Biology Investigatory

Project
Gene Therapy

Name: OV Shashank
Class: XII D
Roll No:

Certificate
Year: 2013-14
This is to certify that OV Shashank a
student of RN Podar School, of class XIID, Roll No:
, has completed his
full semester project in the fulfilment of
curriculum All India Senior Secondary
Examination
The project work entitled Gene

Therapy, is the original work done

by him during his above full
semester project.
Date:

_____________________
___________________
Internal Examiner
Principal

______________________
External Examiner
School Stamp

Acknowledgement
I take this opportunity to express my
sincere gratitude to the honourable Principal
Mrs Avnita Bir of RN Podar School for her
deep interest and guidance provided to me
during the course.
I am most grateful to our Biology teacher
Mrs Padmavathi for her great help in the
completion of this project.

Students Signature
__________________________

Table of Contents
Introduction
Gene Therapy
Targets
Isolation of gene
Gene Targeting
Gene Delivery
Case Study Cystic Fibrosis
The Disease
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 Is it a good Target
Choosing Vectors
History
Challenges
Ethical Issues
Recent Upcoming
CRISPR
Conclusion
Bibliography
Websites

Books

Introduction
Diseases
The term disease broadly refers to any condition that impairs normal function, and is
therefore associated with dysfunction of normal homeostasis. When the functioning
of one or more organs or systems of the body is adversely affected, characterised by
various signs and symptoms, we say that we are not healthy, i.e., we have a
disease.
Health can be defined as a state of complete physical, mental and social well-being.
When people are healthy, they are more efficient at work. This increases productivity
and brings economic prosperity. Health also increases longevity of people and
reduces infant and maternal mortality.

Based on the cause diseases can be broadly classified as:

Infections
These are diseases caused due to invasion of a foreign
parasitic organism. They are temporary because the immune
system of organisms can fight such pathogens (disease
causing organisms) to a certain extent hence helping in
prevention of the disease. The immune system can also be
aided with the use of several drugs. Apart from easy
treatment they can also be easily prevented

Lifestyle Diseases
Lifestyle diseases (also sometimes called diseases of longevity or diseases of
civilization interchangeably) are diseases that appear to increase in frequency as
countries become more industrialized and people live longer. They can
include Alzheimer's disease, asthma, and obesity. Diet and lifestyle are major factors
thought to influence susceptibility to many diseases. Drug abuse, tobacco smoking,
and alcohol drinking, as well as a lack of exercise may also increase the risk of
developing certain diseases, especially later in life. These diseases can be
prevented completely by living a healthy lifestyle.

Genetic Disorders
A genetic disorder is an illness caused by one or more abnormalities in the
genome, especially a condition that is present from birth (congenital). They are
medical disorders related to gene mutation.
Genetic disorders are heritable, and are passed down from the parents' genes.
Other defects may be caused by new mutations or changes to the DNA. In such
cases, the defect will only be heritable if it occurs in the germ line. The
same disease, such as some forms of cancer, may be caused by an inherited
genetic condition in some people, by new mutations in other
people, and by non-genetic causes in still other people.
These diseases are totally random and difficult to
prevent as they are not caused by external agents. Also
as their
root cause lies in the genome of the organism their cure
was thought to be impossible until the breakthrough research unlocking the secrets
of DNA leading to the development of biotechnology and hence gene therapy.

Gene Therapy
We can think of a medical condition or
illness as a "broken window." Many medical
conditions result from flaws, or mutations, in
one or more of a person's genes. Mutations
cause the protein encoded by that gene to
malfunction. When a protein malfunctions,
cells that rely on that protein's function can't
behave normally, causing problems for
whole tissues or organs. Medical conditions
related to gene mutations are called genetic
disorders.
So, if a flawed gene caused our "broken window," can we "fix" it? What are our
options?
1. Stay silent: ignore the genetic disorder and nothing gets fixed.
2. Try to treat the disorder with drugs or other approaches: depending on the
disorder, treatment may or may not be a good long-term solution.
3. Put in a normal, functioning copy of the gene: if you can do this, it may solve
the problem!
If it is successful, gene therapy provides a way to fix a problem at its source. Adding
a corrected copy of the gene may help the affected cells, tissues and organs work
properly. Gene therapy differs from traditional drug-based approaches, which may
treat the problem, but which do not repair the underlying genetic flaw.

