety of cell
types; however, high rates of cell death have limited its use in
clinical applications.1
Gene Gun
The use of particle bombardment, or gene gun, is another
physical method for DNA transfer. In this method, the DNA
is coated with gold particles and then placed inside a device
which provides the required force to enter the cell. However,
if the DNA is located in the wrong place in a genome, e.g., in
a tumor suppressor gene, it can induce a tumor. This method
has been tested in clinical trials on patients with X-linked
severe immunodeficiency (X-SCID), where HSCs were
infected with a retrovirus containing the modifying gene,
resulting in the successful treatment of T cell leukemia in 3
out of 20 patients.14
Figure 3. Liposome for Drug Delivery.13

Sonoporation
Sonoporation uses an ultrasonic frequency to introduce DNA
to a cell. This process is considered an ultrasound cavitation
in the cell membrane and leads to DNA movement in the cell.1
Magnetofection
With magnetofection, DNA is complexed with magnetic
particles, and a magnet is placed under the cellular tissue
culture container in order to expose the DNA-containing
compound to only one cell layer. This method, which is based
on the hypothesis of targeted drug delivery, works by the
therapeutic gene linking to the magnetic nanoparticles. The
electromagnetic field gradient produced by the ground under
the cell culture medium also increases the complex deposition
and the rate of transfection. In vivo, the gene-magnetic
complex is injected intravenously, and with the help of strong
external magnets, it is absorbed and approaches the target.
Ultimately, the gene is isolated from the magnetic particles
by means of intermolecular restriction enzymes, charge
interaction, or degradation of the matrix. This method is
often used in laboratory studies to transfer a gene to primary
cells and other cells to which it is difficult to transfer genes by
other methods.15-17
Chemical Methods for Improving DNA Transfer
Oligonucleotides
Synthetic oligonucleotides are used in gene therapy to disable
and inactivate genes involved in the disease process. The use of
specific antisense for the target gene impairs the transcription
of defective genes. Another method is the use of siRNA that
leads to the breakdown of a specific sequence of the defective
gene mRNA to stop its translation and, thus, its expression.
Lipoplex and Polyplex
A combination of DNA and polymers is called polyplex. Most
polyplexes include cationic polymers which are made based
on particle accumulation due to interactions of polyplexes.
To improve new DNA delivery to the cell, DNA should be
protected from damage and a positive charge. Therefore,
anionic and neutral liposomes are used for the formation of
lipoplexes as synthetic vectors.
Figure 4. Gene Delivery by Direct and Cell-Based Methods.19
International Journal of Medical Reviews. 2018;5(3):106–117
Gene Therapy
Dendrimers
The dendrimer is a spherical branched macromolecule. The
particle surface can be charged by various methods, and
many properties of the final structure of the particle are
determined by this surface. In the presence of genetic material
such as DNA or RNA, the supplementary charge results in a
temporary nucleic acid linkage with the cationic dendrimer.
The nucleic acid-dendrimer complex is passed into the cell
through the endocytosis.1
Hybrid Methods
Each gene transfer method has its own shortcomings; thus,
hybrid methods, which are, in fact, combinations of several
techniques, are being developed. A virosome, a combination
of a liposome with an inactive HIV or an influenza virus, is
an example of a hybrid method.18 This hybrid method of gene
delivery in respiratory epithelial cells is more efficient than
the viral or liposome methods alone. In general, this method
involves the mixing of different viral vectors with cationic
liposomes or hybrid viruses (Figure 4).1
Advantages and Disadvantages of Gene Therapy
The Advantages of Using Gene Therapy
• Gene Silencing: In the case of an HIV-infected person,
gene therapy and gene silencing can protect the patient
from pain and suffering before the disease progresses.
• Gene therapy is potentially used to eliminate and prevent
hereditary diseases such as cystic fibrosis; it is also a
potential way to treat heart disease, AIDS, and cancer.20
Disadvantages of Gene Therapy
• The novelty of gene therapy methods is one disadvantage.
• Stimulation of immune response: The gene injected by a
virus may cause immune responses due to the presence of
the virus inside the body and the pathogenic potential of
viral vectors (in one case, the viral vector could improve
its ability to cause illness).
• Generation of genetic disorders due to the presence of
multigene: The genetic material transferred may not
necessarily enter the target cell; even if it does, it may not
109
Yazdani et al
be placed at an appropriate place in the genome.21
Ethical Issues Regarding Gene Therapy
• Who will decide which attributes are normal and which
cause disability or disorder?
• Will gene therapy be available only for the creation of
wealth (economic issues)?
• Can spreading gene therapy reduce the social acceptability
of individuals who are different?
• Should humans be allowed to use gene therapy to
promote essential features such as height, intelligence, or
sports abilities?
Applications of Gene Therapy
Parkinson’s Disease
According to independent reports, the effectiveness of gene
therapy in Parkinson’s disease (PD) has been proven. For
example, in one of the proposed methods, the level of a
chemical called GABA, the absence of which causes PD, is
