Gene Therapy

WHAT IS GENE THERAPY?

Gene therapy is a procedure that utilizes genes to treat or prevent disease. This technique
involves altering the genes inside the cells by replacing a faulty gene, inactivating or "knocking
out" a mutated gene, or adding a new gene to cure disease or improve the body's ability to fight
illness. The technique is different from traditional drug-based approaches, which may treat
symptoms but not the underlying genetic problems. The idea of gene therapy is to modify the
genetic information of the cell responsible for a disease, and then bring back that cell to normal
conditions.
In the future, gene therapy may allow doctors to treat a disease without using drugs or surgery.
Gene therapy can treat both inherited and acquired diseases. This technique may be a promising
treatment for a wide range of diseases such as cancer, cystic fibrosis, heart disease, diabetes,
hemophilia, and AIDS. But though it may have promising results, the use of this technique is still
on the debate as it poses risks that outweigh the benefits.
BRIEF HISTORY OF GENE THERAPY
Gene therapy is not a new idea. However, it is difficult to pinpoint its beginning. The concepts of
gene therapy arose initially during the 1960s and early 1970s. In 1967, Marshall Nirenberg wrote
a paper about "programming cells with synthetic messages," which recognized the usefulness as
well as the dangers of the procedure. Nirenberg won the Nobel Prize in Physiology in 1968, and
his work was considered as the start of at least the discussion about gene therapy.
In the 1970s, the research on recombinant DNA, a DNA that has been formed artificially by
combining constituents from different organisms, was advancing. The National Institutes of
Health (NIH) lead in regulating recombinant DNA research in 1974. Then a committee to the NIH
Director, a regulatory oversight body called Recombinant DNA Advisory Committee (RAC), was
created. With the Food and Drug Administration (FDA), RAC review gene therapy protocols. In
1984 the RAC created a new committee called the Human Gene Therapy Subcommittee (HGTS),
individually to review clinical gene therapy protocols. Their first task was to produce a reference
document, "Points to Consider for Protocols for the Transfer of Recombinant DNA into the
Genome of Human Subjects." In 1985 HGTS finished and published its “Points to Consider”
document and was waiting for applications for clinical gene therapy protocols for review. It took
almost three years until the first protocol was presented to RAC. It was a gene marking study by
Steven Rosenberg in 1988. He proposed to use gene marking techniques to track the trafficking
of tumor-infiltrating blood cells in cancer patients. However, no actual "gene therapy" was
recommended.
Gene therapy established a foundation for the treatment of specific diseases in 1980s. It is due
to the advances in the science of genetics. In 1990, HGTS received two protocols for review. The
first protocol was from Michael Blaese and W. French Anderson for T lymphocyte-directed gene
therapy for adenosine deaminase - severe combined immunodeficiency (ADA-SCID). The first
person to undergo gene therapy was a four-year-old girl suffering from adenosine deaminase
(ADA) deficiency, a genetic disease that weakens her immune system. She went through the
clinical trial on September 14, 1990, at the National Institute of Health in Bethesda, Maryland.
White blood cells were extracted from her, and the normal genes for making adenosine
deaminase were inserted into them. Then, the corrected cells were injected back to her. Although
the girl showed to produce ADA, her recovery cannot be uniquely attributed to gene therapy
because she also received other drug treatments. Researchers already had developed a cure for
ADA deficiency before the gene therapy trials began. Synthetic ADA was used in a complex with
polyethylene glycol (PEG). Injection of the ADA-PEG complex allowed some immune system
development, although not full immune reconstitution. Nevertheless, gene therapy had gained
fame as more and more scientists conducted clinical trials in this area.
The other protocol was from Steven Rosenberg. He wanted to use the same tumor-infiltrating
blood cells he had previously investigated as a vector to transfer the neomycin resistance marker
gene into lymphocytes from 5 patients with metastatic melanoma. These lymphocytes were then
expanded in vitro and later reinfused into the patients. The procedure showed that retroviral
gene transfer was safe and practical, thus led to many other studies.

TYPES OF GENE THERAPY

Depending on which types of cells are corrected, there are two different types of gene therapy:
the somatic gene therapy and the germline gene therapy.

