Gene Therapy 
Biology Project 
─ 

Sharan S 
XII C  
Bala Vidya Mandir 
 
 
 
 

 
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Gene Therapy

A Brief Introduction:
Gene therapy is an experimental technique that uses genes to treat or prevent disease.
In the future, this technique may allow doctors to treat a disorder by inserting a gene
into a patient’s cells instead of using drugs or surgery. Researchers are testing several
approaches to gene therapy, including:
● Replacing a mutated gene that causes disease with a healthy copy of the gene.
● Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
● Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of diseases

(including inherited disorders, some types of cancer, and certain viral infections), the
technique remains risky and is still under study to make sure that it will be safe and
effective. Gene therapy is currently being tested only for diseases that have no other
cures.

The concept of transferring genes to tissues for clinical applications has been discussed
for nearly half a century, but our ability to manipulate genetic material via recombinant
DNA technology has brought this goal to reality. While originally conceived as a way to
treat life-threatening disorders (inborn errors, cancers) refractory to conventional
treatment, gene therapy now is considered for many non–life-threatening conditions,
including those adversely affecting a patient’s quality of life. The lack of suitable
treatment has become a rational basis for extending the scope of gene therapy.

Despite some well-publicized problems, gene therapy has made substantive progress,
including tangible success, albeit much slower than was initially predicted. Although
gene therapy is still at a fairly primitive stage, it is firmly science based. There is
justifiable optimism that with increased pathobiological understanding and
biotechnological improvements, gene therapy will become a standard part of clinical
practice within 20 years.
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Introduction and History:

Gene therapy typically involves the insertion of a functioning gene into cells to correct a
cellular dysfunction or to provide a new cellular function. For example, diseases such as
cystic fibrosis, combined immunodeficiency syndromes, muscular dystrophy,
hemophilia, and many cancers result from the presence of defective genes.

Gene therapy can be used to correct or replace the defective genes responsible. Gene
therapy has been especially successful in the treatment of combined immunodeficiency
syndromes, showing lasting and remarkable therapeutic benefit.

However, it is important to remember that gene therapy is not a new idea. In 1963,
Joshua Lederberg wrote, “We might anticipate the . . . interchange of chromosomes
and segments. The ultimate application of molecular biology would be the direct
control of nucleotide sequences in human chromosomes, coupled with recognition,
selection and integration of the desired genes. . . . It will only be a matter of time . . .
before polynucleotide sequences can be grafted by chemical procedures onto a virus
DNA.”

Less than 30 years later, the first clinical study using gene transfer was reported.
Rosenberg and his colleagues used a retroviral vector to transfer the neomycin
resistance marker gene into tumor-infiltrating lymphocytes obtained from 5 patients
with metastatic melanoma. These lymphocytes then were expanded in vitro and later
rein-fused into the respective patients. Since this first study showed that retroviral
gene transfer was safe and practical, it led to many other studies. Indeed, since
1989, more than 900 clinical trials have been approved worldwide . What made gene
therapy possible between 1963 and 1990 was the development of recombinant DNA
technology.
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Working/ Principle of Gene therapy:

Gene therapy is designed to introduce genetic material into cells to compensate for
abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary
protein to be faulty or missing, gene therapy may be able to introduce a normal copy of
the gene to restore the function of the protein.

A gene that is inserted directly into a cell usually does not function. Instead, a carrier
called a vector is genetically engineered to deliver the gene. Certain viruses are often
used as vectors because they can deliver the new gene by infecting the cell. The
viruses are modified so they can't cause disease when used in people. Some types of
virus, such as retroviruses, integrate their genetic material (including the new gene) into
a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their
DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

The vector can be injected or given intravenously (by IV) directly into a specific tissue in
the body, where it is taken up by individual cells. Alternately, a sample of the patient's
cells can be removed and exposed to the vector in a laboratory setting. The cells
containing the vector are then returned to the patient. If the treatment is successful, the
new gene delivered by the vector will make a functioning protein.

Researchers must overcome many technical challenges before gene therapy will be a
practical approach to treating disease. For example, scientists must find better ways to
deliver genes and target them to particular cells. They must also ensure that new genes
are precisely controlled by the body.

