Biology Research Report
Gene Therapy
Sashi Amanda Samarathunga
ID No. 00008786
Introduction
As the name suggests, Gene Therapy is a class of treatment procedure that utilizes ‘genes’ (DNA
segments)- instead of drugs or surgery- to treat or prevent disease. In Gene Therapy, an individual's
cells and tissues are inserted with genes that can help treat certain diseases, particularly those that are
hereditary. More specifically, a functional allele is inserted over a defective mutant allele but gene
targeting is also commonly used; this involves gene amplification (Eugene, 2001).
A crucial element in this procedure are proteins as they carry an important role in the cell structure
and function; any mutation or defect in them would have an impact on the structure and homeostasis
in the cells, triggering diseases. The synthesis and secretion of protein in our cells are guided by
genes, hence, by removing and replacing the ‘culprit gene’ with a healthy, functional gene it is
possible to acquire more useful and normal proteins (Ermak, 2015). The most popular vectors used in
Gene Therapy to transplant healthy genes- or more recently, portions of genes- are viruses, this is
because all viruses tend to attack and introduce their genetic material into the host cell as a stage in
their process of replication. Scientists recognized that separating the viral DNA and manipulating the
virus like a ‘vehicle’ to transport the therapeutic DNA to be a sound and feasible strategy in the
procedure of Gene Therapy (Ermak, 2015).
The procedure of gene therapy varies depending on the type of cell that is receiving treatment, as of
now, there are essentially two types of Gene Therapy; Somatic Gene Therapy and Germline Gene
Therapy. The literal definition of the term ‘Somatic’ is pertaining to the body. Therefore Somatic
genetic engineering specifically involves making changes to the cells of the body, excepting ova and
sperm; however this exception forbids these genetic alterations from being passed on to the next
generation. In contrast to Somatic genetic therapy, Germline genetic therapy involves modifications
that are assimilated into the genome of future generations (Sheridan, 2011). In general terms,
geremline treatment includes alterations to ova and sperm prior to or after fertilisation; which is at the
single-cell stage of development.
Even though Gene Therapy appears to be a favourable treatment option for certain types of cancer,
viral infections, inherited disorders and a variety of
other diseases, it is still an experimental
technique and has only been tested on diseases that lack a cure. In the future, doctors might be able to
treat diseases by inserting a gene into patients’ cells rather than following treatment with drugs or
surgery (Rojahn, 2014). But on the bright side, scientists continue to study it in order to confirm it’s
safety and performance, and are examining multiple strategies to gene therapy, a few include:
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Replacing a defective disease-causing gene with a functional copy of the gene.
Inactivating, or “knocking out,” a defective gene which is dysfunctional.
Introducing the body with a new gene to help fight a disease.
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Procedure
As explained, Gene Therapy generally involves changing the genetic format of cells to correct
defective genes; this is generally done by replacing or editing a genetic code by adding missing code.
Somatic Gene Therapy and Germline Gene Therapy are advanced procedures that intend to alter the
genetic code inside a patient's cells to repair, replace or silence a broken gene (Crasto, 2013). In
addition to that, they can also institute code that can synthesise a completely new protein.
Gene Therapy can be done in two ways, ‘Vivo’ and ‘Ex Vitro’. In vivo therapy, an individual is
instituted with a vector (usually a virus) which will then transfer the required therapeutic gene into the
patient’s cells. On the other hand, ex vitro introduces a vector that codes for a specific protein (i.e.;
chimeric antigen receptor) to an individual’s ‘autologous’ (their own cells) or ‘allogeneic’ (donor
cells) cells that are extracted and isolated outside the body which are then modified and instituted
back into the patient (refer to figure 01). When comparing the two methods, vivo seems the most
convenient as there is no need to gather mitotic cells but ex vitro therapy poses less serious immune
risks and is more compatible with the body.
Figure: 01
1.1. Somatic Cell Gene Therapy
In Somatic cell gene therapy (SCGT), therapeutic genes are placed inside ‘somatic’ cells- any cell
(i.e.: bone marrow) other than gamete cells (sperm and ova), these cells do not produce the eggs and
sperms that in turn produce the next generation. What this means is that Somatic Cell Gene Therapy
aims to cure a disease only in the patient and not in the patient’s descendants; any alteration
performed would only have an effect on an individual patient and are not inherited by their offspring.
SCGT constitutes conventional clinical and basic research where therapeutic DNA, that is either
merged as an external plasmid or episome or into the genome, is utilized to treat disorders (Williams
& Orkin, 1986).