Targets for Gene Therapy

But now a question arises, which disorders or diseases can we target using gene
therapy? Many disorders or medical conditions might be treated using gene therapy,

but others may not be suitable for this approach. For a disease to be targeted by
gene therapy it must satisfy the following conditions:
1. The condition must result from mutations in one or more genes
2. To treat a genetic flaw, the knowledge of which gene(s) to pursue is
absolutely necessary. Also a DNA copy of that gene available in the
laboratory. The best candidates for gene therapy are the so-called "singlegene" disorders - which are caused by mutations in only one gene.
3. To design the best possible approach, knowledge about how the gene
factors into the disorder is required. For example:

Which tissues are affected?

What role does the protein encoded by the gene play within the cells of
that tissue?

Exactly how do mutations in the gene affect the protein's function?

4. Adding a normal copy of the gene should fix the problem in the affected
tissue. This may seem like obvious, but it's not. What if the mutated gene
encodes a protein that prevents the normal protein from doing its job? Mutated
genes that function this way are called dominant negative and adding back the
normal protein won't fix the problem.
5. The gene delivery to cells of the affected tissue must be possible. It
depends on:

How accessible is the tissue? Is it fairly easy (skin, blood or lungs), or

more difficult to reach (internal organs)?
What is the best mode of delivery?

The techniques of biotechnology have made it possible to isolate the required gene
in the laboratory and also deliver the gene.

Isolation of Gene of Interest

The first step is to find and isolate the gene that will be inserted into the genetically
modified organism. Finding the right gene to insert usually draws on years of
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scientific research into the identity and function of useful genes. Once that is known
the DNA needs to be cut at specific locations to isolate the gene of interest. This can
be done by using restriction enzymes also known as molecular scissors which cut
DNA at specific sites containing palindromic DNA sequences. But in order to cut the
DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules.

Isolation of DNA
Since the DNA is enclosed within the membranes, we have to break the cell open to
release DNA along with other macromolecules such as RNA, proteins,
polysaccharides and also lipids. This can be achieved by
treating the bacterial cells/plant or animal tissue with
enzymes such as lysozyme (bacteria), cellulase (plant
cells), chitinase (fungus). Genes are located on long
molecules of DNA intertwined with proteins such as
histones. The RNA can be removed by treatment with
ribonuclease whereas proteins can be removed by
treatment with protease. Other molecules can be removed
by appropriate treatments and purified DNA ultimately
precipitates out after the addition of chilled ethanol. This can be seen as collection of
fine threads in the suspension.

Cutting of DNA
Restriction enzyme digestions are performed by
incubating purified DNA molecules with the
restriction enzyme, at the optimal conditions for that
specific enzyme. The cutting of DNA by restriction
endonucleases results in the fragments of DNA.
These fragments can be separated by a technique
known as gel electrophoresis. Since DNA
fragments are negatively charged molecules they
can be separated by forcing them to move towards
the anode under an electric field through a medium/matrix. The separated bands of
DNA are analysed for the required gene and then it is cut out from the agarose gel
and extracted from the gel piece. This step is known as elution.

Multiplication of Gene (PCR)

PCR or polymerase chain reaction is then used to create multiple copies of the gene
of interest. In this reaction, multiple copies of the gene (or DNA) of interest is
synthesised in vitro using two sets of primers (small chemically synthesised
oligonucleotides that are complementary to the regions of DNA) and the enzyme
DNA polymerase. The enzyme extends the primers using the nucleotides provided in
the reaction and the genomic DNA as template. If the process of replication of DNA
is repeated many times, the segment of DNA can be amplified to approximately
billion times, i.e., 1 billion copies are made.