Gene therapy may be classified into two types:

Somatic
In ​somatic cell​ gene therapy (SCGT), the therapeutic genes are transferred into any cell
other than a ​gamete​, ​germ cell​, ​gametocyte​, or undifferentiated ​stem cell​. Any such
modifications affect the individual patient only, and are not inherited by ​offspring​.
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Somatic gene therapy represents mainstream basic and clinical research, in which
therapeutic DNA (either integrated in the ​genome​ or as an external ​episome​ or ​plasmid​)
is used to treat disease. Most focus on severe genetic disorders, including
immunodeficiencies​, ​haemophilia​, ​thalassaemia​, and ​cystic fibrosis​. Such single gene
disorders are good candidates for somatic cell therapy. The complete correction of a
genetic disorder or the replacement of multiple genes is not yet possible. Only a few of
the trials are in the advanced stages.

Germline

In ​germline​ gene therapy (GGT), ​germ cells​ (​sperm​ or ​egg cells​) are modified by the
introduction of functional genes into their genomes. Modifying a germ cell causes all the
organism's cells to contain the modified gene. The change is therefore ​heritable​ and
passed on to later generations. Australia, Canada, Germany, Israel, Switzerland, and
the Netherlands prohibit GGT for application in human beings, for technical and ethical
reasons, including insufficient knowledge about possible risks to future generations and
higher risks versus SCGT.

Vectors:
The delivery of DNA into cells can be accomplished by multiple ​methods​. The two major
classes are ​recombinant viruses​ (sometimes called biological nanoparticles or viral
vectors) and ​naked DNA​ or DNA complexes (non-viral methods).

There are two general approaches for introducing genes into a cell: viral and nonviral .
Viral vectors have been used in about 70% of the clinical trials to date . Viral vectors are
extremely efficient at transferring genes but can create some safety risks. Gene transfer
mediated by viral vectors is referred to as transduction. Nonviral vectors are considered
to be much safer than viral vectors, but at present, they are fairly inefficient at
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transferring genes. Gene transfer mediated by nonviral vectors is referred to as

transfection.

As indicated, viral vectors have the advantage of achieving highly efficient gene transfer
in vivo. Although replication-deficient vectors are used, viral vectors still pose significant
safety concerns.

The two most common viral vectors used in clinical trials have been those derived from
a serotype 5 adenovirus (Ad5; ~26%) and Moloney murine leukemia virus (MoMLV;
~28%), a retrovirus. MoMLV vectors target dividing cells with a reasonably high degree
of efficiency. Importantly, they also lead to stable gene transfer because they integrate
randomly into chromosomes of the target cell. A major disadvantage of MoMLV vectors
is the risk of insertional mutagenesis caused by the integration of the retroviral genome
into the host genome. Also, since retroviral vectors require dividing cells for successful
transduction, they are not useful for targeting gene transfer to well-differentiated,
quiescent cell types, such as in epithelial tissues. Ad5 vectors are able to transduce
both dividing and nondividing cells and facilitate highly efficient gene transfer.

Importantly, Ad5 vectors only very rarely integrate into a chromosome, that is, they
exist in a target cell nucleus in an epi-chromosomal location. Thus, if the target cell
divides, only one daughter cell will receive the transferred gene, and with subsequent
cell-division cycles, the gene will be dramatically diluted. The main disadvantage of Ad5
vectors is that they induce a potent host-immune response. It is also important to
recognize that different viral vectors will vary in their ability to transduce different cell
types. Often, this reflects the presence or absence of cell membrane receptor proteins
that mediate viral entry into the target cell.

Of the developed nonviral gene-transfer approaches, two methods have been used
fairly often in clinical trials. One involves simply the direct injection of plasmids
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containing the transgene (termed “naked DNA”) into a tissue. This has been used in
~14% of approved clinical trials, most often in muscle. The second method uses cationic
lipids (so-called liposomes) to surround the plasmid DNA and is termed lipofection. This
method has been used in ~9% of approved trials. The cationic lipids facilitate plasmid
entry into the cell. Nonviral gene transfer typically does not result in integration of the
transgene.