increased in the brain. In an experiment conducted on 45
volunteers with severe PD, tubes were placed in the areas of
the brain associated with movement. Half of the participants
were injected with viruses carrying the gene that increases
GABA production, and the other half were given an innocuous
saline solution (as the control group). After 6 months, those
who underwent gene therapy showed a 23% improvement in
movement ability, which was twice the improvement observed
for those in the control group.
The study discussed was a randomized controlled trial
to investigate the improvement of advanced symptoms of
PD using gene therapy. In the research, genes producing
the chemical agent glutamic acid carboxylase (GAD) were
transferred into the base ganglia cells, which are a set of
cerebral areas controlling movement. The transferred GAD
gene increased the level of a chemical messenger called
GABA. The level of GABA in some parts of the basic ganglia
is reduced in people with Parkinson’s disease.1
In addition to the disease-causing pathways that have so
far been discovered, cell-to-cell transfer of the protein mass
has been recognized as a mechanism of damage that extends
throughout the central nervous system. There is also a growing
awareness of the function of the immune system as the main
cause of PD in controlling transcription and translation at the
onset of the disease.22 Increasing our understanding of PD
will make it possible to understand the underlying causes of
the disease. It is highly critical to cross the blood-brain barrier
as a barrier to the introduction of drugs with specific target
cells within the brain.23 According to a recent study of LRRK2
inhibitor in mammals other than humans,24 the long-term use
of the inhibitor or genetic manipulation of the target tissue of
the Parkinson’s drug may have serious consequences.23
Alzheimer’s Disease
Mental disorders are among the most common nervous
system disorders, and Alzheimer’s disease (AD) is the most
common cause of dementia worldwide for which there are
no effective therapies. AD and a number of frontotemporal
dementias (FTDs) are collectively known as tauopathies,
110 International Journal of Medical Reviews. 2018;5(3):106–117
which are caused by the abundant accumulation of defective
tau in the brain. Recent developments in gene therapy-based
approaches, recombinant AAVs (rAAVs) in particular, have
provided new tools for the study of AD and other neurological
disorders.25
In 2001, a clinical trial of nervous growth factor (NGF)
gene therapy was conducted on Alzheimer’s disease patients
to determine whether decaying neuronal cells in AD, after
the onset of the disease, still had the ability to respond to the
neural growth factor. In the first attempt, a dysfunctioning
nerve gene was transferred to an adult. Based on the results,
the decaying neuronal cells of the brain in AD responded
to NGF. All patients showed a nutritional response to NGF;
they grew axonal sprouts toward the source of NGF. The
brains of 3 patients who underwent one-way gene transfer
were examined in terms of the degree of treatability, and it
was found that cholinergic neuronal hypertrophy occurred
on the side treated with NGF (P>0.05). In the cases of the 2
patients treated with AAV2-mediated and NGF gene transfer,
functional markers and cellular signaling were activated.
Neurons with Tau damage (proteins that cause microtubule
stability) as well as neurons without Tau damage expressed
NGF, suggesting that decaying cells can be treated with gene
therapy, resulting in the activation of the cellular signaling
pathway. No NGF-related side effects were observed. NGF-
induced sprouting continued for more than 10 years, longer
than the expected assurance period.26
According to the Daily Mirror, scientists silenced a gene
involved in AD using the new method of direct drug delivery
to the brain. Researchers used small particles called exosomes
which are released to the cells and led the drug delivery into
the brain of rats. Based on the results of experiments on rats,
it has been proven that exosomes have numerous potential
applications for carrying specific genes in the brain and
can thus be used in gene therapy. One such gene is BACE1.
Although this study paved the way for future research and was
paid a great amount of attention by the scientific community, it
was a preliminary study, and this technology has not yet been
tested on human cells for certain reasons that include ethical
issues. Many neurological diseases include the degradation
and loss of cells. Neurotrophic factors are proteins that
increase cell growth in the developmental stage and have a
neuroprotective role in a range of neurological diseases; they
are ideal candidates for gene therapy in both the central and
peripheral nervous systems.
GDNF is a neurotrophic factor in the brain that has attracted
considerable attention and is recognized as a neuroprotective
agent for the subset of dopaminergic (dopamine-producing)
neurons in the midbrain. These neurons decay in patients
with PD. GDNF production is a possible therapeutic goal.
Several studies have shown that GDNF induction into the
brain has been successful in eliminating the behavioral
symptoms of PD in animals and preventing the degradation
of nigrostriatal dopaminergic pathways. The authors of the
study suggested that gene therapy is more effective in that it
causes the relatively limited release of GDNF in the cerebral
cortex and prevents further degradation of brain structures.
Another lentiviral vector has been developed based on

