In normal gene into the cells or disrupting
In the middle of the 20th century and with
the discovery of DNA by Watson and Crick, this discovery led to a revolution in
the world of biology and medicine because of the great importance of this
compound. DNA is a molecule that carry genetic information found in all living
organisms and viruses, have important role in growth, development, reproduction
and regulate of many biological processes. DNA in living organisms composed
from two strands, each one made up of four chemical bases: Adenine, Thymine,
Cytosine and Guanine, and each one of the strand is complementary to the other
by base pairing which show DNA as double helix.
A mutation is any change that occurs in DNA
sequence that happen during DNA replication or as result of exposure to
chemical and physical factors ,this change might happen in somatic or germ
cells during replication and it’s related with many hereditary diseases ( e.g.
sickle cell anemia) or non-hereditary diseases (e.g. cancer).
The term of gene therapy emerged during the
1960s .In 1972, Theodore Friedmann and Richard Roblin published a paper in
Science called “Gene therapy for human genetic disease?” which cited
Stanfield Roger’s proposal in 1970 that “good DNA” could be used to
replace defective DNA in people with genetic disorders. A 4-year-old girl was
the first to be treated for congenital disease called adenosine deaminase (ADA)
deficiency using a gene therapy technique.
Gene therapy is a process to repair this
change in the DNA sequence in an attempt to restore the normal function of the
mutant genes, by inserting the normal gene into the cells or disrupting the
gene in which the mutation occurred to overcome these diseases, by viral or
non-viral vectors. This article will focus on viral vectors due to their
efficiency advantages in gene delivery.
What is The Gene
Gene therapy is defined by insertion
new DNA into a patients with hereditary or non-hereditary diseases to treat
these disease. This is done by viral or non-viral vectors in order to replacing
mutated genes, inactivation mutated genes or insert new DNA fragment usually
contains a functioning gene to correct the effects of a disease that result
from mutation in genes1.
Viruses could be used as vectors to transfer
‘good’ genes into a human cells in vitro and in vivo. First, it must remove
viral genes which cause the diseases. Then it should replace those genes with
genes encoding the desired effect (e.g. insulin production in the case of
diabetics), the insertion of foreign DNA into viral vectors done by many
techniques, one of them done by using restriction enzymes to cut the viral
genome at specific sites and insert the foreign DNA into viral genome and
ligate it by using DNA ligase which produce recombinant DNA (viral genes and
insertion genes). This procedure must be done in such a way that the genes
which allow the virus to insert its genome into its host’s genome 1.
Target cells like the patient’s nerve or germ
cells are infected with the vectors. The vectors then transfer its genome
containing the normal gene into the target cells. The production of a
functional protein product from the normal gene restores the target cell to a
normal state 1.
There are two types of gene therapy according
to target cells treatment:
1. Somatic gene
DNA fragments into all cells body except germ cells which produce eggs and
sperms and it is effect confined to the patient and didn’t pass to children, many
genes are transferred to somatic cells including hematopoietic cells, liver
cells, cancer cells and muscle cells.
2. Germline gene
DNA fragments into germ cell and it is effect pass to children patients, this
type of gene therapy gives great hope to treat some rare genetic diseases .There are no
clinical trials done in this area, because they are related to ethical issues.
A viruses is an infectious particles
that don’t reproduces only within host body, contain genetic material (DNA or
RNA genome) inside a protein shell called a capsid, genetic material can be
single or double strands. Some viruses have an internal or external membrane
envelope. Viruses don’t have organelles and that means they can’t make ATP or
energy for themselves 1.
Viral replication starts when the viruses
enter to host and attach to receptors integrated in host cell membrane, then
viruses inject genetic material into host cell. When viruses enter into host
cells, it must transfer its genetic material into the nucleus of the host. The
virus can enter to the nucleus directly, or inject its genetic material into
the nuclear envelop without entrance into the nucleus. The virus then uses the
own cell materials (enzymes) and organelles to produce capsid proteins and
replicate the viral genome. These parts then self-assemble into new virus
particles, which can exit the cell and infect healthier host cells 1.
There are types of viruses that classified
according to genetic material, size, shape or mode of replication. Many of
viruses’ type used as vectors in gene therapy but There are four types of
viruses used mainly as vectors in gene therapy – Retroviruses, Adenoviruses,
Herpes-simplex virus and Adeno-associated virus 2.