Problems and Prospects

Since the first clinical gene-therapy trial was conducted , much attention and
considerable promise has been given to the field. There has been substantial public-
and private-sector investment, as well as increasingly higher levels of research activity.
Numerous preclinical animal-model studies have provided proofs of concept for multiple
potential clinical applications. Also, major advances have been made in understanding
vector biology and improving vector design and production.

However, clinical progress has been slow. A major setback for the field occurred in
September 1999, when a widely publicized death resulting from a gene-therapy trial
was reported . Jesse Gelsinger, an 18-year-old man, died in a clinical trial at the
University of Pennsylvania, which used a modified Ad5 vector to deliver the gene for
ornithine decarboxylase, a deficient hepatic enzyme.

According to an investigation by the university, Gelsinger died from a massive immune

reaction to the Ad5 vector. This widely publicized case led to congressional and Food
and Drug Administration (FDA) hearings on the conduct of clinical gene-therapy trials as
well as a transient hold, subsequently lifted, on all adenoviral-vector clinical trials. An
investigation by the FDA found numerous possible violations in the way that this clinical
trial had been conducted and monitored. After 5 years of investigations, in February
2005, the case was settled. As a result of the Gelsinger case, gene therapy experienced
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an intense phase of criticism and skepticism. Clearly, mistakes were made in that
particular clinical trial, and as an appropriate outcome, all gene-therapy trials are now
subject to much tighter regulation.

Fortunately for the gene therapy field, less than 1 year after Gelsinger died, the first
report of a dramatically successful gene-therapy trial was published. In 2000,
Cavazzana-Calvo and her colleagues in Paris described results from a study involving
two children suffering from a severe combined immunodeficiency disorder (SCID-XI),
which had restricted them to life in an isolated environment .

These investigators used a MoMLV vector to transfer a curative gene (γc cytokine
receptor subunit) into the patients’ lymphocytes ex vivo, and after amplification of the
cells, returned them to the patients. Both patients were able to leave the hospital and
resume normal lives. Subsequently, several other patients were treated and apparently
cured in these studies. However, there was a downside. Of ~11 early patients treated
with the MoMLV vector, 3 developed leukemia directly as a result of the gene-transfer
procedure ).

In all of these patients, the MoMLV vector had integrated apparently in a nonrandom
manner near the LM02 (LIM domain only 2) gene. The LM02 gene was activated by this
integration, and leukemia was the result. The patients were treated for the leukemia,
and a large, collaborative scientific effort began to understand what mechanisms
influence MoMLV integration.

The SCID-XI trial likely reflects the path that gene therapy will follow during the next 1 to
2 decades: success, but with some complications. Last year, a somewhat similar
scenario occurred in a German clinical trial of a MoMLV vector to treat chronic
granulomatous disease—that is, some clinical success, but subsequently, a patient
died.
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Also, a clinical trial to correct a clotting disorder, Factor IX deficiency, by hepatic gene
transfer using an AAV2 vector recently showed that transient correction was possible
but quite limited in time because of subsequent immune reactivity . These experiences
add further credence to the general viewpoint offered by Leiden in a 1995 editorial that
gene therapy is a field in its infancy, and despite some pitfalls, it is well grounded in
fundamental scientific principles with real clinical promise for the future (​Leiden, 1995​).

Immune Deficiencies:

Several inherited immune deficiencies have been treated successfully with gene therapy.
Most commonly, blood stem cells are removed from patients, and retroviruses are used to
deliver working copies of the defective genes. After the genes have been delivered, the stem
cells are returned to the patient. Because the cells are treated outside the patient's body, the
virus will infect and transfer the gene to only the desired target cells.

Severe Combined Immune Deficiency (SCID)​ ​was one of the first genetic disorders to be
treated successfully with gene therapy, proving that the approach could work. However, the
first clinical trials ended when the viral vector triggered leukemia (a type of blood cancer) in
some patients. Since then, researchers have begun trials with new, safer viral vectors that
are much less likely to cause cancer.