equine infectious anemia virus (EIAV) and specifically used
to infect the brain and spinal cord. Most of the infected cells
have a neurological morphology, and VSVG-EIAV-mediated
GDNF delivery has been successful in animals with PD.
Research on treating pain areas is mostly focused on the use
of HSV vectors that can be used in posterior and peripheral
transfers to the posterior root of the ganglia cell. A study on
the use of HSV as a GDNF vector reported successful pain
relief and low neuro-chemical changes. Although this delivery
method has numerous advantages with minimal invasion, the
virus can infect the neural cells even through a skin scratch.
Prospects for using the HSV system are as follows:
1. Viruses are associated with toxicity and immune
responses; nonetheless, using these new methods,
viruses can be upgraded, and those parts of the viruses
that produce toxicity and immune responses can be
eliminated.
2. The replication of the virus in the infected cells has a
slight expression in the incubation period.
Several spinal cord inducer viral mediator molecules have
been used successfully in a number of methods and models
associated with pain. Eaton et al reported the protective
effects of an AAV-BDNF neuronal agent. GDNF as an intra-
spinal cord inducer has not yet been implemented in the
peripheral neuropathy model. To date, several studies using
the intra-spinal injection of GDNF have been performed by
viral expression vector (Lv and AAV) in ALS models and
ventral root avulsion.1
Cystic Fibrosis
Cystic fibrosis (CF) is a disease which gradually destroys
the lungs. Its symptoms include respiratory tract infection,
inflammation, deformity, and obstruction. Direct delivery
of cystic fibrosis transmembrane regulator (CFTR) gene into
respiratory tract epithelium cells as the target tissue has some
advantages. However, the physical and immunity barriers
in the host’s lung create some challenges for the successful
transfer of the gene to the respiratory tract. Progress in gene
transfer methods, tissue engineering, and animal models have
motivated CF research.27
History of Cystic Fibrosis Gene Therapy
Gene therapy for cystic fibrosis began in 1990, just as scientists
succeeded in modifying CFTR genes by adding normal gene
copies to in vitro cell cultures. In 1993, the first experimental
cystic fibrosis treatment was given to a patient in the form of
gene therapy. The researchers changed a normal cold virus to
act as a vector delivering healthy genes to CFTR cells in the
lungs and respiratory tract.
In subsequent studies, other gene delivery methods were
tested, such as the use of fat capsules, synthetic vectors, or
nose drops, or the drizzling of cells down a flexible tube to
CFTR cells lining the airways of the lung. Researchers are now
testing an arousal delivery using nebulizers.
Finding the best delivery method for the transfer of natural
CFTR genes is just one of the challenges that scientists need
to overcome for the effective treatment of cystic fibrosis.
Scientists should also:
Gene Therapy
• Detect the range of affected lung cells;
• Identify the parent cells that produce the CFTR cells;
• Determine how long the treatment should take and how
often it should be repeated.1
A significant barrier to CF treatment was the lack of an
appropriate model for testing gene therapy results. A rat with
CF will not suffer a pulmonary problem, while the pulmonary
problem is the main cause of disease and mortality in humans.
In 2008, CF researchers at the University of Iowa (UI), led by
Michael Wales, Director of Pappajohn Biomedical Institute
(PBI), with the help of Howard Hughes, a researcher at the
Medical Institute, managed to produce a pig model with CF
with human-like symptoms. In this study, 2 teams, the first
one focusing on the lentivirus and the other on the adeno-
associated virus AAV2, introduced CFTR into the airway
cells of the pigs with CF. The main advantage of lentiviruses