In the U.S. more than six hundred clinical trials promoting Somatic Cell Gene Therapy have been
initiated, most of them focusing on serious hereditary disorders such as haemophilia, cystic fibrosis,
thalassaemia and immunodeficiencies which are single gene mutations- when it comes to SCGT,
single gene disorders appear to be the best and easiest to work with. SCGT in method encompasses
the idea of inserting a corrective, normal gene into the genome of a patient affected by a hereditary
disease, thereby permanently fixing the disorder. Figure 02 represents the basic procedure of inserting
genetic material into an individual’s genome, utilizing. viruses that hold the human genome required
in the place of their own genetic material. Small fat-like molecules, namely ‘liposomes’, can also be
used to transfer DNA into cells (Mawillo & Ferrari, 2008).
For example, the target cells can be bone marrow cells; bone marrow cells are convenient as they can
easily be isolated and re-implanted, they also tend to constantly divide and multiple throughout the
span of an individual's life so as to create blood cells- however, this approach is only limited to genes
that have a biological role in the blood. In the case of transferring a gene with a different biological
role like the liver, lungs, muscle, etc., delivery would have to take place within those target organs. A
major problem in multiple instances is accessing the appropriate tissue or multiple tissues (i.e.:.
muscles all over the body) and making sure it can be delivered where it is needed. (Mawillo & Ferrari,
2008).
Figure: 02
1.2.
Germline Cell Gene Therapy
Germline Gene Therapy (GGT) involves the modification of germ-cells- more commonly known as
gametes (sperm and ova)- by introducing functional and corrective genes through to their genetic
structure. Germ cell modification leads to all of the organism’s cells to accommodate this revised gene
and thus is heritable, and can be inherited by their offspring and sweeped on to later generations- GGT
removes a genetic disorder from a family line forever (refer figure 03). Some advantages of GGT
include the fact that germ cells are situated outside the body and hence are easily accessible, this way
gene delivery is less of an issue unlike with SCGT. This also connotes the idea that this inserted gene
or genes would be present in all of the cells of the patient as it is transferred to progeny cells during
growth and development (Hanna, 2006).
Although, countries like Canada, Israel, Australia, Germany and the Netherlands forbid GGT for
application in humans due to ethical and technical reasons as there is inadequate information and
potential safety threats to future generations and include more drawbacks in comparison to Somatic
Cell Gene Therapy. Speculating the exact location in the genome a gene would culminate in is
impossible, and this suggests risks with normal gene functioning, developing new mutations.
Unfortunately, even if these risks were to be overlooked, there is a possibility of developing new
ethical problems with regard to serious development of GGT; some examples of these ethical
problems are; determining what genetic modifications should be permitted-the main focus would be
on correcting detrimental mutations, but others might be considered enhancements, rather than
treatments (Hanna, 2006).
Figure: 03
Viral And Non-viral Vectors
As previously explained, gene therapy mainly focuses on transferring genetic material into cells, there
are multiple ways to do this but two prime methods stand out; viral vectors and non-viral vectors.
When it comes to viral methods, they utilize viral vectors which are also commonly known as
recombinant viruses or biological nanoparticles. Non-viral methods utilize non-viral vectors- DNA
complexes or Naked DNA (Sheridan, 2011).
1.1
Viral Methods
All viruses tend to attack and insert their genepool into the host cell as a stage in their process of
replication. Primary information or ‘instructions’ to replicate viruses are stored in this genetic material
and possess the ability to manipulate the host-cell or body to address the requirements of the virus; the
host cell is manoeuvred into manufacturing more copies of the virus, inevitably infecting more cells.
Most viruses usually do not enter the host cell and instead tend to inject their genetic material towards
the host’s cytoplasm, but some other viruses prefer to insinuate themselves into the host cell through
the cell membrane by disguising themselves as protein molecules thereby confusing the host cells.
Lytic and Lysogenic are two prime virus infections; viruses that follow the lytic cycle break out from
their host cell and infect more cells once more viruses have been produced. Lysogenic viruses follow
a cycle where they merge their DNA into the genome of the host cells, these viruses will continue to
reproduce at the same pace as the cell does causing no harm to the host body until it is triggered
(Sheridan, 2011).
Retrovirus
Retroviruses have their genetic material structured in RNA molecules unlike their host cells who
structure is in the form of DNA instead and so these viruses contain special enzymes called reverse
transcriptase and integrase to help guide the insertion of their RNA into a host cell. A DNA copy of
this RNA molecule is formed through a process called reverse transcription (using enzyme reverse
transcriptase) in order to be integrated into the host cell’s genetic material. Next, it utilizes the enzyme
integrase to incorporate DNA copy into the genome of the host cell (refer to figure 04). Once the host
cells start dividing, all it’s daughter cells will also contain this new genetic makeup (Eugene, 2001).