Adenosine deaminase (ADA) deficiency​ ​is another inherited immune disorder that has
been successfully treated with gene therapy. In multiple small trials, patients' blood stem
cells were removed, treated with a retroviral vector to deliver a functional copy of the ADA
gene, and then returned to the patients. For the majority of patients in these trials, immune
function improved to the point that they no longer needed injections of ADA enzyme.
Importantly, none of them developed leukemia.
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Hereditary Blindness

Gene therapies are being developed to treat several different types of inherited
blindness—especially degenerative forms, where patients gradually lose the light-sensing
cells in their eyes. Encouraging results from animal models (especially mouse, rat, and dog)
show that gene therapy has the potential to slow or even reverse vision loss.

The eye turns out to be a convenient compartment for gene therapy. The retina, on the
inside of the eye, is both easy to access and partially protected from the immune system.
And viruses can't move from the eye to other places in the body. Most gene-therapy vectors
used in the eye are based on AAV (adeno-associated virus).

In one small trial of patients with a form of degenerative blindness called LCA (Leber
congenital amaurosis), gene therapy greatly improved vision for at least a few years.
However, the treatment did not stop the retina from continuing to degenerate. In another trial,
6 out of 9 patients with the degenerative disease choroideremia had improved vision after a
virus was used to deliver a functional REP1 gene.
Hemophilia

People with hemophilia are missing proteins that help their blood form clots. Those with the
most-severe forms of the disease can lose large amounts of blood through internal bleeding
or even a minor cut.

In a small trial, researchers successfully used an adeno-associated viral vector to deliver a

gene for Factor IX, the missing clotting protein, to liver cells. After treatment, most of the
patients made at least some Factor IX, and they had fewer bleeding incidents.

Blood Disease
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Patients with beta-Thalassemia have a defect in the beta-globin gene, which codes for an
oxygen-carrying protein in red blood cells. Because of the defective gene, patients don't have
enough red blood cells to carry oxygen to all the body's tissues. Many who have this disorder
depend on blood transfusions for survival.

In 2007, a patient received gene therapy for severe beta-Thalassemia. Blood stem cells were
taken from his bone marrow and treated with a retrovirus to transfer a working copy of the
beta-globin gene. The modified stem cells were returned to his body, where they gave rise to
healthy red blood cells. Seven years after the procedure, he was still doing well without blood
transfusions.

A similar approach could be used to treat patients with ​sickle cell disease​.

Fat Metabolism Disorder

In 2012, Glybera became the first viral gene-therapy treatment to be approved in Europe.
The treatment uses an adeno-associated virus to deliver a working copy of the LPL
(lipoprotein lipase) gene to muscle cells. The LPL gene codes for a protein that helps break
down fats in the blood, preventing fat concentrations from rising to toxic levels.

Cancer

Several promising gene-therapy treatments are under development for cancer. One, a
modified version of the herpes simplex 1 virus (which normally causes cold sores) has been
shown to be effective against melanoma (a skin cancer) that has spread throughout the
body.

The treatment, called T-VEC, uses a virus that has been modified so that it will (1) not cause
cold sores; (2) kill only cancer cells, not healthy ones; and (3) make signals that attract the
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patient's own immune cells, helping them learn to recognize and fight cancer cells throughout
the body. The virus is injected directly into the patient's tumors. It replicates (makes more of
itself) inside the cancer cells until they burst, releasing more viruses that can infect additional
cancer cells.

A completely different approach was used in a trial to treat 59 patients with leukemia, a type
of blood cancer. The patients' own immune cells were removed and treated with a virus that
genetically altered them to recognize a protein that sits on the surface of the cancer cells.
After the immune cells were returned to the patients, 26 experienced complete remission.
Parkinson’s Disease

Patients with Parkinson's disease gradually lose cells in the brain that produce the signaling
molecule dopamine. As the disease advances, patients lose the ability to control their
movements.

A small group of patients with advanced Parkinson's disease were treated with a retroviral
vector to introduce three genes into cells in a small area of the brain. These genes gave cells
that don't normally make dopamine the ability to do so. After treatment, all of the patients in
the trial had improved muscle control.
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