In hereditary diseases ( e.g. sickle cell anemia)

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

  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).

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  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 Therapy?

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 genes.

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

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.

are two types of gene therapy according to target cells treatment:

1.      Somatic
gene therapy: transfer 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.

2.      Germline
gene therapy: transfer DNA
fragments into germ cell and it is effect pass to children patients.




  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

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.

  There are types of viruses that classified
according to genetic material, size, shape or mode of replication.  There are four types of viruses used mainly
as vectors in gene therapy – Retroviruses, Adenoviruses, Herpes-simplex virus
and Adeno-associated virus and others.



  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 expressed there. One of the most famous example of
retroviruses is human immunodeficiency virus (HIV).

  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.



  Another 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 adenoviruses.

Herpes-simplex viruses

  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 viruses

  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.

Therapy examples

  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. Patients with
inherited immune deficiencies have been fully treated, by treated of blood stem
cells taken from patients and then infected with retroviruses carry normal
genes in vitro, after this the treated stem cells returned into patients.

   Adeno-associated viral vectors are used in
hemophilia in order to deliver gene for Factor IX, the missing clotting protein,
to liver.

  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 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

  Studies 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. Previous studies showed that
modified herpes-simplex viruses type 1 help in skin cancer treatment, these
vectors carry T-VEC drugs, the viruses injected directly into cancer cells and
replicated inside these cells until they decompose the cells and then infect
more cancer cells. Also, gene therapy used in the blood cancer treatment, specifically
white blood cell cancer (leukemia), by removed patients’ blood cells and
infected with modified viruses that can recognize specific proteins on the
surface of cancer cell and help in kill cancer cells, after the blood cells
were returned to the patients, the results show completely reduction in the

  Also, in the fat metabolism disorder
researchers used adeno-associated viruses to transfer normal copy of lipoprotein
lipase gene, which have important role fats breakdown in the blood, into muscle

  Beta-thalassemia is a blood disorder that
arise from mutation in the beta-globin gene, which codes for beta polypeptides
that are important domains in hemoglobin which responsible about oxygen
transfer into all body tissues. Blood stem cells removed from beta-thalassemia
patient and treated with modified retroviruses contain normal beta globin gene,
then the infected stem cells backed into patient body where they gave rise into
normal red blood cells. After Seven years, the patient was still doing well
without blood transfusions.


Therapy Challenges

  There are 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.

  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.

  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
to occur.



  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.



therapy is a process to repair this change in the DNA sequence 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.




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