Figure: 04
Adenoviruses
Adenoviruses cause infections in the intestines, eyes and respiratory system in humans; they store
their genetic material in the form of double-stranded DNA. Once infecting a host cell, these viruses
have their genetic material in the form of a DNA molecule free in the host cell’s nucleus instead of
incorporating it into the host’s genome. Transcription occurs in this extra new DNA similar to other
genes except the fact that they’re not recreated during the host cell's cell division process thereby
having no effect on its daughter cells.
1.2
Non-viral methods
When compared to viral methods, non-viral methods are more advantageous, for example, they pose
less adverse immune responses and can also be produced on a large scale. In the past, non-viral
methods constituted lower levels of gene expression and transfection and so presented multiple
drawbacks but latest developments in vector technology proffered efficient transfection techniques
and molecules akin to viruses. As of now, the easiest method to non-viral vector method is injection of
naked DNA (Eugene, 2001). Experiments with intramuscular injection of plasmid containing naked
DNA were to some extent successful, but still presented low levels of gene expression when
compared to other transfection techniques.
Figure: 05
Gene gun
The method of gene gun utilizes the idea of particle bombardment and is a physical method of DNA
transfection. Gene gun method or biolistic particle delivery system is usually used in biotechnology
for genetically modifying plants. With the use of a gene gun, the gene gun method delivers vector
DNA or plasmid directly into a host cell’s nucleus. The payload is an elemental particle of a heavy
metal coated with DNA. The method is also commonly called particle acceleration or microprojectile
bombardment.
Sonoporation
This technique transfers DNA by utilizing ultrasonic frequencies to deliver DNA into cells. It is
believed that this process of acoustic cavitation sanctions DNA to advance into the cell by distorting
the cell membrane.
Hydrodynamic delivery
This method involves injecting vasculature swiftly (i.e.; bile duct and tail vein) with a high volume of
solution, rich with molecules like DNA plasmids or siRNA, to create elevated hydrostatic pressure
which facilitates cell penetration (Hanna, 2006).
Adverse effects
When it comes to gene therapy there are a multitude of drawbacks and adverse effects, for example
some of the unanswered issues include: the therapeutic DNA delivered into target cells must be
functional and the cells holding the therapeutic DNA must be stable before gene therapy may become
a permanent treatment for an illness. Long-term success is hampered by issues in integrating
therapeutic DNA into the genome and the constantly dividing nature of many cells. Another issue
isImmune reaction- when a foreign item enters human tissue, the immune system is activated to fight
the intruder. It is possible to stimulate the immune system in a way that decreases the efficacy of gene
therapy. The immune system's increased reaction to previously encountered viruses decreases the
efficacy of repeated treatments.Further more, problems with viral vectors also pose a rather great
threat; viral vectors can cause toxicity, inflammatory reactions, and problems with gene regulation and
targeting. There are also disorders caused by several genes, variations in many genes influence certain
common illnesses, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and
diabetes, complicating treatment. Finally, cost- in 2013, Alipogene tiparvovec, or Glybera, was
claimed to be the world's most costly medicine, with a price tag of $1.6 million per patient ((Mawillo
& Ferrari, 2008).
Reference List
Crasto, AM (2013). Glybera – The Most Expensive Drug in the world & First Approved Gene
Therapy in the West. All About Drugs. [Retrieved 2 November 2013].
Ermak, G. (2015). Emerging Medical Technologies. World Scientific.
Eugene, H. (2001). Gene and Stem Cell Therapies. JAMA. 285 (5): 545–550.
Hanna, K. (2006). Germline Gene Transfer. National Human Genome Research Institute.
Mavilio, F., Ferrari, G. (2008). Genetic modification of somatic stem cells. The progress, problems
and prospects of a new therapeutic technology. EMBO Reports. 9 Suppl 1: S64–69.
Rojahn, S.Y., (2014). Genome Surgery. MIT Technology Review. [Retrieved 17 February 2014].
Sheridan, C. (2011). Gene therapy finds its niche. Nature Biotechnology. 29 (2): 121–128.
Williams, D.A., Orkin, S.H. (1986). Somatic gene therapy. Current status and future prospects. The
Journal of Clinical Investigation. 77 (4): 1053–6.
Zimmer, C. (2013). DNA Double Take. The New York Times.