Group of viruses that belong to Retroviridae
family and contained single strand RNA genome. Retroviruses have unique enzyme,
called reverse transcriptase that help them to transcript their RNA into DNA
after invade host cells, then retroviral
DNA can insert and integrate into the chromosomal DNA of the host cell, to be
One of the most studied species of
retroviruses is Lentiviruses and widely used in gene delivers, human
immunodeficiency virus (HIV) which cause AIDS is most important example of
Retroviruses have many characteristics made
them good vectors in order to gene delivery, some of these characteristics are
the efficient way of these viruses to enter into host cells and its ability to
integration into the host chromosomal DNA without expression of any immunogenic
viral particles. The disadvantages of these viruses include that these vector
require cell division for stable infection and the limited capacity for these
vectors which prevents the delivery of large DNA fragments 2.
group of viruses that used in gene therapy, these viruses belong to
Adenoviridae family, nonenveloped viruses and contained double stranded DNA
genome. The adenoviruses genome encodes nearly 35 proteins that are expressed
in two phases: early phase and late phase. The early phase happens before the
start of viral DNA replication it is take about 7 hours post-infection, and the
late phase which follow the DNA replication. About ~20 early proteins have
regulatory functions that allow the virus to take control of the cell and to
carry out viral DNA replication. The late proteins have structural role in the
virus. Adenoviruses responsible about many of respiratory infections.
The advantages of adenoviruses as vectors are
that they can infect large numbers of cell types, including nondividing cells,
and the genome of the adenoviruses does not integrate into the host cells
chromosomes. The ds DNA of adenoviruses keep free inside nucleus of infected
cells. The presence of viral genes is necessary to replication and spreading of
these viruses and immune system stimulation are Classified as disadvantages of
Herpes-simplex viruses or also known as human
herpesviruses, herpesvirus 1 and 2 (HHV-1 and HHV-2), are two members of the herpesvirus
family, Herpesviridae, belong to the subfamily of Alphaherpesvirinae. Consist
of double stranded DNA genome, contain around 100-200 genes that involved in
capsid and lipid bilayer envelop membrane formation and included in infectivity
and genome replication of these viruses. Previous studies indicate that there
are correlation between HSV-1 and Alzheimer’s disease.
Herpesviruses are currently used in gene
delivery due to some special features over other viral vectors. Some of these
features are the high capacity of these vectors to transfer long sequence of
new DNA into cells and the ability to targets nerve cells. Some of
disadvantages of herpesviruses are that the large number of their genes which
is required to replication and spreading inside the host and difficult to
Adeno-associated virus belong to
parvovirus family, small viruses contain single-stranded DNA genome.
Adeno-associated viruses don’t associated in causes of diseases, and need to
another types of viruses like Herpes-simplex viruses or adenoviruses to act as
helper viruses, help them to replicate.
Adeno-associated virus can integrates at
specific site on chromosomes, these viruses can insert foreign DNA at a
specific site on chromosome 19 with near 100% certainty. In addition, these
viruses don’t not produce immune response. These characteristics made them good
vector for gene delivery. Conversely, There are a few disadvantages to using
adeno-associated virus as vectors, including the small amount of genetic
material it can transfer (low capacity) and the difficult to produce 2.
Many achievements have been
accomplished in the field of gene therapy despite many challenges, studies are
still ongoing and the results are promising, many diseases have been treated.
Adenosine deaminase (ADA) deficiency is
an inherited diseases that cause problems in the immune system and lead to
cause severe combined immunodeficiency (SCID), Adenosine deaminase responsible
for convert some toxic compound (deoxyadenosine) that effect immune system into
nontoxic compound (deoxyinosine), so any mutation in ADA gene lead into high
toxic level in the body. SCID that result from ADA deficiency was treated by
using gene therapy, retroviruses vectors carry the intact gene of ADA gene were
transfer into autologous hematopoietic stem cells, these stem cells
returned into 10 patients after
nonmyeloablative chemotherapy these for patients. The sustained expression of
ADA gene in multiple hematopoietic-cell lineages allowed for the detoxification
of purine metabolites and improvement in the patients’ physical development
Cystic Fibrosis is an inherited disease
caused by mutation in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene that passed into
children, the effect of cystic fibrosis mainly on respiratory system. Viral and
non-viral vectors are used in the treatment of cystic fibrosis with different
techniques. Adeno-associated viruses which is small viruses to carry CFTR gene,
so researchers have attempted to create a functional CFTR “mini gene”, using
techniques such as cutting the CFTR in half and using two complementary AAVs.
Also, Sendai viruses are used as vectors carry CFTR gene to treat cystic
fibrosis because its ability to infect airway epithelial cells due to the
presence of sialic acid and cholesterol receptors on their apical surface.
These recombinant SeV vector carrying the CFTR gene, CFTR chloride channel
activity was confirmed in vitro and in vivo 4.