Somatic Gene Therapy

Somatic gene therapy is a technique wherein a section of DNA is transferred to any cell
of the body except for those that produce sperm or eggs. This type of gene therapy is viewed as
more conservative and safer because it affects only the targeted cells, and the effects will not be
passed onto the patient’s children. However, this type of therapy poses a unique problem. Often
the results are only short-lived because the cells of tissues are continuously replaced by new cells.
Thus, repeated treatments over the patient’s life span are required to maintain the effect. In
essence, somatic gene therapy is just another drug delivery system. It is just a different way to
bring a healthy human protein to the right position in the body.

Germline Gene Therapy

Germline gene therapy is a technique in which a section of DNA is transferred to cells that
produce eggs or sperm. This type of therapy results in permanent changes or therapeutic effects
that are passed onto the patient’s children and subsequent generations. Thus, there is a
possibility of eliminating the disease from a particular family forever. However, this also raises
controversy as some people view this as unnatural and liken it to “acting God”.
MECHANISM OF GENE THERAPY
Genes are part of DNA that controls the cell to produce proteins that perform various
functions in a body. Diseases are developed when a mutated gene causes a protein to be
abnormal or missing. In gene therapy, a functional copy of the gene is delivered into a patient's
cells. Healthy protein that is produced from a functional gene has the potential to correct the
underlying cause of the disease and to provide restorative therapeutic benefit.
Gene therapy uses a carrier called a vector to deliver genes into cells. Genetically
engineered viruses are often used as vectors because they can transport the functional genes by
infecting the cell. The viruses are modified so they can't infect when used in people. In some
cases, bacterium or plasmid can also be used .
During gene therapy, a vector virus containing the therapeutic gene enters the cell with the
bloodstream to the small capillaries, the region where cells exchange matter with the blood.
There are large pores in the capillary walls which contain specialized cells, the macrophages. The
function of these cells is to remove any foreign and harmful particles, including the virus particles.
Thus, the vector virus should be coated with a protein that can only bind to a specific cell. The
vector virus can pass the macrophages and reach a particular cell more quickly. Once the vector
virus particles attach to the cell, the virus' envelop merges with the cell membrane. It enables
the virus's load of healthy or therapeutic DNA to penetrate the cell. Then it will proceed to the
cell’s nucleus. The virus proteins assure that the therapeutic gene is taken into the genetic
material of the cell. Once inside the nucleus, the functional gene can support the production of
a healthy protein and correct the underlying cause of the disease. It will enable the cell to
function once again as a healthy cell [8].
VARIOUS APPROACHES TO CARRY OUT GENE THERAPY

There are several approaches for carrying out gene therapy which includes:

1. Gene augmentation therapy. It is done by replacing a missing gene caused by mutation

with a healthy and functional version of the gene. The healthy gene produces a product sufficient
to replace that was missing. It is only successful if the effects of the disease are reversible or have
not resulted in lasting damage to the body, such as cystic fibrosis. A functional copy of the gene
is introduced to correct the disease, as seen in the illustration.
2. Gene inhibition therapy is done by inactivating or "knocking out" a mutated gene that is
malfunctioning. The product of the introduced gene inhibits the expression of another gene or
inactivates the outcome of another gene. This approach is appropriate for the treatment of
infectious disease, cancer, and other inherited disease caused by incorrect gene activity. For
example, the over-activation of an oncogene (a gene that stimulates cell growth) may result in
cancer. By inhibiting the action of that oncogene, further cell growth will be prevented, and
cancer remission is possible.

3. The killing of specific cells is made by introducing DNA into a diseased cell that causes that
cell to die. It can be achieved in two ways. Either the inserted DNA contains a "suicide" gene that
produces a highly toxic product that kills the diseased cell, or the inserted DNA causes expression
of a protein that marks the cells so that the body's natural immune system attacks the infected
cells. This approach is suitable for diseases such as cancer that can be treated by destroying
certain groups of cells.
CRITERIA IN CHOOSING THE MOST SUITABLE VECTOR

One of the challenges of gene therapy is the delivery of genes, which requires an efficient way of
getting the DNA to the target cells and switching it on. It, in turn, has much to do with choosing
the most suitable vectors as vehicles of a gene for treating a disease. There is no "perfect vector"
that can manage every disorder. A vector must be modified to fit or match the unique features
of the disorder.
To be successful, a vector must:
a. Target the right cells. If the disorder is in the liver cell, the vector must deliver the gene
to that cell and not anywhere else.
b. Integrate the gene into the cells. It is not enough that the vector with the gene binds to
the right cell. The gene must integrate or becomes part of the host cell's genetic material or must
survive in the nucleus without being trashed.
c. Activate the gene. A gene's goal is not over when it enters the nucleus in the cell. It must
be "turned on," meaning it is transcribed or translated to make the protein product it encodes.
For gene delivery to be successful, the produced protein must function correctly.
d. Avoid harmful side effects. Anytime an unknown biological substance is introduced into
the body, this can trigger an immune response that could be harmful to the patient. It can be
avoided by using a vector that is less likely to trigger an immune response.
TYPE OF VECTORS