Gene Targeting
Gene delivery is one of the biggest challenges in the field of gene therapy.
Gene Delivery includes:
1. TARGETING the right cells.
2. ACTIVATING the gene. A gene's journey is not over when it enters the cell. It
must go to the cell's nucleus and be "turned on," meaning that its transcription and
translation are activated to produce the protein product encoded by the gene. For
gene delivery to be successful, the protein that is produced must function properly.
3. INTEGRATING the gene in the cells. The gene must stay put and continue
working in the target cells. If so, it must be ensured that the gene integrates into, or
becomes part of the host cell's genetic material, or that the gene finds another way to
survive in the nucleus without being rejected.
4. AVOIDING harmful side effects. Anytime an unfamiliar biological substance is
introduced into the body, there is a risk that it will be toxic or that the body will mount
an immune response against it. If the body develops immunity against a specific
gene delivery vehicle, future rounds of the therapy will be ineffective.

Choosing the Best Vector

There is no "perfect vector" that can treat every disorder. Like any type of medical
treatment, a gene therapy vector must be customized to address the unique features
of the disorder. We have learnt the lesson, of transferring genes into plants and
animals from bacteria and viruses, which have known this for ages how to deliver
genes to transform eukaryotic cells and force them to do what the bacteria or viruses
want.
Part of the challenge in gene therapy is choosing the most suitable vector for treating
the disorder. Some vectors commonly used are:

Viruses
Usually when we think of viruses, we think of them causing diseases such as the
common cold, the flu, and HIV/AIDS. When faced with the problem of gene delivery,
scientists looked to viruses. Why reinvent the wheel if there's a perfectly good one
out there? If we can modify viruses to deliver genes without making people sick, we
may have a good set of gene therapy tools.
General advantages of viral vectors:
-

They're very good at targeting and entering cells.

-Some viral vectors might be engineered to target

specific types of cells.

destroy the cell.

They can be modified so that they can't replicate and

General drawbacks of viral vectors:

A virus can't "expand" to fit a piece of genetic material larger than it is naturally built
to carry. Therefore, some genes may be too big to fit into a certain type of virus.
Viruses can cause immune responses in patients, resulting in two potential
outcomes:

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 Patients may get sick.

A patient's immunity to a virus may prevent him from responding to repeated
treatments.
However, modern viral vectors have been engineered without most of the proteins
that would cause an immune response.

Non-Viral Vectors
Although viruses can effectively deliver genetic material into a patient's cells, they do
have some limitations. It is sometimes more efficient to deliver a gene using a nonviral vector, which has fewer size constraints and which won't generate an immune
response.
Non-viral vectors are typically circular DNA molecules, also known as plasmids. In
nature, bacteria use plasmids to transfer genes from cell to cell.
Scientists use bacteria and plasmids to easily and efficiently store and replicate
genes of interest from any organism.
Vectors used at present, are engineered in such a way that they help easy linking of
foreign DNA and selection of recombinants from non-recombinants.

These are not the only way to introduce alien DNA

into
host cells. In a method known as micro-injection, recombinant DNA is directly
injected into the nucleus of an animal cell. In another method, suitable for plants,
cells are bombarded with high velocity micro-particles of gold or tungsten coated with
DNA in a method known as biolistics or gene gun.

Delivery to specific tissues

Delivering genes to specific tissues within a patient's body can be very difficult.
Delivering genes into a group of cells in a patient's body can be done in one of two
ways.

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The first way is to inject the vector into the body and specifically target affected cells.
This is called an in vivo approach. The second way, called ex vivo, is to deliver the
gene to cells while they're outside the body by:

Isolating the desired cells from the body.