is the direct combination (integration) with the cell genome,
which means they are permanent. However, the production of
a large amount of lentivirus is challenging, and the virus has
not yet been tested in terms of immunity in the human lung.
Another team at the University of California focused on
adeno-associated virus AAV2. AAVs are safe for use on humans
and are relatively easy to produce in large quantities. The gene
delivered by AAV vectors is not permanently integrated into
the cell genome but often have long-term gene expression.
An important aspect of the AAV study was the molecular
upgrade of the virus, so that it was specified for entering the
pig’s airway cells. Five mutations of the AAV2 virus have been
created that are 240 times more effective in infecting pigs’
airway cells than the AAV2 itself; these developing vectors
can be used as a multi-purpose gene transfer tool in addition
to their application in CF gene therapy (Figure 5).28
Diabetic Neuropathy
In a study on a common disorder resulting from chronic
diabetes, researchers found that gene therapy is promising
in the treatment of diabetic polyneuropathy. Researchers
in Boston found that intramuscular injection of a vascular
endothelial growth factor (VEGF) gene may help diabetic
neuropathy patients. This study included 39 patients who
Figure 5. The red color is the CFTR protein transferred by AAV. The blue
areas are the nucleus of the airway cells. The green areas are cellular
links.28
International Journal of Medical Reviews. 2018;5(3):106–117 111
Yazdani et al
received 3 VEGF injections in one leg, plus 11 patients
who received placebos. Legs and plantar pain, weakness,
and balance problems are signs of diabetic neuropathy. A
reduction in the tactile sense means that a foot ulcer may not
be detected, and this can result in amputation. Most patients
had relatively severe neuropathy and not much hope for
recovery. “The VEGF gene used in this study was active and
present without any packaging in the virus, which is a great
and solid benefit,” said Dr. Allan Ropper, Executive Director
of the Department of the Neurology at Brigham and Boston
Hospitals. The study showed that this form of gene transfer
could be relatively safe, but before it can be introduced as a
major treatment, further research is needed using a larger
study population.1
Gene Therapy for Cancer Treatment
Progress in the genomics of humans over the past 2 decades has
shown that cancer is caused by anomalies in the somatic cells
of the host genome. These achievements have led many cancer
researchers to use treatments based on genetic manipulation
and modification to treat cancer and find a potential cure
for the disease. Examples include gene therapy using viral
(or bacterial) vectors or non-viral vectors, stimulating the
immune system (immunomodulation) against tumor cells,
manipulation of the tumor cell in order to reduce the tumor
tissue, and increasing antigens for better detection of tumors
by the immune system of the host. In general, the number
of treatments with few side effects has been limited. New
generation viral or non-viral vectors significantly decrease
risks associated with previous methods of cancer treatment,
such as the integration of a retrovirus with a host genome with
the risks of mutagenicity or malignancy, immune response
against viruses, formation of tumors, drug resistance, or
disease relapse.
Several tumor-specific antibodies, genetically-modified
immune cells, and vaccines have been developed and are
currently available on the market; many others are being tested
in clinical trials. Gene therapy is expected to play an important
role as part of a multi-faceted treatment for cancer, together
with other cancer treatments such as surgery, radiotherapy,
and chemotherapy. The type and state of gene therapy are
determined based on individual genome components, tumor
characteristics, genetics, and host immune status in order to