Scientists refer vectors as DNA delivery vehicles. Each vector is intended to target specific cells.
There are two general types of vectors used in gene delivery into a cell: viral and nonviral.6
a. Viral vectors. When we think of viruses, we usually think of the ones that cause diseases.
But scientists have been able to genetically engineered viruses to deliver DNA to cells for gene
therapy. To date, about 70% of clinical trials used viruses. Gene transfer mediated by viral vectors
is referred to as transduction.
Advantages of viral vectors:
• They’re highly efficient at targeting and entering cells.
• Some target specific types of cells.
• They can be engineered so that they can't replicate and destroy.
Disadvantages of viral vectors:

• They can carry a limited amount of genetic material. Therefore, some viruses will not be
big enough to accommodate genes.
• They can cause immune responses in patients, leading to two potential problems:
- Patients may get sick.
- The immune system may block the virus from delivering the gene to the patient's cells, or
it may kill the cells once the gene is delivered.

b. Non-viral vectors. These include any method of gene transfer that does not involve the
production of a viral particle. It is divided into two classes: (1) RNA or DNA that can be amplified
in bacteria or eukaryotic cells, and whose transfer into a cell does not involve a viral particle, or
(2) oligodeoxynucleotides or related molecules synthesized chemically. Gene transfer mediated
by non-viral vectors is referred to as transfection.
Advantages of non-viral vectors:
• They can carry enormous genes.
• Most do not trigger an immune response, thus much safer than viral vectors.
• They do not carry the risk of recombining to generate wild-type virus.
Disadvantages of non-viral vectors:
• They are less efficient than viruses at delivering genes into cells.
• They integrate at a low frequency into the chromosome, therefore, pose some risk of
insertional mutagenesis.
• Gene expression has a transient nature.

BASIC WAYS OF DELIVERING GENES INTO THE CELLS

Gene therapy will only work if we can deliver a healthy gene into the right cells in the correct
tissue. Remember, there are large numbers of cells – say, several million – in a tissue. Not only
that. Once the gene reaches its target, it must be activated or turned on to produce the protein
encoded by the gene. Thus, not only gene delivery but also its activation is one of the challenges
in gene therapy.
There are two ways to deliver a gene into a group of cells in a patient's body: in vivo and ex vivo.

a. In vivo involves injecting the vector carrying the therapeutic gene directly into the
patient’s body, and specifically aiming to target the affected cells.
b. Ex vivo involves the harvesting of cells from a patient, followed by subsequent viral
transduction, then delivery of the gene to the cells that have been removed from the body and
are grown in culture. After the gene is delivered, integration and activation are confirmed, and
the cells are put back into the patient. The process involved the following:

1. Isolation of desired cells from the body

2. Culturing the gene in a petri dish, in the laboratory
3. Delivering the gene to the cells (using an appropriate vector), activating it, and integrating
it properly into the cells.
4. Placement of the genetically modified cells back into the proper place in the body,
ensuring that they survive there, and letting them get to work
POTENTIAL BENEFITS, DETRIMENTS, AND ISSUES OF GENE THERAPY
POTENTIAL BENEFITS OF GENE THERAPY
For many years genetic disorders and gene-related diseases have been responsible for
high mortality and reduced quality of life. Some of these may manifest at a very early stage, say
days after birth. But with little option to treat such conditions, families are in much pain as they
are left hopeless. However, research in gene therapy has been ongoing, and several protocols
have been successful. Despite its complexity, gene therapy has the following potential benefits:

1. Long-lasting and Timeless Effects

Once the defective gene is replaced with a functional gene in disease, there are limited
chances of remission. Thus, the therapeutic effect has the advantage of being long-lasting or
sometimes permanent, although this benefit doesn't apply in every situation. Furthermore, there
is a possibility that a therapeutic effect could be inherited to the next generation. When you
remove the defective gene from a patient, he/she will not transfer the defective gene to his/her
offspring but the new functional gene. It will reduce the risk of suffering a similar disease in the
future by his/her descendants.