Culturing the cells in a Petri dish in the laboratory.

Delivering the genes to the cells (using one of the vector options described
on this page), activating them, and making sure that the cells integrate them
properly.

Case Study
Cystic Fibrosis
The Disease A Genetic Disorder
Cystic fibrosis (CF), also known as mucoviscidosis, is an autosomal recessive
genetic disorder that affects most critically the lungs, and also the pancreas, liver,
and intestine. It is characterized by abnormal transport of chloride and sodium across
an epithelium, leading to thick, viscous secretions, preventing the cilia from clearing
debris which cause symptoms such as coughing, poor digestion and increased
vulnerability to infection.

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CF is caused by a mutation in the gene for the

protein cystic fibrosis transmembrane conductance
regulator (CFTR) gene on chromosome 7. Most
commonly, the mutation in the CFTR gene is a
three-base-pair deletion. This protein is required to
regulate the components of sweat,
digestive fluids, and mucus.
CFTR regulates the
movement of chloride
and sodium ions
across epithelial
membranes, such as
the alveolar epithelia
located in the lungs. Since all of the cells of a CF
patient have the defective protein, large quantities of
thick, sticky mucus build up throughout the lungs
and other organs. This results in the severity of symptoms seen in CF patients.

Is It A Good Target For Gene

Therapy?
To check this some questions must be answered:

Does the condition result from mutation? Yes.

Is the biology of the disorder known? Yes.

Will adding a normal copy of the gene fix the problem in the
affected tissue? Yes. While the mutated CFTR gene encodes a non-functional ion
channel protein, it will not prevent a normal CFTR channel protein from working
properly. Therefore, adding a normal copy of the CFTR gene should fix the problem

Is it feasible to deliver the gene to the cells of the affected tissue?

Yes, in part. Treating the lungs of patients with CF might be feasible, since the lung
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surfaces are exposed to the air and somewhat easy to reach. Because the digestive
system is less accessible, however, it might be a more difficult region to treat.
Hence we can conclude that it is a perfect disease to be treated by gene therapy.

Choosing vectors
The vectors that are most suitable for gene therapy are:

Retrovirus
Retroviruses are enveloped viruses that replicate in a host cell through the process of
reverse transcription. It is a single-stranded RNA virus that stores its nucleic acid in
the form of an mRNA genome targets. Once inside the host cell cytoplasm the virus
uses its own reverse transcriptase enzyme to produce DNA from its RNA genome,
the reverse of the usual pattern, thus retro (backwards). This new DNA is then
incorporated into the host cell genome by an integrase enzyme, at which point the
retroviral DNA is referred to as a provirus. The host cell then treats the viral DNA as
part of its own genome, translating and transcribing the viral genes along with the
cell's own genes, producing the proteins required to assemble new copies of the
virus.
One drawback of retroviruses, such as the Moloney retrovirus, involves the
requirement for cells to be actively dividing for transduction. As a result, cells such as
neurons are very resistant to infection and transduction by retroviruses.
But the airway cells which are affected by the disease cystic fibrosis and must be
targeted divide infrequently. Hence Retrovirus is not a suitable vector for this disease.

Adenovirus
Adenoviruses (members of the family Adenoviridae) are medium-sized (90100 nm),
nonenveloped (without an outer lipid bilayer) viruses with anicosahedral
nucleocapsid containing a double stranded DNA genome.
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They have a broad range of vertebrate hosts and have been

cause a wide range of illnesses, from mild
respiratory infections in young children to lifethreatening multi-organ disease in people with a
weakened immune system.
But they can cause/induce an immune response in the
body hence not suitable for gene delivery.

found to

human

Herpes Simplex Virus

Herpes simplex viruses, also known as Human
herpes virus, are members of the herpes virus
family, Herpesviridae, that infect humans. They
can be spread when an infected person is
producing and shedding the virus. Herpes
Simplex can be spread through contact with
saliva, such as sharing drinks.
But these viruses only affect the cells of the nervous system and cannot infect the
airway cells and hence not suitable.