design a multifaceted treatment that is unique to the needs of
each individual.29
Viral vectors are the main means of gene transfer in
gene therapy for cancer. Particularly, the oncolytic viruses
that selectively infect and kill cancer cells provide a more
promising prospect. Technology for editing and modifying
hereditary mutated genes, the interaction with stem cells for
tissue regeneration, and the effective use of powerful immune
responses to fight cancer also contribute to gene therapy
revivification.30
Gene Therapy in Pancreatic Cancer
Pancreatic cancer (PC) is highly lethal and difficult to treat.
Only a few patients with pancreatic cancer qualify for surgery,
112 International Journal of Medical Reviews. 2018;5(3):106–117
while conventional chemoradiotherapy has considerable
toxicity and little effect. Gene therapy has been widely studied
as a new method for the treatment of pancreatic cancer; it is
considered a new and promising way of treating PC patients
in the future. In all types of pancreatic cancers, the P53 tumor
inhibitor gene and the mutated K-RAS gene are 2 important
samples that have been precisely studied and have provided
promising results in in vitro and animal models. Genetic
changes such as K-RAS mutations, especially epigenetic
disorders of genes associated with a tumor (e.g., P53), are the
main symptoms of pancreatic cancer; however, they have not
yet been used in clinical trials. Efficient therapeutic targets for
gene therapy include the P53 tumor inhibitor gene, K-RAS
oncogene, VEGFR anti-angiogenic genes, HSK-TK suicide
gene, cytosine deaminase and cytochrome p450, and multiple
cytokine genes. A clinical experiment on cytochrome
P450 has shown interesting results when combined with
ifosfamide; further experiments are being carried out. As
an anti-angiogenesis approach, VEGFR is the best choice.
Clinical trials of the peptide vaccine derived from VEGFR-2
and the deoxynucleotides vaccine that targets VEGFR-2 have
shown some advantages; however, just like anti-angiogenesis,
they are not effective for all patients as a single treatment.
Thus, they are considered as a complementary therapy. Low
gene transfer efficiency is the most important barrier to the
use and generalization of gene therapy. The selection of highly
efficient vectors that target only cancerous tissues should be
taken into consideration. In general, viral vectors have higher
transfer efficiency and long-term gene expression, explaining
why more than two-thirds of clinical trials are reported to be
carried out by viral vectors.
Recently, oncolytic viruses that selectively infect cancerous
cells and reproduce have shown promising prospects in this
regard. Oncolytic viruses can spread from infected cancerous
cells to adjacent and further tissues. Therefore, intratumoral
injection can be beneficial even for the treatment of spreading
tumors. Systemic applications of oncolytic viruses have
shown to be safe for patients with pancreatic cancer, but
direct injection of the virus into the primary ulcer is difficult
in patients with pancreatic cancer.30
Breast Cancer
Several methods of gene therapy have been developed for
breast cancer, the most common cancer among women.
Among them are neutralization of the mutation, molecular
chemotherapy, proapoptotic gene therapy, anti-angiogenesis
gene therapy, immunopotentiation (enhancement of the
immune response by increasing the speed and extent of
its development and prolonging its duration), and genetic
modulation of resistance-sensitivity.
Clinical trials on breast cancer have begun to evaluate
the immunity, toxicity, and efficacy of gene therapy. Hybrid
methods of gene therapy with chemotherapy or radiotherapy
have provided promising results. New gene therapy approaches
and improvements in vector design have all led gene therapy
to play an important role in the treatment of breast cancer.
Most clinical trials focus on TSG P53. The preferred method
 Gene Therapy