Hemophilia is bleeding disease that affect
blood clotting in patients, two major types of hemophilia: hemophilia A (factor
VIII deficiency) and hemophilia B (factor IX deficiency). Adenovirus-associated
virus vectors carry human factor IX in a peripheral vein in six patients with
hemophilia B showed improve in the level of bleeding phenotype without any
toxicity in the patient’s bodies 5.
Duchenne muscular dystrophy (DMD) is an
inherited disease one of nine type muscular dystrophy result by miss function
of DMD gene which responsible for dystrophin production, any mutation in DMD
gene lead to muscle weakness. Recombinant adeno-associated virus (rAAV) vectors
carry intact copy of DMD gene transferred to skeletal muscle showed decrease in
the muscular dystrophy symptoms 6.
are ongoing in the field of gene therapy on cancer, there are great hopes for
effective treatment of various types of cancer, some studies have shown
positive results, understanding how cancer is produced is very helpful in
research. Tumor necrosis factor alpha (TNF?) is type of cytokines have
anti-cancer properties, sarcoma patients are candidates for the TNF treatment.
The TNFerade construct represents such an approach. TNFerade was constructed as
a second generation adenovector, expressing the human TNF? cDNA. To increase
the effective of this treatment and decrease the toxicity, the researchers used
radiation-inducible immediate response Egr-1 (early growth response) gene
promoter which ligated upstream to the transcriptional start site of the human
TNF cDNA. TNFerade vectors give maximum expression of TNF gene. The activity of TNFerade in combination with
radiation has been evaluated in a number of different human models, including
human prostate cancer, human malignant glioma, radio resistant human laryngeal
carcinoma, and human esophageal adenocarcinoma. This approach of gene therapy
showed acceptable range of the toxicity and the tumor response ranged from
complete, partial and simple response by effect this approach affect the
apoptosis and cellular necrosis 7.
Choroideremia (CHM) is a hereditary
disease that cause progressive vision loss due to CHM genes mutation which is
coding gene for the Rab escort protein 1 (REP1). Adeno-associated viruses carry
normal copy of REP1 gene transferred into the retina of six Choroideremia
patients. Patients were tested show
improved in vision and gradually regained the ability to see 8.
Gene Therapy Challenges
many challenges to gene therapy, which are related to transport, toxicity, and
efficacy and gene delivery. The most important challenge in gene therapy is the
lack of a 100% effective way to ensure that the genes reach the nuclei of the
targeted cells to be expressed. This risk is concentrated in that these viral
vectors are transferred to abnormal cells and healthy cells alike, and this may
lead to problems that may occur in healthy cells 9.
Another challenge is that there is also no
ways ensure to transfer the new genes to target sites in target cells
chromosomes and this may lead to other more complex diseases, such as cancer.
This risk was observed in clinical trials where hematopoietic stem cells were
transferred to X-linked severe combined immunodeficiency (X-SCID) patients by
retroviruses containing intact genes, some of these patients have appeared T
cell leukemia. There are other concerns that these viral vectors can reach the
cells responsible for producing the gametes and cause changes in the DNA they
contain, and the possibility of transferring these changes to the children 9.
Other danger of gene therapy based on viral
vectors is that the inability to control the amount of the transferred gene
expression, it might be overexpressed, producing large amount of new protein
and this could be harmful, or it might be downexpressed, so the purpose is not
Because viruses stimulate the immune system,
viral vectors may encounter a large defense of immune cells in the body.
Changes in the genetic material of these viruses may lead to the weakening of
their resistance and thus to their destruction before the target is achieved.
Another challenge is that these viruses may
transported from patients who are undergo to gene therapy into healthy persons
or into the environment.
However, gene therapy techniques are
currently subject to many studies in different fields, and results show many
hopes for patients around the world to overcome these challenges. Experiments
are initially conducted on animals to better understand and overcome these
risks, before any application occurs in clinical trials on humans.
is a process to repair any change in the DNA sequence that happen during
replication or Exposure to physical or chemical factors in an attempt to
restore the normal function of the mutant genes, this is done by viral or
non-viral vectors. About 80% of the studies were conducted on viral vectors due
to their efficiency in the gene delivery. Many types of viruses are used in
gene therapy, each type has advantages that make it a good vectors and at the
same time have disadvantages that make it impossible to use it in all
experiments. Experiments are still ongoing in the field of gene therapy, some
of which have been effective in treating some diseases, and other studies are
still ongoing on some diseases, notably cancer. There are also challenges to
gene therapy, one of the most prominent of these challenges is the lack of an
effective way to ensure that DNA is transferred to the target location, Current
studies are trying to find appropriate solutions to these challenges.