Adeno-Associated Viruses
Adeno-associated virus (AAV) is a small virus which infects humans and some
other primate species. AAV is not currently known to cause disease and
consequently the virus causes a very mild immune response.AAV can infect both
dividing and quiescent cells and persist in an extra chromosomal state without
integrating into the genome of the host cell. Despite its few disadvantages these
features make AAV a very attractive candidate for creating viral vectors for gene
therapy, and for the creation of isogenic human disease models
Hence it is the best choice for gene delivery in the case of Cystic Fibrosis.
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History of Cystic Fibrosis Gene

Therapy
Gene therapy for cystic fibrosis began in 1990, when scientists successfully
corrected faulty cystic fibrosis transmembrane conductance regulator (CFTR) genes.
They did this by adding normal copies of the gene to laboratory cell cultures.

1993
In 1993, the first experimental CF gene therapy treatment was given to a patient with
cystic fibrosis. Researchers modified a common cold Adenovirus to act as a
delivery vehicle by carrying normal genes to the CFTR cells in the nasal passages.
Researchers chose nasal passages as the site of delivery because they are easier to
access and measure gene activity than the lung airway. Later trials delivered the
vector to patients lung airways.
In the earlier trials, it had looked like the virus had entered cells and that the CTFR
gene was working. But later trials with different patients showed levels of VFTR gene
activity that were too low to make any difference. Researchers came to think that the
adenovirus cant easily enter airway cells, especially in the low doses that were being
given. In the earlier trials, they speculated, gene activity resulted from the damage to
the cells during delivery allowing the virus to enter easily.
Hence when higher doses of the virus were tried, the immune system of the patients
started mounting immune responses and fighting off the virus. This caused a
blockage in the trials until 1998.

1998
Trials using Adeno-associated virus to deliver the CTFR gene began in 1998. Unlike the
adenovirus, the Adeno-associated virus caused no immune response or adverse side effects
in patients.
But unlike the researchers predictions, the adeno-associated virus did not enter cells
efficiently and integrate into calls genomic DNA. They produced only low and fleeting
amounts of CFTR gene activity. Researchers are still working to figure out what caused the
viruses to fail.
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But because it is safe, the virus as we predicted earlier holds promise for being a good
way to deliver the CFTR gene to patients airway cells. But researchers need to learn more
about how the virus infects cells in order or make it an effective delivery method.

Challenges
Some the factors that have kept gene therapy from becoming an effective treatment
for genetic diseases are:

Short-lived nature of gene therapy - Before gene therapy can

become a permanent cure for any condition, the therapeutic DNA introduced
into target cells must remain functional and the cells containing the
therapeutic DNA must be long-lived and stable. Problems with integrating
therapeutic DNA into the genome and the rapidly dividing nature of many cells
prevent gene therapy from achieving any long-term benefits. Patients will have
to undergo multiple rounds of gene therapy.

Immune response -Anytime a foreign object is introduced into human

tissues, the immune system is designed to attack the invader. The risk of
stimulating the immune system in a way that reduces gene therapy
effectiveness is always a potential risk. Furthermore, the immune system's
enhanced response to invaders it has seen before makes it difficult for gene
therapy to be repeated in patients.

Problems with viral vectors - Viruses, while the carrier of choice in

most gene therapy studies, present a variety of potential problems to the
patient --toxicity, immune and inflammatory responses, and gene control and
targeting issues. In addition, there is always the fear that the viral vector, once
inside the patient, may recover its ability to cause disease.

Multigene disorders -Conditions or disorders that arise from mutations

in a single gene are the best candidates for gene therapy. Unfortunately, some
the most commonly occurring disorders, such as heart disease, high blood
pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the
combined effects of variations in many genes. Multigene or multifactorial

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disorders such as these would be especially difficult to treat effectively using

gene therapy.