is intramuscular injection of the p53 adenoviral vector.

Despite observations of the high expression of the transgenic
adenoviral gene, clinical evidence suggests tumor relapse
in a small minority of patients. The results of clinical trials
on gene therapy for breast cancer have shown little toxicity
to date. However, the rate of low clinical response and high-
expression of transgenic genes remain controversial. In this
regard, future research needs to focus not only on the new
transgenic strategy, but also on the development of new gene
transfer vectors to help overcome this inefficiency. Predicted
methods for treating breast cancer include a multifaceted
approach, hybrid therapy, and reduction in tumor size using
a surgery called debulking resections, following auxiliary
therapies such as synchronous or sequential gene therapy,
chemotherapy, and radiotherapy.31

Results
Rapid changes in gene therapy have created numerous
innovative methods for treating patients with cancer. Progress
in the genetic modification of cancer and immune cells and
the use of viruses and bacteria to control cancer cells have led
to multiple clinical trials and product development for cancer
treatment. However, up to the publication date of this article,
no gene therapy product has been approved by the US Food
and Drug Administration (FDA).32

Number of Tests Per Year

The number of tests carried out annually is significantly Figure 6. Number of global clinical trials conducted in the period from 1989
to 2017.33
influenced by reports that indicate risks in this area; for
example, in 2003 and 2007, the number of tests decreased,
but in 2005, 2006, and 2008, considerably high numbers of
clinical trials took place. There has also been a growing trend
in the number of annual trials since 2012. In recent years,
an inadequate number of articles has been submitted to
databases due to timeliness of publication, which has in turn
led to a delay in obtaining information on most experiments
(Figure 6).33

Countries Participating in Gene Therapy Experiments

Clinical gene therapy experiments have been carried out on all
5 continents, measured only in 38 countries. The distribution
of the range of tests has not changed much over the past few
years and largely reflects the cost of research and development
of the experiments. In 2007, 64.9% of the experiments were
carried out in the United States, 23.2% in Europe, and 6.5%
in Asia (Figure 7).
A significant number of multinational experiments have
been conducted, and this number has increased from 16
cases in 2012 to 130 cases in 2017. This indicates a probable
increase in mutual cooperation between research centers due
to the need for access to a population of patients in more than
one country, especially in cases of rare diseases (Table 1).33
Future Works
In the third millennia, gene therapy is considered an
important new approach compared to conventional medicine.
Gene therapy facilitates continuous, stable, and regular
Figure 7. Geographic distribution of gene therapy clinical trials.33
expressions of biological agents by the targeted delivery of
gene cassettes containing genetic information. When gene
therapy is combined with cell therapy, cells become smart
vectors for gene therapy purposes. As shown in studies, gene
therapy guides powerful biological processes toward disease
modification and tissue repair and regeneration. For example,
the sustainability, validity, and reinforcement of the performed
treatment can be guaranteed by transferring information
through genetic mechanisms. Gene therapy utilizes the
high potential of stem cell division and transplantation of
defensive immune cells that are used to remove the treated
cells in specific cases. Other important challenges should be