2. Eradication of Diseases

Some of the previously incurable diseases can be managed or even eradicated using gene
therapy. By using the germline method, the remedy is not just for one generation but also for the
succeeding generations. Although it is not always successful, the transmission of defective genes
to the next generations can be avoided; thus, no further occurrence of the disease. Gene therapy
has the potential to eliminate and prevent hereditary diseases such as cystic fibrosis, Parkinson’s
disease, Huntington’s disease, and Alzheimer’s disease. It is also a possible cure for heart disease,
AIDS, and cancer.

3. Hope for Quality Life

Many people have their lives compromised because of having genetic defects. Congenital
disabilities could also lose the joy of having a child, which leads to struggle in such a hard life.
With gene therapy, parents can be confident that their unborn babies will be delivered safely and
grow to their prime. For example, unborn babies with family histories of cancer can undergo gene
therapy in the womb to remove the cancer gene. Thus the child will not develop cancer genes
and could live a normal happy life unless exposed to lots of carcinogens.
Gene therapy does not only focus on diseases but also on other conditions such as infertility. It
is projected that soon gene therapy will activate reproductive genes and allows infertile people
to have children.

POTENTIAL DETRIMENTS OF GENE THERAPY

 Although gene therapy has the potential benefits for treating diseases and improving life,
its effects are too unpredictable. Even if the treatment is successful, other mutations can happen.
Thus, gene therapy poses potential detriments or risks. Some of the risks are as follows:

1. Unwanted immune system reaction

Our body's immune system is very good in combating invaders, such as bacteria and viruses.
During gene therapy, this system may see the vectors, the newly introduced viruses, as
potentially-harmful invaders and attack them. It can lead to an immune response in the patient,
which can lead to health issues like inflammation, dizziness, and headaches. In severe cases, the
immune response will target the body's organs, causing them to fail, and eventually lead to
death.
An example was Jesse Gelsinger, who had a rare liver disorder. He joined in a gene therapy clinical
trial in 1999. He died shortly after a dose of adenovirus vector due to complications from an
inflammatory response.
The genes must be delivered without the immune system, "noticing." To avoid an immune
response, researchers can hide the vector and its transgene product from the immune system by
decreasing the effective vector dose, delivering the vector to the immune-privileged site, and
preventing the anaphase-promoting complex (APC) expression. Researchers can also hide the
immune system from the vector by immune suppression and immune modulation.
2. Targeting the wrong cells
Targeting a therapeutic gene to the correct cells is crucial to the success of any gene therapy.
Delivering the gene into the wrong cell would be inefficient, which can cause health problems.
Moreover, viruses as vectors have the capability of affecting other cells, not just the cells that
contain the mutated or missing genes, the target cells. In this case, the healthy cells could be
damaged, which will result in disease development, illness sensitivity, or even cancer.
The therapeutic gene introduced during gene therapy will ideally integrate into the genome of
the patient and will continue working for the rest of their lives. However, if the gene inserted into
the path of another gene, it will disrupt its activity. If this interferes with a critical gene, the one
involved in regulating cell division, it could result in other diseases.
It happened in gene therapy clinical trials aiming to treat children with severe combined immune
deficiency (SCID). Children with this disorder have no immune protection against bacteria and
viruses. The gene therapy treatment targets to restore the function of the gamma c gene in the
cells of the immune system. The treatment may seem very successful, but later some of the
patients developed leukemia. It was found that the newly transferred gamma c gene had
attached to a gene that usually helps regulate the rate at which cells divide. It results in
uncontrolled cell division, causing leukemia.
3. Infection caused by the virus vector
The vector, usually a virus, is genetically engineered to perform the task of carrying the
therapeutic gene. Yet it is possible that the virus may recover to its original ability to cause disease
once introduced into the patient's cell. Although researchers would not use life-threatening
illnesses for the method, there is still a risk of becoming sick from other concerns other than the
target genetic disease. For example, current researchers use adeno-associated viruses to deliver
genes. Although it is unknown to cause any disease in humans, it can cause colds.

4. Potential for developing resistance

The benefits of gene therapy might be brief. Just like the antibiotic resistance, the human body
could develop resistance to the information transfer. So there is no guarantee that gene therapy
will always fulfill its expectation to treat diseases. It means that today gene therapy may be useful
for a specific condition but will not have the same effect in the next generation.

