Gene

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

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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

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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.

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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.
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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.

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 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 "single-gene" disorders - which are caused by
mutations in only one gene.

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 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.

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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
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 macro-molecules.

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

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(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

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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
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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.

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 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.
 -They can be modified so that they can't replicate
and destroy the cell.
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.

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 Viruses can cause immune responses in patients,
resulting in two potential outcomes:
 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 non-
viral 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,
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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.
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

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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.

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

15
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
surfaces are exposed to the air and somewhat easy
to reach. Because the digestive system is less

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 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

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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 (90–100 nm),
nonenveloped (without an
outer lipid bilayer) viruses
with anicosahedral
nucleocapsid containing a
double stranded DNA
genome.
They have a broad range of vertebrate hosts and have
been found to cause a wide range of illnesses, from mild
respiratory infections in young children to life-threatening
multi-organ disease in people with a weakened immune
system.
But they can cause/induce an immune response in the
human body hence not suitable for gene delivery.

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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

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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.

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
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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 can’t
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.
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.

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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
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 --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 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?

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 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.

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?

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 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
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

25
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
that’s 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 won’t 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 patient’s 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.

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Thank
you

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