Issues regarding
Gene Therapy
What are the possible implications of gene therapy research to society? All of us researchers, policymakers and the public - have a responsibility to explore the
potential effects of gene therapy research on our lives so that we can make informed
decisions.
For each new application of gene therapy research, we must consider:

What are the benefits?

What are the risks?

Whom will the technology help? Who will it potentially hurt?

What does gene therapy mean for us?

There are several types of issues to consider as we think about gene therapy:

Ethical issues ask us to consider the potential moral outcomes of gene

therapy research.

Legal issues require researchers and the public to help policymakers decide
whether and how gene therapy research should be regulated by the
government.

Social issues involve the impact of gene therapy research on society as a

whole.

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Some questions to ponder

When should gene therapy be used? Should it be used to treat critically ill
patients? Should it be used to treat babies and children?

What effect would gene therapy have on future generations if

germline (reproductive) cells were genetically altered? How might this
alteration affect human variation?

Who should decide what are "good" or "bad" uses of genetic

modifications? How do you define "normal" with regard to human beings?

What if we could alter human traits not associated with

disease? Would it be okay to use gene therapy to improve or enhance a person's
genetic profile?

Who will have access to gene therapy, treatments and long-term

follow-ups? Will gene therapy and genetic enhancements create an advantage for
those who can afford it?

Recent Upcoming
CRISPR

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CRISPR stands for clustered regularly

interspaced short palindromic repeats.
These RNA sequences serve an immune
function in archaea and bacteria, but in
the last year or so, scientists have seized
upon them to rewrite genes. The RNA
sequence serves as a guide to target a
DNA sequence in, say, a zygote or a
stem cell. The guide sequence leads an
enzyme, Cas9, to the DNA of interest.
Cas9 can cut the double strand, nick it, or
even knock down gene expression. After Cas9 injures the DNA, repair systems fix
the sequence - or new sequences can be inserted.
It isn't the first or only method of gene repair therapy thats been developed, but the
CRISPR technology, says Ramesar, is so special because, unlike previous methods
which were more laborious and could only target one kind of cell in the body, it
appears to be a "one size fits all delivery", adaptable for different tissues. The
procedure also seems relatively simple to perform.
Exciting as the development may be, CRISPR wont be delivering instant cures just
yet.
Ramesar says, from his initial impressions of the literature, that it would seem that
localised, accessible abnormal tissue (as in the retina or skin) could be targeted
more easily.
Conditions affecting the body more systemically, however, such as certain
developmental syndromes, or central nervous system disorders, might be
problematic in terms of getting the repair technology into enough of the target cells in
that tissue to make an effective difference.
"It may also depend on the stage one attempts to carry out the therapy, in terms of
the patients age and level of advancement of the disease," says Ramesar.

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Conclusion
Although early clinical failures led many to dismiss gene therapy as over-hyped,
clinical successes since 2006 have bolstered new optimism in the promise of gene
therapy. These include successful treatment of patients with the retinal
disease Leber's congenital amaurosis, X-linked SCID, ADA-SCID,
adrenoleukodystrophy, chronic lymphocytic leukaemia (CLL),acute lymphocytic
leukaemia (ALL),multiple myeloma, haemophilia and Parkinson's disease. These
recent clinical successes have led to a renewed interest in gene therapy, with several
articles in scientific and popular publications calling for continued investment in the
field.

Bibliography
Websites

http://en.wikipedia.org/wiki/Gene_therapy
http://www.trip2medi.com/treatmentCGeneTherapy.php
http://learn.genetics.utah.edu/content/tech/genetherapy/
http://ghr.nlm.nih.gov/handbook/therapy/
http://cystic-fibrosis.emedtv.com/cystic-fibrosis/cystic-fibrosis-genetherapy.html
http://en.wikipedia.org

Books
12th NCERT Biology
Stryer Biochemistry

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