International Journal of Medical Reviews. 2018;5(3):106–117 113

Yazdani et al

Table 1. Important Clinical Experiments in Gene Therapy

No. of Follow-up Patient Status and Biologic and Clinical

Disease Vector and Strategy Clinic ID Source
Patientsa (mon) Efficiency
All patients AAW; Stabilized by
Wiskott-Aldrich Lentiviral vector; in vitro
7 10-60 transferred cells; clinical advantages and NCT01515462 34 and L.N.b
syndrome gene transfer to CD34+ cells
continuous safety
6 patients AAW; 1 patient died of NCT01347242
Wiskott-Aldrich Lentiviral vector; In vitro
7 9-42 infection. Stabilized by transferred cells; NCT01347346 35
syndrome gene transfer to CD34+ cells
clinical advantages and continuous safety NCT02333760
36 and M.
2 patients: continuous injection of the
Cavazzana
Lentiviral vector; In vitro transferred cells; 1 patient: independent
β-Thalassemia major 3c
24-72 N/A and
gene transfer to CD34+ cells injection; 1 patient: failed injection, then
BlueBird
received rescue cell
Biob
M. Cavazzana
Continuous injection of transferred cells;
Lentiviral vector; In vitro and
β-Thalassemia major 2c 15 independent injection. Both patients NCT02151526
gene transfer to CD34+ cells BlueBird
healthy
Biob
Continuous injection of transferred cells;
Lentiviral vector; In vitro BlueBird
β-Thalassemia major 5d 1-6 independent injection. Both patients NCT01745120
gene transfer to CD34+ cells Biob
healthy
Continuous injection of transferred cells,
Adrenoleukodystrophy Lentiviral vector; In vitro 37, 38 and P.
4 54-101 safety in all patients; Stable clinical N/A
gene transfer to CD34+ cells Aubourgb
advantages in 3 patients
Continuous injection of transferred cells,
Metachromatic
Lentiviral vector; In vitro safety in all patients; Stable clinical
leukodystrophy 20 3-60 NCT01560182 39 and L.N.b
gene transfer to CD34+ cells advantages in all patients who underwent
treatment before onset of symptoms

No inhibitor; stable expression of FIX; in

AAV8 vector
Hemophilia B 10 16-48 high-dose group, FIX levels of 5.1±1.7% NCT00979238 40
Intravenous induction
observed in 6 treated patients
AAV8 vector No inhibitor; stable expression of FIX in
Hemophilia B 7 Up to 12 NCT01687608 41
Intravenous induction 1 patient
γ-RV; in vitro gene transfer
B-cell lymphoma or
to T-cells; CAR-modified 15 1-23 8 CRs, 4 PRs; ORR 80% NCT00924326 42
CLL
anti-CD19 cells

5 CRs; ORR 100%;

γ-RV; in vitro gene transfer 4 patients under allo-HSC transplant;
B-cell ALL to T-cells; CAR-modified 5 1-4 studied as a general method; 1 patient NCT01044069 43
anti-CD19 cells was not qualified for HSC transplant and
returned to initial state.

27 CRs; ORR 90%; 171/5000;

Lentiviral vector; in vitro
9 patients were recovering; 3 of them NCT01626495
gene transfer to T-cells; CAR-
B-cell ALL 30 1-24 e
underwent HSC transplant; 7 cases of NCT01029366 44
modified
relapse, 3 of them occurred after T-cells
anti-CD19 cells
loss

γ-RV; in vitro gene transfer Median

4 CRs (at day 28); ORR 67%; 10 patients
B-cell or lymphoma to T-cells; CAR-modified 21 = NCT01593696 45
underwent HSC transplant
anti-CD19 cells 10f

GAD gene by AAV2; in vivo NCT00195143

PD 46,47
intrathecal injection NCT00454818

LPL gene by AAV2; in vivo

LPLD 48,49
intramuscular injection

RPE65 gene by AAV2; in

LCA Vision improvement 50-52
vivo injection

Squamous cell P53 gene by Adenovirus

carcinoma of head (Gendicine); in vivo 53
and neck injection

Abbreviations: AAW: alive and well; ALL: acute lymphocytic leukemia; CLL: chronic lymphocytic leukemia; CR: complete response; N/A: not
applicable; ORR: overall response rate; PR: partial response; PD, Parkinson’s disease.
a
As stated in the referenced publications or updated by personal communication.
b
Personal communication.
c
β0/βE genotype.
d
2β0/βE, 2β0/β0, 1β0/β + genotypes.
e
Median = 7.
f
51 days to HSC transplantation media.