ETHICAL AND SOCIAL ISSUES OF GENE THERAPY

Gene therapy is very complex and has many variables. Despite the promising results in several
clinical trials, more techniques or protocols are still needed to be developed to ensure the safety
and efficacy of the procedure. Furthermore, diseases are required to be understood more fully
before gene therapy can be used appropriately. Since gene therapy involves altering the body's
genetic setup, it raises ethical and social issues.
Two conceptual distinctions are vital to an understanding of the ethical and social issues of gene
therapy: the therapy vs. enhancement and somatic vs. germline gene therapy.

Therapy vs. enhancement

Gene therapy should be therapy, such as the treatment of a valid disease, rather than
enhancement, which would mean "improving humankind." But the questions here are: who
decides which traits are typical and which constitute a disability or disorder? How are disorders
or traits determined to warrant gene therapy? Unfortunately, there is a thin line separating or
distinguishing a gene therapy for disease and gene therapy for enhancement desired traits, such
as hair or eye color. Everybody would agree that conditions that cause suffering, disability, and
possibly death are the right candidate for gene therapy. However, in the case of dwarfism
disorder achondroplasia, there is no clear-cut distinction, whether it's a disease or a trait. For
many, it is considered a "disease," while others thought it a "trait" of a healthy individual with
short stature. It is a fact that successful gene therapy for correcting socially unacceptable traits
or enhancing the desirable ones will improve an individual's quality of life. But there is a
possibility that the widespread use of gene therapy for improving a trait would have a negative
social impact as it will promote discrimination toward those that have “undesirable traits." Also,
with the continuous discovery of the function of many genes, it would be increasingly difficult to
classify which gene traits can be considered a disease and which are physical, mental, or
psychological traits. It would be challenging to identify who is suffering. Is it a child who cannot
shoot the ball as ideally as a professional athlete? No. Physically the child is not suffering from a
genetic disease but is suffering from social pressure to be the best basketball player. Another
issue against the procedure is the possibility of producing superior humans who will reduce the
nonenhanced to menial servitude.

Somatic vs. germline gene therapy

To date, acceptable clinical trials involving humans deal with somatic gene therapy, which
uses techniques that will prevent the spread of corrected genes to the germ cells. Since it will not
affect the germline or, consequently, the patient's children, all effects of the therapy will end
with the life of the patient. It makes the somatic cell gene therapy relatively uncontroversial.
However, the argument about germline therapy has not been resolved. Many ethicists worry that
if germline gene therapy becomes more feasible and legalized and more genes causing different
traits are discovered, there will be a riffling effect of whichever genes are to be used in future
therapy. The acceptance of germline therapy would become equal to the approval of gene
therapy for genetic enhancement.
The ethical debate on germline therapy has usually revolved around two kinds of issues:
1. Germline therapy is an "open-ended” therapy. The main objective of germline therapy
which is to correct the genetic defect once and for all lays an ethical problem. Since its effect will
extend indefinitely into the future generation, germline therapy may or may not consider an
individual's human rights. On the one hand, no unborn child can choose their genetics and
whether they are born with or without a genetic problem. But on the other hand, the unborn
child of persons who underwent gene therapy cannot give their consent for their genetic makeup
to be modified.
2. Germline therapy may involve invasive experimentation on human embryos. Germline
therapy is carried out in gametes or pre-embryos, which may require a reproductive process that
involves in vitro fertilization (IVF) to identify a defective gamete. This process is too risky for the
patient. If a potential disease is found out in a gamete, then one can avoid the illness by merely
discarding the gamete. Once again, there is no guarantee that this will be safe. Although germline
therapy could spare future generations from a genetic disorder, it might also affect the
development of a fetus in unpredictable ways or have long-term side effects that are not yet
known.
Another essential issue of gene therapy is the cost or its affordability. The procedure is
expensive. In countries with no free health service, only parents who can afford can avail. More
impoverished parents cannot access this therapy. And a more likely scenario that would possibly
happen is the gradual worsening of existing class distinctions. But because germline therapy has
the potential of completely eradicate the disease; it will reduce the long term healthcare cost of
treating the disease. Nonetheless, an initially expensive technology usually becomes available to
all people, computers, for example. It will eventually happen to gene therapy.