114 International Journal of Medical Reviews. 2018;5(3):106–117


overcome before the effects of this therapeutic approach are
fully realized. For example, the efficacy and safety of gene
transfer vectors should be improved by formulas and advanced
engineering designs which include combining the biological
properties of different viruses with synthetic molecules. These
enhancements will increase the precision and accuracy of the
vector in reaching target tissue and cellular models and reduce
cellular constraints in gene transfer and sensors passing
through exogenous nucleic acids. Researchers also prevent
the activation of inherent and acquired immune systems in
gene therapy by upgrading vectors. Ultimately, these changes
will ensure the high expression and reproducibility of the
transferred gene over a long period of time, and the gene
expression will take place in a way similar to the internal
pattern (when the gene is replaced).
Progress in the manipulation of genes and their characteristics
has led to standardization and comparative evaluation of
the performance of vectors in various experiments. The
magnitude and specificity of gene modification in its original
or natural position, the integration of vectors in genomic safe
locations, and the exclusive silencing of alleles by synthetic
nucleases and epigenetic modifications provide more
opportunities for improving gene therapy strategies. A deeper
understanding of the pathology of hereditary, multi-genic,
or acquired diseases will lead to the development of new
gene therapy strategies. Since gene transfer strategies use live
biological agents that can induce long-term effects on patients
and their sex cells, long-term care and preventive measures
should be considered when using them. Furthermore, due
to the limited understanding of stem cell configurations,
tissue regeneration, and immune response inspections, there
is increasing and undeniable concern about the undesirable
effects of genetic manipulation. From a clinical point of
view, the clinical delivery of genes and cell therapy requires
multidisciplinary specialists and, in some cases, advanced cell
processing in the clinical setting. Biological readouts should
also be performed to monitor the safety and efficacy of the
treatment. Since the first advancements, from registration
to marketing, were made in gene therapy, pharmaceutical
departments and regulatory agencies have provided quality
standards for the production and distribution of these highly
specialized medicines. In social terms, the complexity and
cost of producing and delivering live biological medicine in
conventional healthcare systems challenge the sustainability
of these treatments and require the establishment of
reimbursable policies so that all patients can benefit from it.
Ultimately, whether medical science surrenders to technology
laws or takes on more responsibility for these developments
is an important ethical issue. Of course, the development of
strategies to treat and reduce the suffering of patients justifies
our efforts from an ethical point of view.54
Conclusions
Clinical gene therapy has made a number of achievements
in the last decade. Several significant successes such as
treatments now available for diseases such as cystic fibrosis,
diabetes, Alzheimer’s disease, Parkinson’s disease, various
cancers, etc, can be mentioned. In other words, gene therapy
Gene Therapy
can be applied to a wide range of diseases and includes many
methods of gene transfer. Initially, LV and AAV vectors were
used in experiments, while other vector systems are expected
to provide further advancement in their clinical applications.
Lessons learned from the successes, problems, and barriers in
recent experiments will guide clinical gene therapy practices
toward innovation. Next-generation protocols that will help
expand the range of diseases treatable by gene therapy are
currently being developed. For some of the hardest goals,
such as muscular dystrophy and some lysosomal storage and
nervous system disorders, there is little chance for immediate
success. However, ongoing efforts will lead to the discovery of
other treatments in the future. Gene therapy will be a more
accurate method of treatment if it is integrated with gene
editing tools, as HSC genome editing has recently shown.
Authors’ Contributions
AY and ZA, Collected data and wrote manuscript; MJM;
Edited the manuscript, reviewed and approved the final
manuscript; JA; supervised the research, reviewed and
approved the final manuscript.
Conflict of Interest Disclosures
The authors declare they have no conflicts of interest.
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